Datasets:

LA1512

/

train_pubmed_ORC_4096_20k

like 0

carboxylic acids are pervasive in nature and are produced industrially on a large scale . therefore , methods of employing them in organic preparations have received a lot of attention . hard uv ( 250 nm ) irradiations generate carbon - centered radicals but also induce much degradation . conventionally , decarboxylative alkylations have relied on preparations of unappealing precursors such as peroxides , barton esters , or hunsdieker salts . recently attention shifted toward discovering catalytic systems capable of utilizing them in target - oriented syntheses . decarboxylative copper - catalyzed reactions of carboxylic acids with carbonyls and with aldimines , and of amino acids with alkynes have been reported . photoredox methods have also been used for this purpose but can suffer from difficulties in removing the catalyst . interest in heterogeneous photoredox methods for carboxylates dates back to the 1970s and the photo - kolb reaction . the chosen semiconductor can be nontoxic and can easily be removed by filtration , and when visible or soft uva light is used , the procedure can be convenient and benign . most applications of tio2 scpc have utilized aerobic conditions , resulting in selective oxidations of organic substrates or more complete substrate mineralizations . under anhydrous , anaerobic conditions and with certain substrates , tio2 scpc can lead to the generation of specific radicals adapted for molecular assembly applications . to date , however , only a modest number of such processes have been identified and developed . notably , scpc additions of enol ethers to various acceptors have been investigated , and successful additions of tertiary amines to electron - deficient alkenes , some bearing chiral auxiliaries , have been described . we recently discovered that under dry , anaerobic conditions , tio2 mediation with carboxylic acid precursors could result in carbon alkylations and annulations were achieved when photolyses of certain acid precursors were carried out in the presence of suitable electron - deficient alkenes . this paper reports advances in crucial aspects of this process in the following areas : ( 1 ) structural features in the carboxylic acids that are necessary and sufficient ; ( 2 ) the range of functionality in the alkene acceptors that can be tolerated ; ( 3 ) how the reactions respond to modifications to the catalyst form and the catalyst support ; and ( 4 ) the mechanism followed by the reaction . in our preliminary study , we discovered an experimental protocol with reasonably wide applicability utilizing degussa ( evonik ) p25 as a 15 mg ml dispersion in dried acetonitrile . this dispersion , with added carboxylic acid and substrate , in an oven - dried pyrex tube was purged with argon and then irradiated by two face - to - face sunlamp arrays ( uva ) at ambient temperature . after photolysis , the catalyst was removed by filtration and the products were isolated by conventional organic techniques . during initial investigations , n - phenylmaleimide ( 2 ) was found to be an ideal radical acceptor in this system , as it is symmetrical and photostable , its photodimerization is negligible ( under our experimental conditions ) , and its electron - withdrawing character favors addition by weakly nucleophilic radicals . this alkene was therefore chosen to test the applicabliity of our process with simple aliphatic carboxylic acids 1 . standard unoptimized conditions were adopted so a clear comparison of behavior from acid to acid could be obtained ( table 1 ) . in all instances , 0.1 mmol of 1 and 0.2 mmol of 2 were photolyzed in a suspension of the p25 catalyst ( 12 mg , 0.15 mmol ) in ch3cn ( 12 ml ) for 18 h. the reactions did indeed lead to the formation of adducts 3 incorporating the r moiety of the carboxylic acid and an additional h atom , together with significant amounts of succinimide 4 ( scheme 1 ) . for acids that decarboxylated to give methyl ( entry 1 ) or primary radicals ( entries 2 and 3 ) , the yields were low ; however , increased yields were obtained with secondary and tertiary radicals ( entries 4 and 5 ) . negligible adduct was detected for the destabilized , -type cf3 radical ( entry 6 ) . the efficiency of the process evidently increased as the stabilization energy of the released radical increased ( see the supporting information for a plot of radical stabilization energy vs yield ) . the observation of a significant amount of 4 in each case suggested that alkene 2 plays the role of an electron sink , consuming electrons in two sequential reduction protonations . we next examined a set of addition reactions with n - phenylmaleimide involving more diverse carboxylic acids ( scheme 2 ) . primary alkyl radicals stabilized by -alkoxy groups proved to be a good deal more reactive than the simple alkyl radicals . methoxy- and tert - butoxyacetic acids gave the corresponding adducts ( 3 g and 3h ) in reasonable yields of 54% and 57% , respectively , while 2-tetrahydrofuroic acid furnished a 1:1 mixture of the two diastereomers of 3i in a very pleasing yield of 75% . however , methylthioacetic acid returned only a disappointing 34% yield of the desired product 3j . moderate and poor yields were obtained with benzyl ( 57% ) and 2-thienylmethyl acids ( 22% ) , but these adducts ( 3k and 3l ) were accompanied by the dimers bibenzyl ( 18% ) and 1,2-bis(thiophen-2-yl)ethane ( 27% ) , respectively . accompanied by dimers ( see the text ) . decarboxylative additions also worked with boc - protected -amino acids , and the bottom row in scheme 2 displays the adducts and the useful yields obtained . while boc - proline was transformed into adduct 3n in a fine yield of 75% , boc - alanine and the unnatural -amino acid boc - piperidinecarboxylic acid gave much more modest yields of 38% and 29% , respectively . no retention of chirality was observed when optically pure amino acids were employed because the released radicals have planar , or close to planar , configurations at their reactive centers . surprisingly , vinylacetic , ethynylacetic , and cyanoacetic acids failed to react , even though the released radicals would be strongly stabilized . to compare the efficiencies of different forms of the titania catalyst , the reaction of phenoxyacetic acid with acrylamide , which was clean and efficient ( 82% adduct isolated ) was chosen . for each photolysis , suboptimum conditions of phenoxyacetic acid ( 0.1 mmol ) and acrylamide ( 0.2 mmol ) with irradiation for 17 h in anhydrous ch3cn ( 12 ml ) were employed . where applicable , tio2 ( 12 mg ) was employed in a 1 mg ml dispersion ( see the supporting information for full details ) . with p25 itself ( table 2 , entry 1 ) , a 47% yield of the 4-phenoxybutanamide adduct was recorded . with millenium pc500 titania , having about 6 times the surface area , the yield dropped slightly ( entry 2 ) . reactions were also carried out in schlenk tubes coated with a fine internal layer of tio2 by a sol gel process ( see the supporting information for details ) . these proved to be very efficient and led to the best yield ( entry 3 ) . tio2 photospheres consisting of hollow pyrex beads coated externally with rutile titania delivered lower conversion and adduct yields ( entry 4 ) . this was attributed to difficulties in dispersing the photospheres satisfactorily throughout the reaction flask . because of their buoyancy , they tended to accumulate at the top , and this was compounded by the fact that the high rate of magnetic stirring used in attempts to overcome this tended to result in their becoming damaged . an isolated yield of 68% was achieved upon scale - up of the reaction in the coated tube ( entry 3 ) . incorporation of 4-tert - butyl and 4-trifluoromethyl substituents in the aryl ring of the acid component furnished the corresponding adducts in similarly pleasing yields of 68% and 66% , respectively . although the sol gel - coated tubes gave the best yields and conversions , the coatings tended to detach , so they could only be used about three times . we therefore concluded that for ease of handling with conventional organic techniques , scale - up , and efficient product formation , the orthodox p25 catalyst was the best compromise . isolated yield from 5:1 acid / acrylamide irradiated for 62 h. millenium pc500 ( anatase , surface area 300 m g , particle size 510 nm ) . hollow glass spheres ( mean diameter 45 m , density 0.22 g / ml ) coated with tio2 , from microsphere technology . the reaction of aryloxyacetic acids ( 5 ) with n - substituted maleimides ( 2 ) led to n - substituted-3,4-dihydrochromenopyrrole-1,3-dione derivatives 6 , each accompanied by a significant amount of the expected adduct 7 ( scheme 3 ) . tricyclics 6 presumably resulted from cyclization of the initial adduct radical onto the aryl ring followed by rearomatization . good to excellent overall yields were obtained under all conditions ( table 3 ) . a moderate amount of succinimide ( 1143% yield ) was also formed in each case . dispersion of 1 mg ml except for dd = dense dispersion of 5 mg ml . table 3 shows that the process worked well with both electron - releasing and electron - withdrawing substituents in the aromatic ring and for methyl- and phenyl - substituted maleimides . the reactions with just p25 generally gave a slight excess of the chromenopyrrole 6 ( entries 1 , 4 , 6 , 8 , and 10 ) , except in the case of the cf3 substituent ( entry 12 ) , for which an excess of adduct 7 was observed . the selectivity for chromenopyrroles 6 was improved by using a more dense dispersion of p25 ( entries 2 , 7 , and 11 ) . on the other hand , gel tio2-coated tubes ( entries 3 , 5 , 9 , and 13 ) led to a reversal in selectivity with predominant formation of adduct 7 . the possibility to tune h coupling constants between the two protons at the junction of the pyran and dihydropyrrole rings within the range of 9.29.3 hz . this indicated a cis arrangement , in agreement with the conclusion that the reaction selectively formed the cis isomer in every case . very few syntheses of dihydrochromenopyrrole-1,3-diones 6 have been reported , but compounds with the corresponding reduced ring system , chromenopyrrolidines , are well - known biologically active species . three types of acid framework with different acceptor groups were prepared to test the applicability of p25-mediated ring closures . 2-(2-vinylphenoxy)acetic acids having alkene acceptors with ester ( 8a ) , nitrile ( 8b ) , and ketone ( 8c ) substituents ( scheme 4 ) were obtained by treatment of 2-formylphenoxyacetic acid with the appropriate phosphorane ( see the experimental section ) . c having aromatic acceptors were prepared by coupling of the appropriate 1,1-biphenyl-2-ol with methyl bromoacetate and subsequent hydrolysis of the ester with lioh in meoh / h2o . acids 13a and 13b having oxime ether acceptor groups were prepared by condensation of 2-formylphenoxyacetic acid with the appropriate alkoxylamine hydrochloride . the second used 1 ml aliquots of acid solutions ( 10 mm ) in cd3cn ( 5 ml ) in sol gel - coated nmr tubes . the tubes were purged with argon and then placed in an overhead stirrer ( turned on its side ) , which was then spun at 250 rpm . the tube was held at a slight angle with respect to horizontal in the chuck of the overhead stirrer so that , when rotated , it described a small ( ca . 2 cm ) circle at its non - chuck - held end so as to agitate the reaction solution . irradiation was performed using a photoreactor with twelve 8 w black light blue ( blb ) uv lamps ( max = 365 nm ) ( see the supporting information for a graphic ) . periodically the tube was removed for nmr analysis . this was repeated until all of the starting material had been consumed or the product concentration plateaued . for acids 8a c with alkene acceptors , control photolyses in the absence of tio2 showed that e / z isomerization was significant ( 5279% ) . in conventional p25-mediated reactions , 8a c all gave rather complex product mixtures containing unreacted alkene as a mixture of e and z isomers ( 8/9 ) as well as the 5-exo ring - closed dihydrobenzofurans 10 . nmr and gc ms analyses showed complex product mixtures containing possible trace amounts of the cyclized 6h - benzo[c]chromenes 12a c . the 2,3-dihydrobenzofuran core of 10 is found in many natural products and pharmaceutically relevant molecules and has generated much interest . yields in brackets are maximum yields from nmr monitoring of sol gel tube reactions . the reaction profile from irradiation of acid 8a in a sol gel - coated tube obtained by nmr monitoring ( figure 1 ) shows the sharp decrease in 8a ( the e isomer ) , which was all consumed in < 150 min . the z isomer 9a initially increased , passed through a maximum at about 25 min , and then steadily decreased as it too was converted to cyclic product 10a . analogous reaction profiles were obtained for the sol gel tube reactions of 8a c , 11b , and 13a . gel tube reactions of 11a and 11b . a significant yield ( 28% ) of the cyclized product n-(2,3-dihydrobenzofuranyl)-o - methylhydroxylamine ( 14a ) was found in the sol gel tube reaction of oxime ether - functionalized acid 13a ( scheme 4 ) . however , attempts to isolate and fully characterize this alkoxyamine were not successful . reaction profile for acid 8a obtained by nmr monitoring . hole capture at the tio2 surface by a carboxylate creates the corresponding acyloxyl radical . radicals of this type were directly observed by cw x - band epr spectroscopy of frozen suspensions of p25 and t - buco2h in ch3cn . transient t - bu radicals and phoch2 radicals were observed during uv irradiation of the acids t - buco2h and phoch2co2h , respectively , with pc-500 in fluid phh at 300 k. in similar epr experiments with vinylacetic acid and 2,2,2-triphenylacetic acid , we were also able to detect and characterize allyl and triphenylmethyl radicals , respectively ( see the supporting information ) . the isotropic character of the solution epr spectra of all the rxch2 radicals established that in the main they were freely tumbling and not attached to the tio2 surface ( scheme 5 ) . literature precedents imply that weakly nucleophilic rxch2 radicals should add rapidly to the electron - deficient double bonds of maleimides ( and similar acceptors ) . most likely the resulting adduct radicals 15 will be converted to enolates 16 by electron transfer from the tio2 particles . , addition produces electrophilic imidoalkyl radicals 15 , for which a competition exists between reduction to adducts 7 or homolytic closure ( 6-endo - trig ) onto the aryl ring . as radicals 15 are weakly electrophilic in character , this annulation should be favored by increasing the electron density in the ring and , conversely , disfavored by decreasing it . in agreement with this , the proportion of 6 increased when an electron - releasing tert - butyl group was introduced to the ring , whereas it decreased when an electron - withdrawing trifluoromethyl group was introduced . the resonance - stabilized cyclohexadienyl - type radicals 17 will rearomatize to yield the functionalized chromenes 6 . this aromatization could result from hole capture from tio2 by the radicals 17 and subsequent proton loss as shown . alternatively , electron transfer to maleimide might take place , as suggested in related work by hoffmann . protonation of the resulting maleimide radical anions , followed by further electron capture and protonation steps , would explain the significant yields of succinimides obtained in our reactions . however , as greater amounts of succinimides than cyclized products 6 were generally formed , it is thought that direct reduction of the maleimide acceptor by the conduction band of the p25 also initiates this process . from the results described in schemes 1 and 2 and table 1 , it can be deduced that the stability of the initial radicals is an important factor in this system . carboxylic acids dissociate on the tio2 surface to give the corresponding surface - bound carboxylate and a proton . hole pair , which can migrate through the bulk of the semiconductor to the surface . the tio2 surface is extensively hydroxylated , and it is thought that these species act as surface trapping sites for the valence - band holes . electron transfer from the system of the carboxylate to a hole trap site furnishes the surface - bound rxch2co2 radical . we propose that a competition exists between -scission of this surface - bound species to yield the desired free rxch2 radical and back transfer of the electron , either from the trap site or from trapped conduction - band electrons , to the carboxylate . for acid precursors that generate stabilized rxch2 radicals , such as those depicted in scheme 2 , the decarboxylation step is more favorable , thus explaining the improved yields and conversions recorded . conversely , for the simple aliphatic acids of table 1 , the radical stabilization energy from the rx groups is minimal , so back transfer of an electron to the tio2 will take precedence over the loss of co2 . it should be noted that while we believe radical stability to be a key aspect in this system , it is clear that there are other factors at play as well and that these take precedence in deciding the reaction outcome in a few instances . for example , vinylacetic acid would be expected to generate the allyl radical very readily in the presence of photoexcited tio2 . while we did observe the allyl radical in our epr experiments , no products at all were observed in the preparative photolysis involving vinylacetic acid and 2 . in this case , other as yet unknown factors at the tio2 surface must come into play . a crucial feature of the process is that in the formation of adducts 3 and 7 a hydrogen atom is gained from some source within the dispersion . this is not the proton lost from the aromatic ring because reduced adducts were formed equally well from acids lacking aromatic rings . the reaction of phenoxyacetic acid with acrylamide was again chosen as a test - bed process ( scheme 7 ) because it was clean and practically quantitative . when this reaction was carried out in d3-ch3cn solvent , the isolated adduct was screened for d incorporation by h and h nmr spectroscopy and gc ms . reaction with d1-phenoxyacetic acid also yielded adduct with no detectable d incorporation . thus , unless the acidic d atom of 5a rapidly and completely exchanges , the source of the additional h atom is neither the acid nor the solvent . however , it is well - established that h2o molecules and ho groups are attached to the surface of the tio2 particles . drying p25 in a vacuum at 150 c removes a significant amount of the surface h2o while leaving the chemically attached ho groups . when p25 dried in this way was used , the yield of 18 was scarcely reduced , suggesting that the oh groups were likely the proton donors . attempts were made to deuterate the h2o and oh groups on the p25 surface by refluxing in degassed d2o in a glovebox . however , it was found that back exchange occurred immediately upon exposure to air and rapidly with any moisture traces in solvents or on surfaces . when the partly deuterated p25d was photolyzed with the undeuterated acid , the phenoxybutanamide product contained no deuterium . it seemed possible , however , that p25 surface od groups could have rapidly exchanged with protons from the cooh groups of 5a . experiments with partly deuterated p25d and 5a - cood were also carried out , but deuterated 19d was again not found . in view of the partially deuterated nature of the p25d and the ease with which it reverts to the protiated form , making handling difficult , our tentative conclusion is still that the p25 is the source of the h atoms . we have found that photochemical reactions employing p25 and carboxylic acids under dry anaerobic conditions generate c - centered radicals that can be deployed in several types of c c bond - forming processes . acids that yielded alkoxyalkyl , alkylthioalkyl , and benzyl radicals alkylated electron - deficient alkenes in moderate to good yields . boc - protected amino acids furnished aminoalkyl radicals that alkylated n - phenylmaleimide in useful yields . the reactions of aryloxyacetic acids with maleimides resulted in a cascade process in which a pyrrolochromene derivative accompanied the alkylated succinimide . selectivity for one or other of these products could be tuned to some extent by employing the tio2 catalyst under different conditions . aryloxyacetic acids adapted for intramolecular ring closures by the inclusion of 2-alkenyl , 2-aryl , or 2-oximinyl functionality reacted rather poorly , especially the 2-aryl acceptor types . the mechanism ( scheme 5 ) is supported by epr spectroscopic evidence of the intermediates . experiments with deuterium - labeled solvent and reactants implied that this was the hydroxyl groups on the surface of the p25 . it appears that the titania supplies electrons and holes upon photostimulation but also donates protons from surface hydroxyl groups , thus acting as both a catalyst and a reaction partner . these photoredox reactions are cleanly , safely , and cheaply carried out in the laboratory , and the heterogeneous catalyst is simply filtered off during workup . thus , with the exception mentioned above , they constitute a useful synthetic protocol . to a suspension of semiconductor ( 15 mg ml ) in mecn ( freshly distilled over cah2 ) in an oven - dried pyrex tube were added known amounts of the desired carboxylic acid and alkene . the mixture was then irradiated with eight 29 cm 15 w philips cleo tubes ( = 350 nm ) for the desired reaction time at ambient temperature . following irradiation , the solvent was removed under reduced pressure , and the reaction mixture was purified by chromatography . yields were determined from the amounts of isolated products and/or from the h nmr spectra by reference to ch2br2 as an internal standard . a photoreactor consisting of two lots of six 8 w blb uv lamps ( emission max = 365 nm ) , each arranged in a semicylinder with an aluminum reflector , was used to irradiate the contents of the nmr tube . the two photoreactor semicylinders were brought together to surround the nmr tube ( see the supporting information for a graphic ) . for a typical reaction , a stock solution comprising acid ( 10 mm ) in d3-ch3cn was prepared . this solution was purged with argon for 5 min , and then 1 ml was pipetted into an argon - flushed sol gel - coated nmr tube . the loaded nmr tube was placed in an overhead stirrer ( turned on its side ) , which was spun at 250 rpm . the tube was held at a slight angle with respect to horizontal in the chuck of the overhead stirrer so that , when rotated , it described a small ( ca . 2 cm ) circle at its non - chuck - held end so as to agitate the reaction solution . once the nmr tube was in place , the irradiation was started , and the tube was periodically removed and analyzed by nmr . these experiments were carried out in a similar fashion to the photoredox experiments outlined above . following photolysis , the reaction mixtures were scrutinized for deuterium incorporation by h and h nmr spectroscopy and gc ms . further details of these experiments are provided in the supporting information . acetic acid ( 6 mg , 0.1 mmol ) , n - phenylmaleimide ( 34.7 mg , 0.2 mmol ) , and tio2 ( 12 mg , 0.15 mmol ) were reacted in accordance with the general procedure to yield 3a ( 24% by nmr ) as a white solid . h nmr ( 400 mhz , cdcl3 , 296 k ) : 1.44 ( d , j = 7.2 hz , 3h ) , 2.472.54 ( m , 1h ) , 2.973.14 ( m , 2h ) , 7.277.30 ( m , 2h ) , 7.367.42 ( m , 1h ) , 7.447.50 ( m , 2h ) . c nmr ( 100 mhz , cdcl3 , 297 k ) : 16.9 , 34.9 , 36.7 , 126.4 , 128.6 , 129.2 , 132.0 , 175.5 , 179.6 . hr - esims : m / z 190.0861 ( calcd for c11h12no2 , 190.0863 ) . also observed was n - phenylsuccinimide ( 41% by nmr ) . n - hexanoic acid ( 11.6 mg , 0.1 mmol ) , n - phenylmaleimide ( 34.7 mg , 0.2 mmol ) , and tio2 ( 12 mg , 0.15 mmol ) were reacted in accordance with the general procedure . following irradiation , a yield of 4% ( 0.04 mmol ) was determined for 3b . yields were determined by assigning peaks a and b by analogy with the previously characterized 3-ethyl adduct.h nmr ( 500 mhz , cdcl3 , 295 k ) : 2.60 ( dd , j = 3.3 , 17.0 hz , 1h ) , 2.973.07 ( m , 1h ) . also observed were n - phenylsuccinimide ( 4% by nmr ) and unreacted n - hexanoic acid ( 65% conversion by nmr ) . tert - butylacetic acid ( 11.6 mg , 0.1 mmol ) , n - phenylmaleimide ( 34.7 mg , 0.2 mmol ) , and tio2 ( 12 mg , 0.15 mmol ) were reacted in accordance with the general procedure to yield 3c ( 18% by nmr ) as a white solid . h nmr ( 400 mhz , cdcl3 , 296 k ) : 0.94 ( s , 9h ) , 1.36 ( dd , j = 10.7 , 14.0 hz , 1h ) , 2.12 ( dd , j = 2.0 , 13.9 hz , 1h ) , 2.54 ( dd , j = 5.3 , 18.1 hz , 1h ) , 3.03 ( dd , j = 9.1 , 18.1 hz , 1h ) , 2.842.92 ( m , 1h ) , 7.197.22 ( m , 2h ) , 7.297.34 ( m , 1h ) , 7.377.42 ( m , 2h ) . c nmr ( 100 mhz , cdcl3 , 297 k ) : 29.6 , 29.7 , 31.0 , 37.6 , 45.9 , 126.4 , 128.6 , 129.2 , 132.1 , 175.8 , 179.8 . hr - esims : m / z 246.1486 ( calcd for c15h20no2 , 246.1489 ) . also observed was n - phenylsuccinimide ( 40% by nmr ) . isobutyric acid ( 8.1 mg , 0.1 mmol ) , n - phenylmaleimide ( 34.7 mg , 0.2 mmol ) , and tio2 ( 12 mg , 0.15 mmol ) were reacted in accordance with the general procedure to yield 3d ( 25% by nmr ) as a white solid . h nmr ( 400 mhz , cdcl3 , 297 k ) : 0.99 ( d , j = 6.8 hz , 3h ) , 1.07 ( d , j = 6.9 hz , 3h ) , 2.322.40 ( m , 1h ) , 2.56 ( dd , j = 4.4 , 18.3 hz , 1h ) , 2.79 ( dd , j = 9.4 , 18.3 hz , 1h ) , 2.872.92 ( m , 1h ) , 7.187.21 ( m , 2h ) , 7.297.34 ( m , 1h ) , 7.377.43 ( m , 2h ) . c nmr ( 100 mhz , cdcl3 , 300 k ) : 17.3 , 20.0 , 29.2 , 30.6 , 45.8 , 126.5 , 128.6 , 129.2 , 131.9 , 175.9 , 178.4 . hr - esims : m / z 218.1178 ( calcd for c13h16no2 , 218.1176 ) . also observed was n - phenylsuccinimide ( 25% by nmr ) . pivalic acid ( 10.2 mg , 0.1 mmol ) , n - phenylmaleimide ( 34.7 mg , 0.2 mmol ) , and tio2 ( 12 mg , 0.15 mmol ) were reacted in accordance with the general procedure to yield 3e ( 38% by nmr ) as a white solid . h nmr ( 400 mhz , cdcl3 , 300 k ) : 1.12 ( s , 9h ) , 2.672.80 ( m , 2h ) , 2.89 ( dd , j = 8.8 , 17.6 hz , 1h ) , 7.247.26 ( m , 2h ) , 7.367.41 ( m , 1h ) , 7.457.49 ( m , 2h ) . c nmr ( 100 mhz , cdcl3 , 300 k ) : 27.2 , 32.0 , 33.8 , 49.9 , 126.6 , 128.6 , 129.2 , 132.0 , 175.6 , 177.4 . hr - esims : m / z 232.1326 ( calcd for c14h18no2 , 232.1332 ) . also observed was n - phenylsuccinimide ( 31% by nmr ) . trifluoroacetic acid ( 11.4 mg , 0.1 mmol ) , n - phenylmaleimide ( 34.7 mg , 0.2 mmol ) , and tio2 ( 12 mg , 0.15 mmol ) were reacted in accordance with the general procedure to yield 3f ( 1% by nmr ) . h nmr ( 400 mhz , cdcl3 , 295 k ) : 2.86 ( dd , j = 5.5 , 18.6 hz , 1h ) , 2.99 ( dd , j = 9.5 , 18.6 hz , 1h ) , 3.61 ( m , 1h ) , 7.10 ( m , 2h ) , 7.39 ( m , 3h ) . f nmr ( 375 mhz , cdcl3 , 296 k ) : 69.3 ( s , 3f ) methoxyacetic acid ( 54.4 mg , 0.63 mmol ) , n - phenylmaleimide ( 433.6 mg , 2.50 mmol ) , and tio2 ( 75 mg , 0.94 mmol ) were reacted in accordance with the general procedure . following irradiation for 18 h , the reaction mixture was purified by column chromatography on silica gel ( eluent : 30% etoac in petroleum ether 4060 ) to yield 3 g as a white powder ( 74.4 mg , 54% ) . h nmr ( 400 mhz , cdcl3 , 299 k ) : 2.862.99 ( m , 2h ) , 3.083.15 ( m , 1h ) , 3.38 ( s , 3h ) , 3.36 ( dd , j = 3.3 , 9.1 hz , 1h ) , 3.91 ( dd , j = 3.9 , 9.1 hz , 1h ) , 7.267.30 ( m , 2h ) , 7.367.41 ( m , 1h ) , 7.447.49 ( m , 2h ) . c nmr ( 75 mhz , cdcl3 , 298 k ) : 31.9 , 41.2 , 59.3 , 70.8 , 126.6 , 128.6 , 129.1 , 132.1 , 175.8 , 177.3 . tert - butoxyacetic acid ( 87.3 mg , 0.66 mmol ) , n - phenylmaleimide ( 462.3 mg , 2.67 mmol ) , and tio2 ( 80 mg , 1.0 mmol ) were reacted in accordance with the general procedure . following irradiation for 17 h , h nmr analysis revealed that n - phenylmaleimide had been consumed entirely while a significant quantity of tert - butoxyacetic acid remained . a further 462.3 mg of n - phenylmaleimide was hence added , and the reaction was photolyzed for another 18 h. the reaction mixture was then purified by column chromatography on silica gel ( eluent : 25% etoac in pentanes ) to yield 3h as a yellow oil ( 97.8 mg , 57% ) . h nmr ( 500 mhz , cdcl3 , 295 k ) : 1.18 ( s , 9h ) , 2.852.89 ( m , 2h ) , 3.073.11 ( m , 1h ) , 3.59 ( dd , j = 2.9 , 8.5 hz , 1h ) , 3.91 ( dd , j = 3.5 , 8.5 hz , 1h ) , 7.27 ( d , j = 7.4 hz , 2h ) , 7.39 ( t , j = 7.4 hz , 1h ) , 7.48 ( t , j = 7.4 hz , 2h ) . c nmr ( 75 mhz , cdcl3 , 297 k ) : 27.4 , 32.3 , 41.3 , 60.7 , 73.3 , 126.6 , 128.6 , 129.2 , 134.2 , 176.3 , 178.0 . hr - esims : m / z 262.1440 ( calcd for c15h20no3 , 262.1438 ) . 2-tetrahydrofuroic acid ( 76.6 mg , 0.66 mmol ) , n - phenylmaleimide ( 462.3 mg , 2.67 mmol ) , and tio2 ( 80 mg , 1.00 mmol ) were reacted in accordance with the general procedure . following irradiation for 18 h , the reaction mixture was purified by column chromatography on silica gel ( eluent : et2o ) to yield a 1:1 mixture of the two diastereoisomers of 3i as a yellow oil ( 121.8 mg , 75% ) . diastereomer 1 : h nmr ( 400 mhz , cdcl3 , 295 k ) : 1.922.02 ( m , 2h ) , 2.022.12 ( m , 2h ) , 2.792.84 ( m , 1h ) , 2.99 ( dd , j = 9.3 , 18.3 hz , 1h ) , 3.263.30 ( m , 1h ) , 3.763.84 ( m , 2h ) , 4.234.27 ( m , 1h ) , 7.267.32 ( m , 2h ) , 7.377.42 ( m , 1h ) , 7.457.51 ( m , 2h ) . c nmr ( 100 mhz , cdcl3 , 297 k ) : 25.9 , 27.9 , 32.7 , 43.4 , 68.8 , 79.1 , 126.6 , 128.6 , 129.1 , 132.0 , 175.7 , 175.9 . diastereomer 2 : h nmr ( 500 mhz , cdcl3 , 295 k ) : 1.631.71 ( m , 1h ) , 1.922.02 ( m , 2h ) , 2.102.19 ( m , 1h ) , 2.792.84 ( m , 2h ) , 3.083.12 ( m , 1h ) , 3.863.94 ( m , 2h ) , 4.394.44 ( m , 1h ) , 7.267.32 ( m , 2h ) , 7.377.42 ( m , 1h ) , 7.457.51 ( m , 2h ) . c nmr ( 100 mhz , cdcl3 , 297 k ) : 25.9 , 29.7 , 29.8 , 44.4 , 68.8 , 77.3 , 126.6 , 128.6 , 129.1 , 132.0 , 176.5 , 177.4 . gc ms : for tr = 15.75 min , m / z ( % ) 245 ( 38 ) , 217 ( 10 ) , 202 ( 58 ) , 175 ( 100 ) , 147 ( 10 ) , 119 ( 28 ) , 111 ( 11 ) , 71 ( 98 ) ; for tr = 16.20 min , m / z ( % ) 245 ( 51 ) , 217 ( 11 ) , 202 ( 33 ) , 175 ( 81 ) , 147 ( 6 ) , 119 ( 22 ) , 111 ( 12 ) , 71 ( 100 ) . hr - esims : m / z 246.1127 ( calcd for c14h16no3 , 246.1125 ) . methylthioacetic acid ( 70.8 mg , 0.67 mmol ) , n - phenylmaleimide ( 462.3 mg , 2.67 mmol ) , and tio2 ( 80 mg , 1.00 mmol ) were reacted in accordance with the general procedure . following irradiation for 42 h , the reaction mixture was purified by column chromatography on silica gel ( eluent : gradient of 2040% etoac in petroleum ether 4060 ) to yield 3j as a white powder ( 53.6 mg , 34% ) . h nmr ( 500 mhz , cdcl3 , 299 k ) : 2.14 ( s , 3h ) , 2.84 ( dd , 1h , j = 5.0 , 18.4 hz ) , 2.92 ( dd , 1h , j = 7.4 , 13.5 hz ) , 3.01 ( dd , 1h , j = 9.2 , 18.4 hz ) , 3.04 ( dd , 1h , j = 4.1 , 13.5 hz ) , 3.213.28 ( m , 1h ) , 7.227.27 ( m , 2h ) , 7.357.39 ( m , 1h ) , 7.427.47 ( m , 2h ) . c nmr ( 75 mhz , cdcl3 , 297 k ) : 16.5 , 33.7 , 35.5 , 40.1 , 126.5 , 128.7 , 129.2 , 131.9 , 175.2 , 177.5 . hr - esims : m / z 236.0741 ( calcd for c12h14no2s , 236.0740 ) . 2-thiopheneacetic acid ( 93.9 mg , 0.66 mmol ) , n - phenylmaleimide ( 462.3 mg , 2.67 mmol ) , and tio2 ( 80 mg , 1.00 mmol ) were reacted in accordance with the general procedure . following irradiation for 17 h , the reaction mixture was purified by column chromatography on silica gel ( eluent : 20% etoac in pentanes ) to yield 3k as a yellow oil ( 45.9 mg , 26% ) . h nmr ( 500 mhz , cdcl3 , 295 k ) : 2.72 ( dd , j = 4.8 , 18.4 hz , 1h ) , 2.98 ( dd , j = 9.2 , 18.4 hz , 1h ) , 3.303.35 ( m , 1h ) , 3.373.45 ( m , 2h ) , 6.89 ( m , 1h ) , 6.98 ( m , 1h ) , 7.20 ( m , 2h ) , 7.39 ( m , 1h ) , 7.46 ( m , 2h ) . c nmr ( 75 mhz , cdcl3 , 297 k ) 30.7 , 33.4 , 41.3 , 125.0 , 126.5 , 127.0 , 127.3 , 128.7 , 129.2 , 131.8 , 138.2 , 175.2 , 177.8 , 177.5 . hr - esims : m / z 272.0743 ( calcd for c15h14no2s , 272.0740 ) . 1,2-bis(thiophen-2-yl)ethane was also isolated as a clear oil ( 14.1 mg , 22% ) . h nmr ( 500 mhz , cdcl3 , 295 k ) : 3.20 ( s , 4h ) , 6.80 ( d , j = 3.4 hz , 2h ) , 6.916.94 ( m , 2h ) , 7.14 ( d , j = 5.2 hz , 2h ) . data are consistent with literature values . phenylacetic acid ( 89.9 mg , 0.66 mmol ) , n - phenylmaleimide ( 462.3 mg , 2.67 mmol ) , and tio2 ( 80 mg , 1.00 mmol ) were reacted in accordance with the general procedure . following irradiation for 22 h , the reaction mixture was purified by dry flash chromatography on silica gel ( eluent : gradient of 1020% etoac in ch2cl2 ) to yield 3l as a white powder ( 99.3 mg , 57% ) . h nmr ( 500 mhz , cdcl3 , 295 k ) : 2.65 ( dd , j = 4.7 , 18.5 hz , 1h ) , 2.88 ( dd , j = 9.2 , 18.5 hz , 1h ) , 3.08 ( dd , j = 8.0 , 13.8 hz , 1h ) , 3.25 ( dd , j = 4.4 , 13.8 hz , 1h ) , 3.303.35 ( m , 1h ) , 7.17 ( m , 2h ) , 7.22 ( m , 2h ) , 7.29 ( m , 1h ) , 7.34 ( m , 2h ) , 7.39 ( m , 1h ) , 7.46 ( m , 2h ) . c nmr ( 75 mhz , cdcl3 , 297 k ) : 33.5 , 36.7 , 41.4 , 77.2 , 126.6 , 127.4 , 128.8 , 129.0 , 129.3 , 129.4 , 132.0 , 136.8 , 175.4 , 178.4 . data are consistent with literature values . bibenzyl ( 10.9 mg , 18% ) was observed by nmr prior to purification of the reaction mixture . h nmr ( 500 mhz , cdcl3 , 298 k ) : 2.95 ( s , 4h ) , 7.217.24 ( m , 6h ) , 7.307.33 ( m , 4h ) . n - boc - l - alanine ( 126.2 mg , 0.67 mmol ) , n - phenylmaleimide ( 462.3 mg , 2.67 mmol ) , and tio2 ( 80 mg , 1.00 mmol ) were reacted in accordance with the general procedure . following irradiation for 18 h , the mixture was purified by column chromatography on silica gel ( eluent : gradient of 2040% etoac in petroleum ether 4060 ) to yield two diastereomers of 3 m . diastereomer 1 was isolated as a white powder ( 36.7 mg , 18% ) . h nmr ( 400 mhz , cdcl3 , 296 k ) : 1.29 ( d , j = 6.9 hz , 3h ) , 1.38 ( s , 9h ) , 2.78 ( dd , j = 4.8 , 18.6 hz , 1h ) , 2.89 ( dd , j = 9.1 , 18.5 hz , 1h ) , 3.153.20 ( m , 1h ) , 4.084.18 ( m , 1h ) , 4.53 ( bs , 1h ) , 7.187.22 ( m , 2h ) , 7.307.35 ( m , 1h ) , 7.387.43 ( m , 2h ) . c nmr ( 75 mhz , cdcl3 , 297 k ) : 18.7 , 28.3 , 32.2 , 45.3 , 47.7 , 80.2 , 126.5 , 128.7 , 129.2 , 131.8 , 155.5 , 175.3 , 176.6 . hr - esims : m / z 341.1467 ( calcd for c17h22o4n2na , 341.1472 ) . diastereomer 2 ( 20% wrt ch2br2 standard ) was obtained as a 1:1 mixture with n - phenylsuccinimide . h nmr ( 500 mhz , cdcl3 , 297 k ) : 1.25 ( d , j = 6.8 hz , 3h ) , 1.44 ( s , 9h ) , 2.63 ( dd , j = 4.5 , 18.6 hz , 1h ) , 3.00 ( dd , j = 9.6 , 18.5 hz , 1h ) , 3.173.23 ( m , 1h ) , 4.134.17 ( m , 1h ) , 5.20 ( bs , 1h ) , 7.277.30 ( m , 2h ) , 7.397.42 ( m , 1h ) , 7.467.50 ( m , 2h ) . c nmr ( 75 mhz , cdcl3 , 297 k ) : 17.1 , 28.5 , 31.9 , 45.1 , 47.1 , 80.0 , 126.6 , 128.8 , 129.3 , 131.8 , 155.2 , 175.2 , 177.5 . hr - esims : m / z 341.1475 ( calcd for c17h22o4n2na , 341.1472 ) . n - boc - l - proline ( 144.3 mg , 0.67 mmol ) , n - phenylmaleimide ( 462.3 mg , 2.67 mmol ) , and tio2 ( 80 mg , 1.00 mmol ) were reacted in accordance with the general procedure . following irradiation for 17 h , the crude mixture was purified by dry flash chromatography on silica gel ( eluent : gradient of 2060% etoac in petroleum ether 4060 ) to yield an inseparable mixture of two diastereomers of 3n ( 171 mg , 75% , 1:1 ratio ) that also contained 7% n - phenylsuccinimide . h nmr ( 500 mhz , cdcl3 , 326 k ) : diastereomer 1 1.44 ( s , 9h ) , 1.941.99 ( m , 2h ) , 2.182.24 ( m , 2h ) , 2.732.75 ( m , 1h ) , 2.83 ( dd , j = 3.8 , 9.4 hz , 1h ) , 3.53 ( dd , j = 7.3 , 11.1 hz , 1h ) , 3.793.85 ( m , 2h ) , 4.344.39 ( m , 2h ) , 7.277.47 ( m , 5h ) ; diastereomer 2 1.49 ( s , 9h ) , 2.142.17 ( m , 2h ) , 1.661.71 ( m , 2h ) , 2.692.71 ( m , 1h ) , 2.86 ( dd , j = 4.3 , 9.7 hz , 1h ) , 3.30 ( dd , j = 3.4 , 7.8 hz , 1h ) , 3.953.99 ( m , 2h ) , 4.314.35 ( m , 2h ) , 7.277.47 ( m , 5h ) . c nmr ( 75 mhz , cdcl3 , 300 k ) : 23.324.0 , 27.9 , 28.4 , 28.5 , 29.730.1 , 31.8 , 43.744.1 , 47.247.8 , 57.157.8 , 79.980.5 , 126.5126.8 , 128.4129.2 , 131.2132.0 , 155.0156.0 , 174.1177.3 . gc ms : for tr = 19.81 min , m / z ( % ) 344 ( 2 ) , 288 ( 19 ) , 271 ( 17 ) , 243 ( 48 ) , 175 ( 16 ) , 114 ( 50 ) , 70 ( 100 ) 57 ( 41 ) ; for tr = 20.09 min , m / z ( % ) 344 ( 2 ) , 288 ( 19 ) , 271 ( 17 ) , 243 ( 48 ) , 175 ( 16 ) , 114 ( 50 ) , 70 ( 100 ) 57 ( 41 ) . hr - esims : m / z 367.1632 ( calcd for c19h24n2o4na , 367.1628 ) . n - boc - d / l-2-piperidinecarboxylic acid ( 151.4 mg , 0.67 mmol ) , n - phenylmaleimide ( 462.3 mg , 2.67 mmol ) , and tio2 ( 80 mg , 1.00 mmol ) were reacted in accordance with the general procedure . following irradiation for 18 h , the crude mixture was purified by column chromatography on silica gel ( eluent : 30% etoac in pentanes ) to yield two diastereomers of 3o . diastereomer 1 was isolated as an off - white solid ( 32.7 mg , 14% ) . h nmr ( 500 mhz , cdcl3 , 295 k ) : 1.39 ( s , 9h ) , 1.461.59 ( bm , 2h ) , 1.671.79 ( bm , 4h ) , 2.52 ( dd , j = 3.0 , 18.2 hz , 1h ) , 2.98 ( dd , j = 8.9 , 18.2 hz , 1h ) , 3.04 ( t , j = 13.2 hz , 1h ) , 3.263.30 ( m , 1h ) , 4.06 ( d , j = 13.2 hz , 1h ) , 4.344.37 ( m , 1h ) , 7.327.36 ( m , 3h ) , 7.417.45 ( m , 2h ) . c nmr ( 75 mhz , cdcl3 , 297 k ) : 18.9 , 24.8 , 27.0 , 28.4 , 34.2 , 38.1 , 40.2 , 53.3 , 79.9 , 126.7 , 128.4 , 128.9 , 132.1 , 155.5 , 174.9 , 176.1 . hr - esims : m / z 381.1787 ( calcd for c20h26n2o4na , 381.1785 ) . diastereomer 2 ( 15% wrt ch2br2 standard ) was obtained as a 1:2 mixture with n - phenylsuccinimide . h nmr ( 500 mhz , cdcl3 , 295 k ) : 1.42 ( s , 9h ) , 1.53 ( m , 1h ) , 1.681.72 ( m , 1h ) , 1.901.96 ( m , 1h ) , 2.212.23 ( m , 1h ) , 2.402.45 ( m , 1h ) , 2.542.58 ( m , 1h ) , 2.95 ( dd , j = 4.4 , 19.0 hz , 1h ) , 3.05 ( dd , j = 9.6 , 17.6 hz , 1h ) , 3.26 ( dd , j = 9.1 , 19.0 hz , 1h ) , 3.35 ( dd , j = 6.3 , 17.6 hz , 1h ) , 3.373.42 ( m , 1h ) , 3.943.98 ( m , 1h ) , 7.387.42 ( m , 3h ) , 7.447.49 ( m , 2h ) . c nmr ( 125 mhz , cdcl3 , 295 k ) : 19.6 , 24.4 , 24.6 , 28.3 , 33.4 , 37.8 , 39.5 , 46.1 , 82.5 , 126.4 , 128.5 , 129.1 , 131.4 , 152.6 , 175.2 , 175.5 . hr - esims : m / z 381.1787 ( calcd for c20h26n2o4na , 381.1785 ) . cyanoacetic acid ( 56.2 mg , 0.67 mmol ) , n - phenylmaleimide ( 462.3 mg , 2.67 mmol ) , and tio2 ( 80 mg , 1.00 mmol ) were reacted in accordance with the general procedure . following irradiation for 18 h , the reaction mixture was scrutinized by h nmr and gc ms , but none of the desired product was obtained . phenoxyacetic acid ( 100 mg , 0.65 mmol ) , n - methylmaleimide ( 145 mg , 1.3 mmol ) , and tio2 ( 75 mg , 0.98 mmol ) were reacted in accordance with the general procedure . following irradiation for 20 h , the crude mixture was purified by column chromatography on silica gel ( eluent : 20% etoac in petroleum ether 4060 ) to yield 7a as a colorless oil ( 32.6 mg , 23% ) . h nmr ( 400 mhz , cdcl3 , 297 k ) : 2.882.90 ( m , 2h ) , 3.04 ( s , 3h ) , 3.173.23 ( m , 1h ) , 4.17 ( dd , j = 3.4 , 9.2 hz , 1h ) , 4.40 ( dd , j = 4.4 , 9.2 hz , 1h ) , 6.87 ( d , j = 8.8 hz , 2h ) , 6.98 ( t , j = 7.4 hz , 1h ) , 7.28 ( t , j = 7.4 hz , 2h ) . c nmr ( 75 mhz , cdcl3 , 297 k ) : 25.4 , 32.1 , 41.1 , 66.4 , 115.1 , 122.0 , 130.0 , 158.5 , 177.0 178.0 . 6a was also obtained as a white powder ( 77.8 mg , 55% ) . h nmr ( 400 mhz , cdcl3 , 296 k ) : 3.00 ( s , 3h ) , 3.363.40 ( m , 1h ) , 4.01 ( dd , j = 4.2 , 11.4 hz , 1h ) , 4.07 ( d , j = 9.2 hz , 1h ) , 4.62 ( dd , j = 2.9 , 11.4 hz , 1h ) , 6.89 ( d , j = 8.2 hz , 1h ) , 7.05 ( t , j = 7.5 hz , 1h ) , 7.21 ( t , j = 7.7 hz , 1h ) , 7.58 ( d , j = 7.4 hz , 1h ) . c nmr ( 75 mhz , cdcl3 , 297 k ) : 27.8 , 40.1 , 42.7 , 64.3 , 118.1 , 118.3 , 123.2 , 129.4 , 130.5 , 155.7 , 176.6 , 177.2 . data are consistent with literature values . phenoxyacetic acid ( 100 mg , 0.65 mmol ) , n - phenylmaleimide ( 230 mg , 1.3 mmol ) , and tio2 ( 75 mg , 0.98 mmol ) were reacted in accordance with the general procedure . following irradiation for 11 h , the crude mixture was purified by column chromatography on silica gel ( eluent : 20% etoac in petroleum ether 4060 ) to yield 7b as a yellow oil ( 71.5 mg , 39% ) . h nmr ( 400 mhz , cdcl3 , 297 k ) : 3.013.07 ( m , 2h ) , 3.333.39 ( m , 1h ) , 4.28 ( dd , j = 3.2 , 9.0 hz , 1h ) , 4.55 ( dd , j = 4.1 , 9.1 hz , 1h ) , 6.91 ( d , j = 8.8 hz , 2h ) , 7.00 ( t , j = 7.3 hz , 1h ) , 7.287.33 ( m , 2h ) , 7.41 ( t , j = 7.4 hz , 1h ) , 7.48 ( d , j = 7.2 , 2h ) . c nmr ( 75 mhz , cdcl3 , 297 k ) : 32.4 , 41.1 , 66.9 , 115.1 , 122.1 , 127.0 , 129.2 , 129.7 , 130.0 , 132.4 , 158.5 , 175.9 , 177.1 . 6b was also obtained as a white powder ( 72.8 mg , 40% ) . h nmr ( 400 mhz , cdcl3 , 297 k ) : 3.543.58 ( m , 1h ) , 4.11 , ( dd , j = 4.4 , 11.4 hz , 1h ) , 4.23 ( d , j = 9.3 hz , 1h ) , 4.7 ( dd , j = 3.3 , 11.4 hz , 1h ) , 6.94 ( d , j = 8.2 hz , 1h ) , 7.07 ( t , j = 7.5 hz , 1h ) , 7.227.27 ( m , 2h ) , 7.37 ( t , j = 7.4 hz , 2h ) , 7.44 ( t , j = 7.1 hz , 1h ) , 7.63 ( d , j = 7.4 hz , 1h ) . c nmr ( 75 mhz , cdcl3 , 297 k ) : 40.2 , 42.6 , 64.4 , 77.1 , 77.5 , 77.9 , 117.7 , 118.3 , 123.3 , 126.7 , 129.2 , 129.5 , 129.6 , 130.7 , 132.1 155.7 175.5 , 176.1 . 4-(tert - butyl)phenoxyacetic acid ( 130.3 mg , 0.62 mmol ) , n - methylmaleimide ( 275.6 mg , 2.48 mmol ) , and tio2 ( 75 mg , 0.98 mmol ) were reacted in accordance with the general procedure . following irradiation for 22 h , the reaction mixture was purified by column chromatography on silica gel ( eluent : 20% etoac in petroleum ether 4060 ) to yield 7c as a clear oil ( 18.4 mg , 11% ) . h nmr ( 400 mhz , cdcl3 , 296 k ) : 1.29 ( s , 9h ) , 2.842.89 ( m , 2h ) , 3.03 ( s , 3h ) , 3.213.16 ( m , 1h ) , 4.16 ( dd , j = 3.4 , 9.2 hz , 1h ) , 4.39 ( dd , j = 4.4 , 9.2 hz , 1h ) , 6.81 ( d , j = 8.8 hz , 2h ) , 7.29 ( d , j = 8.8 hz , 2h ) . c nmr ( 100 mhz , cdcl3 , 296 k ) : 25.0 , 29.7 , 31.5 , 34.1 , 40.7 , 66.1 , 114.2 , 126.4 , 144.4 , 155.8 , 176.6 , 177.6 . hr - esims : m / z 276.1599 ( calcd for c16h22no3 , 276.1594 ) . 6c was also obtained as a white powder ( 77.2 mg , 46% ) . h nmr ( 400 mhz , cdcl3 , 296 k ) : 1.32 ( s , 9h ) , 2.98 ( s , 3h ) , 3.333.37 ( m , 1h ) , 3.98 ( dd , j = 4.1 , 11.3 hz , 1h ) , 4.05 ( d , j = 9.2 hz , 1h ) , 4.59 ( dd , j = 3.0 , 11.3 hz , 1h ) , 6.82 ( d , j = 8.6 hz , 1h ) , 7.23 ( dd , j = 2.4 , 8.6 hz , 1h ) , 7.58 ( d , j = 2.4 hz , 1h ) . c nmr ( 75 mhz , cdcl3 , 296 k ) : 25.8 , 31.9 , 34.8 , 40.3 , 42.7 , 64.3 , 117.1 , 117.6 , 126.5 , 127.3 , 146.0 , 153.3 , 176.7 , 177.3 . hr - esims : m / z 274.1442 ( calcd for c16h20no3 , 274.1438 ) . 4-(tert - butyl)phenoxyacetic acid ( 70 mg , 0.33 mmol ) , n - phenylmaleimide ( 117 mg , 0.66 mmol ) , and tio2 ( 40 mg , 0.5 mmol ) were reacted in accordance with the general procedure . following irradiation for 19 h , the crude mixture was purified by column chromatography on silica gel ( eluent : 20% etoac in petroleum ether 4060 ) to yield 7d as a clear oil ( 31.2 mg , 28% ) . h nmr ( 300 mhz , cdcl3 , 295 k ) : 1.30 ( s , 9h ) , 2.983.13 ( m , 2h ) , 3.303.37 ( m , 1h ) , 4.23 ( dd , j = 3.2 , 9.1 hz , 1h ) , 4.53 ( dd , j = 3.9 , 9.1 hz , 1h ) , 6.85 ( d , j = 8.9 hz , 2h ) , 7.277.33 ( m , 3h ) , 7.407.51 ( m , 4h ) . c nmr ( 75 mhz , cdcl3 , 297 k ) : 29.7 , 31.5 , 34.2 , 40.8 , 66.6 , 114.2 , 126.4 , 126.6 , 128.7 , 129.2 , 132.0 , 144.6 , 155.8 , 175.6 , 176.8 . hr - esims : m / z 338.1755 ( calcd for c21h24no3 , 338.1751 ) . 6d was also obtained as a clear oil ( 50.3 mg , 46% ) . h nmr ( 300 mhz , cdcl3 , 296 k ) : 1.32 ( s , 9h ) , 3.513.56 ( m , 1h ) , 4.10 ( dd , j = 4.3 , 11.4 hz , 1h ) , 4.23 ( d , j = 7.3 hz , 1h ) , 4.66 ( dd , j = 3.3 , 11.5 hz , 1h ) , 6.87 ( d , j = 8.1 hz , 1h ) , 7.247.29 ( m , 2h ) , 7.337.48 ( m , 4h ) . c nmr ( 75 mhz , cdcl3 , 297 k ) : 31.5 , 34.4 , 40.0 , 42.3 , 64.1 , 116.4 , 117.3 , 126.3 , 127.0 , 128.7 , 129.1 , 131.8 , 134.2 , 145.7 , 153.0 , 175.2 , 175.7 . hr - esims : m / z 336.1598 ( calcd for c21h22no3 , 336.1600 ) . 4-(trifluoromethyl)phenoxyacetic acid ( 143 mg , 0.65 mmol ) , n - methylmaleimide ( 145 mg , 1.3 mmol ) , and tio2 ( 75 mg , 0.98 mmol ) were reacted in accordance with the general procedure . following irradiation for 16 h , the reaction mixture was purified by two rounds of column chromatography on silica gel ( eluent : 20% etoac in petroleum ether 4060 and subsequently 10% etoac in ch2cl2 ) to yield 7e as a clear oil ( 54.7 mg , 29% ) . h nmr ( 400 mhz , cdcl3 , 300 k ) : 2.822.96 ( m , 2h ) , 3.05 ( s , 3h ) , 3.203.26 ( m , 1h ) , 4.22 ( dd , j = 3.4 , 9.2 hz , 1h ) , 4.46 ( dd , j = 4.3 , 9.2 hz , 1h ) , 6.94 ( d , j = 8.5 hz , 2h ) , 7.54 ( d , j = 8.5 hz , 2h ) . c nmr ( 75 mhz , cdcl3 , 300 k ) : 25.1 , 31.6 , 40.5 , 66.1 , 114.6 , 123.9 ( q , jc f = 32.9 hz , 1c ) , 124.4 ( q , jc f = 271.2 hz , 2c ) , 127.0 ( q , jc f nmr ( 376 mhz , cdcl3 , 300 k ) : 62.1 . hr - esims : m / z 288.0838 ( calcd for c13h13no3f3 , 288.0842 ) . 6e was also obtained as a clear oil ( 60.1 mg , 32% ) . h nmr ( 400 mhz , cdcl3 , 298 k ) : 3.01 ( s , 3h ) , 3.403.44 ( m , 1h ) , 4.044.10 ( m , 2h ) , 4.64 ( dd , j = 3.2 , 11.5 hz , 1h ) , 6.98 ( d , j = 8.6 hz , 1h ) , 7.46 ( d , j = 8.5 hz , 1h ) , 7.87 ( s , 1h ) . c nmr ( 75 mhz , cdcl3 , 298 k ) : 25.5 , 39.4 , 41.8 , 64.0 , 117.9 , 118.4 , 123.9 ( q , jc f = 271.7 hz , 1c ) , 125.1 ( q , jc f = 33.1 hz , 1c ) , 127.2 ( q , jc f = 3.5 hz , 1c ) , 127.6 ( q , jc f = 3.8 hz , 1c ) , 157.7 , 175.3 , 176.1 . f nmr ( 376 mhz , cdcl3 , 298 k ) : 62.3 . hr - esims : m / z 286.0687 ( calcd for c13h11no3f3 , 286.0686 ) . 4-(trifluoromethyl)phenoxyacetic acid ( 143 mg , 0.65 mmol ) , n - phenylmaleimide ( 230 mg , 1.3 mmol ) , and tio2 ( 75 mg , 0.98 mmol ) were reacted in accordance with the general procedure . following irradiation for 16 h , the reaction mixture was purified by column chromatography on silica gel ( eluent : 25% etoac in petroleum ether 4060 ) to yield 7f as a colorless solid ( 52.9 mg , 40% ) . h nmr ( 400 mhz , cdcl3 , 297 k ) : 2.983.13 ( m , 2h ) , 3.353.40 ( m , 1h ) , 4.28 ( dd , j = 3.1 , 9.1 hz , 1h ) , 4.57 ( dd , j = 4.0 , 9.1 hz , 1h ) , 6.97 ( d , j = 8.5 hz , 2h ) , 7.29 ( d , j = 7.2 hz , 2h ) , 7.41 ( t , j = 7.3 hz , 1h ) , 7.49 ( t , j = 7.2 hz , 2h ) , 7.56 ( d , j = 8.6 hz , 2h ) . c nmr ( 75 mhz , cdcl3 , 297 k ) : 31.9 , 40.6 , 66.6 , 114.7 , 124.0 ( q , jc f = 32.8 hz , 1c ) , 124.3 ( q , jc f = 271.1 hz , 1c ) , 126.5 , 127.1 ( q , jc f = 3.7 hz , 2c ) , 128.8 , 129.3 , 131.9 , 160.4 , 175.2 , 176.3 . f nmr ( 376 mhz , cdcl3 , 297 k ) : 62.1 . hr - esims : m / z 367.1269 ( calcd for c18h18n2o3f3 , 367.1275 ) . h nmr ( 400 mhz , cdcl3 , 297 k ) : 3.573.61 ( m , 1h ) , 4.15 ( dd , j = 4.2 , 11.4 hz , 1h ) , 4.25 ( d , j = 9.3 hz , 1h ) , 4.71 ( dd , j = 3.4 , 11.4 hz , 1h ) , 7.04 ( d , j = 8.5 hz , 1h ) , 7.247.27 ( m , 2h ) , 7.367.51 ( m , 4h ) , 7.93 ( s , 1h ) . c nmr ( 75 mhz , cdcl3 , 297 k ) : 39.5 , 41.7 , 64.1 , 117.7 , 118.5 , 123.9 ( q , jc f = 271.8 hz , 1c ) , 125.2 ( q , jc f = 33.0 hz , 1c ) , 126.2 , 126.4 ( q , jc f = 3.5 hz , 1c ) , 127.7 ( q , jc f = 3.7 hz , 1c ) , 128.9 , 129.2 , 131.5 , 157.8 , 174.3 , 175.0 . f nmr ( 376 mhz , cdcl3 , 297 k ) : 62.3 . hr - esims : m / z 365.1114 ( calcd for c18h16n2o3f3 , 365.1108 ) . a thf ( 60 ml ) solution of methyl bromoacetate ( 0.62 ml , 6.54 mmol , 1.0 equiv ) and triphenylphosphine ( 1.715 g , 6.54 mmol , 1.0 equiv ) was heated at reflux for 4 h until a white precipitate formed . the suspension was allowed to cool to room temperature before filtration ; the precipitate was washed with et2o ( 3 50 ml ) , and the precipitate was then redissolved in ch2cl2 ( 30 ml ) and h2o ( 20 ml ) . then 2 m naoh ( 10 ml ) was added , and the biphasic mixture was stirred overnight . the reaction mixture was extracted with ch2cl2 ( 3 100 ml ) , and the combined organic layers were washed with brine , dried ( mgso4 ) , and concentrated under reduced pressure to yield the phosphorane as a colorless powder ( 1.923 g , 88% ) . h nmr ( 400 mhz , cdcl3 , 295 k ) : 2.90 and 3.51 ( br , 3h ) , 7.437.48 ( m , 6h ) , 7.527.57 ( m , 3h ) , 7.627.70 ( m , 6h ) . c nmr ( 100 mhz , cdcl3 , 298 k ) : 49.9 , 128.5 ( d , j = 12.0 hz ) , 128.8 ( d , j = 12.2 hz ) , 131.9 ( d , j = 2.7 hz ) , 132.1 ( d , j = 9.8 hz ) , 132.9 , 133.0 ( d , j = 10.2 hz ) . hr - esims : m / z 335.1200 ( calcd for c21h20o2p , 335.1195 ) . a chcl3 ( 200 ml ) solution of phosphorane ( 1.893 g , 5.66 mmol , 1.1 equiv ) and 2-formylphenoxyacetic acid ( 0.927 g , 5.15 mmol , 1.0 equiv ) was heated at reflux for 18 h. the solvent was removed under reduced pressure , and the crude residue was purified by column chromatography ( eluent : gradient of petroleum ether to 1:1 etoac / petroleum ether ) to yield 8a as a colorless powder ( 1.037 g , 85% ) . h nmr ( 300 mhz , cdcl3 , 295 k ) : 3.80 ( s , 3h ) , 4.77 ( s , 2h ) , 6.61 ( d , j = 16.2 hz , 1h ) , 6.81 ( d , j = 8.3 hz , 1h ) , 7.03 ( t , j = 7.6 hz , 1h ) , 7.33 ( td , j = 1.7 , 7.5 hz , 1h ) , 7.54 ( dd , j = 1.6 , 7.7 hz , 1h ) , 8.04 ( d , j = 16.2 hz , 1h ) , 8.899.30 ( br , 1h ) . c nmr ( 75 mhz , cdcl3 , 298 k ) : 52.2 , 65.4 , 112.5 , 119.5 , 122.5 , 124.4 , 129.8 , 131.8 , 140.4 , 156.7 , 168.6 , 173.8 . hr - esims : m / z 235.0610 ( calcd for c12h11o5 , 235.0612 ) . prepared in the same manner as 8a . bromoacetonitrile ( 0.88 ml , 12.5 mmol ) and pph3 ( 3.28 g , 12.5 ml ) were reacted to give the salt ( 4.580 g , 96% ) . the phosphorane was prepared as before to give a colorless powder ( 3.422 g , 91% ) . h nmr ( 300 mhz , cdcl3 , 297 k ) : 7.457.70 ( m , 15h ) . c nmr ( 121 mhz , cdcl3 , 297 k ) : 128.9 ( d , j = 12.1 hz ) , 129.5 ( d , j = 12.4 hz ) , 132.3 ( d , j = 2.6 hz ) , 132.5 ( d , j = 9.9 hz ) , 133.0 ( d , j = 2.1 hz ) , 133.2 ( d , j = 10.1 hz ) . 8b was prepared from the phosphorane ( 3.346 g , 11.11 mmol ) and 2-formylphenoxyacetic acid ( 1.819 g , 10.10 mmol ) to give a colorless powder ( 1.477 g , 72% ) . mp : 118 c. h nmr ( 400 mhz , cdcl3 , 297 k ) : 4.67 ( s , 3h ) , 6.31 ( d , j = 16.8 hz , 1h ) , 6.83 ( d , j = 1.1 hz , 1h ) , 7.01 ( t , j = 7.6 hz , 1h ) , 7.337.39 ( m , 2h ) , 7.62 ( d , j = 16.8 hz , 1h ) . c nmr ( 100 mhz , cdcl3 , 297 k ) : 65.1 , 98.0 , 112.2 , 119.1 , 121.9 , 123.0 , 129.8 , 132.2 , 146.5 , 156.5 , 170.5 . hr - esims : m / z 202.0511 ( calcd for c11h8o3n1 , 202.0510 ) . prepared in the same manner as 8a . 1-bromo-2-butanone ( 1.0 g , 6.62 mmol ) and pph3 ( 1.737 g , 6.62 mmol ) were reacted to give the salt ( 2.613 g , 96% ) . h nmr ( 500 mhz , cdcl3 , 295 k ) : 1.18 ( t , j = 7.6 hz , 3h ) , 2.33 ( qd , j = 1.3 , 7.6 hz , 2h ) , 7.437.47 ( m , 6h ) , 7.527.56 ( m , 3h ) , 7.627.67 ( m , 6h ) . 8c was prepared from the phosphorane ( 1.712 g , 5.15 mmol ) and 2-formylphenoxyacetic acid ( 0.843 g , 4.70 mmol ) to give a colorless powder ( 0.829 g , 75% ) . h nmr ( 400 mhz , cdcl3 , 295 k ) : 1.17 ( t , j = 7.3 hz , 3h ) , 2.73 ( q , j = 7.3 hz , 2h ) , 4.78 ( s , 2h ) , 6.82 ( d , j = 6.82 hz , 1h ) , 6.90 ( d , j = 16.4 hz , 1h ) , 7.04 ( t , j = 7.1 hz , 1h ) , 7.35 ( td , j = 1.7 , 7.4 hz , 1h ) , 7.58 ( dd , j = 1.7 , 7.7 hz , 1h ) , 7.96 ( d , j = 16.4 hz , 1h ) . c nmr ( 100 mhz , cdcl3 , 298 k ) : 8.3 , 33.5 , 65.4 , 112.2 , 121.8 , 124.0 , 127.2 , 128.9 , 131.5 , 137.8 , 156.7 , 170.6 , 202.5 . hr - esims : m / z 233.0820 ( calcd for c13h13o4 , 233.0819 ) . 2-bromophenol ( 6.4 ml , 60.5 mmol ) , tert - butyldimethylsilyl chloride ( 10.0 g , 66.5 mmol ) , and imidazole ( 8.23 g , 121.0 mmol ) were dissolved in the minimum volume of dmf , and the mixture was allowed to stir at room temperature overnight . the dmf was removed under reduced pressure , and the resultant residue was taken up in etoac and washed with 1 m hcl , sat . the solvent was then removed under reduced pressure to yield ( 2-bromophenoxy)(tert - butyl)dimethylsilane ( 17.29 g , 99% ) . h nmr ( 400 mhz , cdcl3 , 296 k ) : 0.25 ( s , 6h ) , 1.05 ( s , 9h ) , 6.806.84 ( m , 1h ) , 6.87 ( dd , j = 1.5 , 8.1 hz , 1h ) , 7.147.19 ( m , 1h ) , 7.51 ( dd , j = 1.7 , 7.9 hz , 1h ) . c nmr ( 100 mhz , cdcl3 , 297 k ) : 4.2 , 18.4 , 25.8 , 115.4 , 120.3 , 122.4 , 128.2 , 133.4 , 152.6 . hr - esims : m / z 287.0461 ( calcd for c12h20obrsi , 287.0461 ) . the aforementioned silane ( 0.54 g , 1.9 mmol ) was coupled with phenylboronic acid ( 0.27 g , 2.2 mmol ) by refluxing overnight in toluene ( 20 ml ) and ethanol ( 5 ml ) with pd[p(ph)3]4 ( 0.2 g , 0.2 mmol ) and k2co3 ( 0.9 g , 6.5 mmol ) . the solvent was then removed under reduced pressure , and the reaction mixture was taken up in h2o before being extracted with etoac . the resultant mixture was subjected to column chromatography on silica gel ( eluent : 25% ch2cl2 in petroleum ether 4060 ) to yield [ 1,1-biphenyl]-2-yloxy)(tert - butyl)dimethylsilane ( 0.39 g , 73% ) . h nmr ( 400 mhz , cdcl3 , 296 k ) : 0.05 ( s , 6h ) , 0.83 ( s , 9h ) , 6.92 ( dd , j = 1.2 , 8.0 hz , 1h ) , 7.04 ( td , j = 1.2 , 7.5 hz , 1h ) , 7.217.24 ( m , 1h ) , 7.307.33 ( m , 2h ) , 7.367.40 ( m , 2h ) , 7.50 ( dd , j = 1.5 , 8.5 hz , 1h ) . c nmr ( 100 mhz , cdcl3 , 297 k ) : 4.6 , 18.1 , 25.6 , 120.4 , 121.6 , 126.7 , 127.8 , 128.3 , 129.8 , 130.9 , 133.6 , 139.2 , 152.6 . the aforementioned biphenylsilane ( 0.36 g , 1.3 mmol ) was treated overnight with acetyl chloride ( 0.3 ml , 0.4 mmol ) in methanol ( 15 ml ) . following removal of the solvent under reduced pressure , the reaction mixture was subjected to column chromatography on silica gel ( eluent : ch2cl2 ) to yield [ 1,1-biphenyl]-2-ol ( 0.19 g , 86% ) . h nmr ( 400 mhz , cdcl3 , 296 k ) : 5.13 ( bs , 1h ) , 6.886.92 ( m , 2h ) , 7.147.19 ( m , 2h ) , 7.287.32 ( m , 1h ) , 7.367.42 ( m , 4h ) . the aforementioned biphenyl alcohol ( 0.19 g , 1.11 mmol ) , methyl bromoacetate ( 0.16 ml , 0.26 mmol ) , and k2co3 ( 0.76 g , 5.5 mmol ) were refluxed overnight in thf ( 20 ml ) . the solvent was then removed under reduced pressure , and the resultant residue was taken up in ch2cl2 before being washed with h2o and dried over mgso4 . the solvent was removed under reduced pressure , and the resultant mixture was subjected to column chromatography on silica gel ( eluent : 25% ch2cl2 in petroleum ether 4060 ) to afford methyl 2-([1,1-biphenyl]-2-yloxy)acetate ( 0.25 g , 94% ) . h nmr ( 300 mhz , cdcl3 , 294 k ) : 3.82 ( s , 3h ) , 4.66 ( s , 2h ) , 6.94 ( d , j = 8.3 hz , 1h ) , 7.15 ( td , j = 1.0 , 7.5 hz , 1h ) , 7.337.51 ( m , 5h ) , 7.69 ( dd , j = 1.4 , 8.4 hz , 1h ) . c nmr ( 75 mhz , cdcl3 , 295 k ) : 52.2 , 65.8 , 112.8 , 122.2 , 127.1 , 128.1 , 128.6 , 129.7 , 131.3 , 131.4 , 138.1 , 154.8 , 169.6 . hr - esims : m / z 243.1020 ( calcd for c15h15o3 , 243.1016 ) . the aforementioned ester ( 0.19 g , 0.8 mmol ) was hydrolyzed with lioh ( 0.13 g , 3.2 mmol ) in a 3:1 mixture of meoh and h2o . following overnight stirring at room temperature , the reaction mixture was concentrated to ca . the combined extracts were dried over mgso4 and concentrated under reduced pressure , and the resultant residue was chromatographed on silica gel ( eluent : ch2cl2 ) to yield 11a ( 0.13 g , 73% ) . h nmr ( 300 mhz , cdcl3 , 294 k ) : 4.63 ( s , 2h ) , 6.92 ( dd , j = 1.1 , 8.2 hz , 1h ) , 7.13 ( td , j = 1.1 , 7.5 hz , 1h ) , 7.297.46 ( m , 5h ) , 7.57 ( dd , j = 1.5 , 8.4 hz , 2h ) , 8.8010.10 ( br , 1h ) . c nmr ( 75 mhz , cdcl3 , 295 k ) : 65.9 , 113.4 , 123.1 , 127.7 , 128.7 , 129.1 , 129.9 , 131.8 , 131.9 , 138.3 , 154.6 , 173.7 . hr - esims : m / z 227.0707 ( calcd for c14h11o3 , 227.0714 ) . prepared in the same manner as 11a . ( 2-bromophenoxy)(tert - butyl)dimethylsilane ( 0.95 g , 3.3 mmol ) , 4-(trifluoromethyl)phenylboronic acid ( 0.75 g , 4.0 mmol ) , pd[p(ph)3]4 ( 0.38 g , 0.33 mmol ) , and k2co3 ( 1.2 g , 9.9 mmol ) were reacted and then columned on silica gel ( eluent : 25% ch2cl2 in petroleum ether 4060 ) to afford 3-(trifluoromethyl)-[1,1-biphenyl]-2-ol ( 0.13 g , 14% ) . h nmr ( 400 mhz , cdcl3 , 296 k ) : 4.985.10 ( br , 1h ) , 6.96 ( d , j = 8.5 hz , 1h ) , 7.03 ( td , j = 1.1 , 7.5 hz , 1h ) , 7.267.31 ( m , 2h ) , 7.577.65 ( m , 2h ) , 7.71 ( d , j = 7.5 hz , 1h ) , 7.78 ( s , 1h ) . c nmr ( 100 mhz , cdcl3 , 297 k ) : 116.2 , 121.3 , 126.9 , 129.3 , 129.7 , 130.5 , 132.5 , 138.3 , 152.4 . hr - esims : m / z 237.0526 ( calcd for c13h8of3 , 237.0533 ) . the aforementioned biphenyl alcohol ( 0.13 g , 0.55 mmol ) was alkylated as before with methyl bromoacetate ( 0.1 ml , 1.1 mmol ) and k2co3 ( 0.38 g , 2.7 mmol ) and columned on silica gel ( eluent : 50% ch2cl2 in petroleum ether 4060 ) to afford methyl 2-((3-(trifluoromethyl)-[1,1-biphenyl]-2-yl)oxy)acetate ( 0.14 g , 84% ) . h nmr ( 400 mhz , cdcl3 , 296 k ) : 3.79 ( s , 3h ) , 4.65 ( s , 2h ) , 6.89 ( d , j = 7.7 hz , 1h ) , 7.12 ( td , j = 1.0 , 7.5 hz ) , 7.327.39 ( m , 2h ) , 7.54 ( t , j = 7.7 hz , 1h ) , 7.60 ( d , j = 7.8 hz , 1h ) , 7.81 ( d , j = 7.6 hz , 1h ) , 7.92 ( s , 1h ) . f nmr ( 376 mhz , cdcl3 , 296 k ) : 62.9 . c nmr ( 100 mhz , cdcl3 , 297 k ) : 52.3 , 65.5 , 112.3 , 122.3 , 123.8 ( q , jcf = 3.7 hz ) , 124.4 ( q , jcf = 272.2 hz ) , 126.5 ( q , jcf = 3.7 hz ) , 128.5 , 129.4 , 129.7 , 130.4 ( q , jcf = 32.1 hz ) , 131.2 , 133.0 , 138.8 , 154.6 , 169.2 . hr - esims : m / z 328.1162 ( calcd for c16h17no3f3 , 328.1155 ) . the aforementioned ester ( 0.14 g , 0.5 mmol ) was hydrolyzed with lioh ( 0.08 g , 1.8 mmol ) to afford 11b ( 0.13 g , 95% ) . h nmr ( 400 mhz , cdcl3 , 296 k ) : 4.68 ( s , 2h ) , 6.91 ( d , j = 8.2 hz , 1h ) , 7.14 ( td , j = 1.0 , 7.5 hz , 1h ) , 7.347.39 ( m , 2h ) , 7.54 ( t , j = 7.7 hz , 1h ) , 7.60 ( d , j = 7.8 hz , 1h ) , 7.76 ( d , j = 7.6 hz , 1h ) , 7.87 ( s , 1h ) . c nmr ( 100 mhz , cdcl3 , 297 k ) : 65.0 , 112.4 , 122.6 , 123.9 ( q , jcf = 3.3 hz ) , 124.2 ( q , jcf = 272.4 hz ) , 126.4 ( q , jcf = 3.6 hz ) , 128.5 , 129.4 , 129.7 , 130.6 ( q , jcf = 32.5 hz ) , 131.2 , 132.8 , 138.6 , 154.1 , 173.0 . hr - esims : m / z 314.1001 ( calcd for c15h15no3f3 , 314.0999 ) . prepared in the same manner as 11a . ( 2-bromophenoxy)(tert - butyl)dimethylsilane ( 0.63 g , 2.2 mmol ) , 4-(trifluoromethyl)phenylboronic acid ( 0.5 g , 2.6 mmol ) , pd[p(ph)3]4 ( 0.22 g , 0.22 mmol ) , and k2co3 ( 0.9 g , 6.5 mmol ) were reacted and then columned on silica gel ( eluent : 25% ch2cl2 in petroleum ether 4060 ) to afford 4-(trifluoromethyl)-[1,1-biphenyl]-2-ol ( 0.43 g , 82% ) . h nmr ( 400 mhz , cdcl3 , 296 k ) : 4.99 ( s , 1h ) , 6.97 ( dd , j = 0.5 , 8.1 hz , 1h ) , 7.04 ( td , j = 1.1 , 7.5 hz , 1h ) , 7.267.32 ( m , 2h ) , 7.64 ( d , j = 8.6 hz , 2h ) , 7.74 ( d , j = 8.6 hz , 2h ) . f nmr ( 376 mhz , cdcl3 , 296 k ) : 63.05 . c nmr ( 100 mhz , cdcl3 , 297 k ) : 116.2 , 121.3 , 124.1 ( q , jcf = 272.0 hz ) , 125.9 ( q , jcf = 3.7 hz ) , 126.9 , 129.4 ( q , jcf = 32.4 hz ) , 129.6 , 129.8 , 130.4 , 141.1 , 152.3 . hr - esims : m / z 237.0539 ( calcd for c13h8of3 , 237.0533 ) . the aforementioned biphenyl alcohol ( 0.4 g , 1.66 mmol ) was alkylated as before with methyl bromoacetate ( 0.24 ml , 2.5 mmol ) and k2co3 ( 1.15 g , 8.3 mmol ) and columned on silica gel ( eluent : 25% ch2cl2 in petroleum ether 4060 ) to afford methyl 2-((4-(trifluoromethyl)-[1,1-biphenyl]-2-yl)oxy)acetate ( 0.529 g , 99% ) . h nmr ( 400 mhz , cdcl3 , 296 k ) : 3.78 ( s , 3h ) , 4.64 ( s , 2h ) , 6.88 ( d , j = 8.2 hz , 1h ) , 7.10 ( td , j = 1.0 , 7.5 hz , 1h ) , 7.327.36 ( m , 2h ) , 7.67 ( d , j = 8.1 hz , 2h ) , 7.73 ( d , j = 8.1 hz , 2h ) . f nmr ( 376 mhz , cdcl3 , 296 k ) : 62.9 . c nmr ( 100 mhz , cdcl3 , 297 k ) : 52.2 , 65.5 , 112.3 , 122.2 , 124.4 ( q , jcf = 271.6 hz ) , 124.9 ( q , jcf = 3.7 hz ) , 129.1 ( q , jcf = 32.4 hz ) , 129.4 , 129.8 , 129.9 , 131.2 , 141.7 , 154.6 , 169.2 . hr - esims : m / z 328.1158 ( calcd for c16h17no3f3 , 328.1155 ) . the aforementioned ester ( 0.49 g , 1.6 mmol ) was hydrolyzed with lioh ( 0.26 g , 6.3 mmol ) to afford 11c ( 0.44 g , 95% ) . h nmr ( 400 mhz , cdcl3 , 296 k ) : 4.68 ( s , 2h ) , 6.91 ( dd , j = 1.1 , 8.9 hz , 1h ) , 7.13 ( td , j = 1.0 , 7.6 hz , 1h ) , 7.337.39 ( m , 2h ) , 7.667.72 ( m , 4h ) . c nmr ( 100 mhz , cdcl3 , 297 k ) : 65.5 , 112.9 , 123.0 , 125.0 ( q , jcf = 273.5 hz ) , 125.4 ( q , jcf = 3.9 hz ) , 129.7 ( q , jcf = 31.2 hz ) , 129.9 , 130.3 ( 2 ) , 131.7 , 142.0 , 154.6 , 173.2 . hr - esims : m / z 295.0592 ( calcd for c15h10o3f3 , 295.0588 ) . a suspension of 2-formylphenoxyacetic acid ( 0.515 g , 2.86 mmol , 1.0 equiv ) , methoxylamine hydrochloride ( 0.478 g , 5.72 mmol , 2.0 equiv ) , and sodium acetate ( 0.469 g , 5.72 mmol , 2.0 equiv ) in ethanol ( 30 ml ) was heated at reflux for 18 h. the solvent was removed under reduced pressure , and the crude residue was redissolved in h2o ( 100 ml ) and extracted into ch2cl2 ( 3 100 ml ) . the combined organic layers were dried ( mgso4 ) , concentrated under reduced pressure , and purified by column chromatography ( 5% etoac / ch2cl2 ) to yield 13a as a colorless solid ( 0.390 g , 65% ) . h nmr ( 300 mhz , cdcl3 , 294 k ) : 4.01 ( s , 3h ) , 4.72 ( s , 2h ) , 6.91 ( d , j = 8.2 hz , 1h ) , 7.12 ( m , 1h ) , 7.34 ( d , j = 7.6 hz , 1h ) , 7.40 ( m , 1h ) , 8.15 ( s , 1h ) . c nmr ( 75 mhz , cdcl3 , 295 k ) : 61.9 , 66.6 , 114.2 , 120.6 , 123.0 , 131.7 , 132.4 , 148.8 , 155.4 , 169.5 . hr - esims : m / z 208.0614 ( calcd for c10h10no4 , 208.0615 ) . a suspension of 2-formylphenoxyacetic acid ( 1.00 g , 5.55 mmol , 1.0 equiv ) , benzoxylamine hydrochloride ( 1.773 g , 11.11 mmol , 2.0 equiv ) , and sodium acetate ( 0.911 g , 11.11 mmol , 2.0 equiv ) in ethanol ( 60 ml ) was heated at reflux for 18 h. the solvent was removed under reduced pressure , and the crude residue was redissolved in h2o ( 100 ml ) and extracted into ch2cl2 ( 3 100 ml ) . the combined organic layers were dried ( mgso4 ) , concentrated under reduced pressure , and purified by column chromatography ( 5% etoac / ch2cl2 ) to yield 13b as a colorless solid ( 0.422 g , 26% ) . h nmr ( 300 mhz , cdcl3 , 294 k ) : 4.70 ( s , 2h ) , 5.25 ( s , 2h ) , 6.90 ( d , j = 8.2 hz , 1h ) , 7.11 ( t , j = 6.6 hz , 1h ) , 7.327.44 ( m , 7h ) , 8.26 ( s , 1h ) . c nmr ( 75 mhz , cdcl3 , 295 k ) : 66.5 , 76.2 , 114.0 , 120.6 , 122.9 , 128.2 , 128.5 , 128.5 , 131.7 , 132.2 , 136.6 , 149.3 , 155.3 , 169.5 . hr - esims : m / z 284.0923 ( calcd for c16h14no4 , 284.0928 ) . phenoxyacetic acid ( 304 mg , 2.0 mmol ) and socl2 ( 0.74 ml , 10.0 mmol ) were refluxed in chcl3 for 72 h. the volatiles were removed under reduced pressure to yield phenoxyacetyl chloride . h nmr ( 400 mhz , cdcl3 , 296 k ) : 4.95 ( s , 2h ) , 6.93 ( m , 2h ) , 7.09 ( m , 1h ) , 7.35 ( m , 2h ) . c nmr ( 75 mhz , cdcl3 , 297 k ) : 64.9 , 77.2 , 114.8 , 122.3 , 129.9 , 157.5 , 174.2 . this compound was immediately treated with d2o ( 0.9 ml , 5.0 mmol ) in anhydrous ch3cn ( 10 ml ) , and the mixture was allowed to stir overnight under an argon atmosphere . the solvent was removed under reduced pressure , and the resultant residue was recrystallized from anhydrous et2o to yield 5ad as a white crystalline solid ( 141 mg , 46% ) . h nmr ( 500 mhz , cdcl3 , 295 k ) : 4.70 ( s , 2h ) , 6.93 ( m , 2h ) , 7.03 ( m , 1h ) , 7.32 ( m , 2h ) . h nmr ( 76 mhz , chcl3 , 315 k ) : 10.13 ( bs , 1d ) . c nmr ( 75 mhz , cdcl3 , 297 k ) : 64.9 , 114.8 , 122.4 , 129.9 , 157.4 , 172.8 . hr - esims : m / z 153.0543 ( calcd for c8h8o3d , 153.0513 ) . phenoxyacetic acid ( 100 mg , 0.66 mmol ) and acrylamide ( 93.9 mg , 1.32 mmol ) in mecn ( 45 ml ) were reacted in accordance with the general procedure in a tio2 sol gel - coated tube . following irradiation for 24 h , the reaction mixture was purified by dry flash chromatography on silica gel ( eluent : gradient of 210% meoh in ch2cl2 ) to yield 19h as an off - white solid ( 79.8 mg , 68% ) . h nmr ( 300 mhz , cdcl3 , 294 k ) : 2.13 ( m , 2h ) , 2.45 ( t , j = 7.2 hz , 2h ) , 4.02 ( t , j = 6.0 hz , 2h ) , 5.63 ( bs , 1h ) , 5.82 ( bs , 1h ) , 6.90 ( d , j = 8.8 hz , 2h ) , 6.95 ( t , j = 7.4 hz , 1h ) , 7.29 ( t , j = 7.4 hz , 2h ) . c nmr ( 75 mhz , cdcl3 , 295 k ) : 25.4 , 32.6 , 67.1 , 114.9 , 121.2 , 129.9 , 159.2 , 175.4 . 4-(tert - butyl)phenoxyacetic acid ( 137.5 mg , 0.66 mmol ) and acrylamide ( 93.9 mg , 1.32 mmol ) in mecn ( 45 ml ) were reacted in accordance with the general procedure in a tio2 sol gel - coated tube . following irradiation for 30 h , the reaction mixture was purified by dry flash chromatography on silica gel ( eluent : gradient of 27.5% meoh in ch2cl2 ) to yield the title compound as an off - white solid ( 105.4 mg , 68% ) containing 5% 4-(tert - butyl)phenol . h nmr ( 400 mhz , cd3od , 297 k ) : 1.30 ( s , 9h ) , 2.042.11 ( m , 2h ) , 2.43 ( t , j = 7.3 hz , 2h ) , 4.00 ( t , j = 6.3 hz , 2h ) , 6.85 ( d , j = 8.9 hz , 2h ) , 7.30 ( dd , j = 3.2 , 11.5 hz , 1h ) . c nmr ( 75 mhz , cdcl3 , 297 k ) : 27.0 , 32.4 , 33.4 , 35.3 , 68.5 , 115.5 , 127.6 , 144.8 , 158.5 , 179.0 . hr - esims : m / z 236.1647 ( calcd for c14h22no2 , 236.1645 ) . 4-(trifluoromethyl)phenoxyacetic acid ( 145.3 mg , 0.66 mmol ) and acrylamide ( 93.9 mg , 1.32 mmol ) in mecn ( 45 ml ) were reacted in accordance with the general procedure in a tio2 sol gel - coated tube . following irradiation for 24 h , the reaction mixture was purified by dry flash chromatography on silica gel ( eluent : 5% meoh in ch2cl2 ) to yield the title compound as an off - white solid ( 108.4 mg , 66% ) . h nmr ( 500 mhz , d6-acetone , 295 k ) : 2.062.11 ( m , 2h ) , 2.41 ( t , j = 7.2 hz , 2h ) , 4.14 ( t , j = 5.9 hz , 2h ) , 6.30 ( bs , 1h ) , 6.86 ( bs , 1h ) , 7.14 ( d , j = 8.7 hz , 2h ) , 7.63 ( d , j = 8.7 hz , 2h ) . c nmr ( 75 mhz , d6-acetone , 297 k ) : 29.9 , 36.3 , 72.7 , 120.0 , 127.2 ( q , jc f = 33.1 hz , 1c ) , 130.0 ( q , jc f nmr ( 470 mhz , d6-acetone , 295 k ) : 61.8 . hr - esims : m / z 248.0896 ( calcd for c11h13no2f3 , 248.0896 ) . a suspension of 8c ( 0.050 g , 0.21 mmol , 1.0 equiv ) and tio2 ( p25 , 0.025 g , 0.32 mmol , 1.5 equiv ) in mecn ( 20 ml ) was reacted in accordance with the general procedure for 9 h. following removal of the solvent under reduced pressure , the mixture was subjected to analysis by h nmr and gc ms . gc ms indicated four major components , and one of these ( tr = 10.7 min ) had the correct mass and fragmentation pattern for the cyclized product 10c : m / z ( % ) : 190 ( 40 ) , 161 ( 36 ) , 146 ( 18 ) , 133 ( 23 ) , 119 ( 38 ) , 118 ( 100 ) , 91 ( 41 ) . h nmr ( 400 mhz , cd3cn , 298 k ) : 0.98 ( t , j = 7.3 hz , 3h ) , 2.47 ( q , j = 7.3 hz , 2h ) , 3.263.29 ( m , 1h ) , 4.37 ( dd , j = 2.3 , 8.4 hz , 1h ) , 4.94 ( dd , j = 5.2 , 9.4 hz , 1h ) , 7.037.17 ( m , 2h , ) , 7.507.54 ( m , 2h ) . attempts to isolate and thoroughly characterize 10c were not successful . photoisomerization of 8c to its z isomer was observed during the course of this experiment . h nmr ( 400 mhz , cd3cn , 298 k ) : 7.38 ( d , j = 7.3 hz , 1h ) , 7.30 ( t , j = 7.3 hz , 1h ) , 7.02 ( d , j = 12.0 hz , 1h ) , 6.95 ( t , j = 7.6 hz , 1h ) , 6.88 ( d , j = 8.3 hz , 1h ) , 6.28 ( d , j = 12.5 hz , 1h ) , 4.69 ( s , 2h ) , 2.47 ( q , j = 7.3 hz , 2h ) , 0.97 ( t , j = 7.3 hz , 3h ) . a suspension of 11a ( 0.052 g , 0.23 mmol , 1.0 equiv ) and tio2 ( p25 , 0.027 g , 0.34 mmol , 1.5 equiv ) in mecn ( 20 ml ) was reacted in accordance with the general procedure for 18 h. following removal of the solvent under reduced pressure , h nmr analysis of the reaction mixture showed mainly unreacted 11a together with a small amount of the cyclized product 12a ( 0.01 mmol , 5% wrt ch2br2 ) . h nmr ( 400 mhz , cdcl3 , 296 k ) : 5.05 ( s , 2h ) , 6.906.95 ( m , 2h ) , 7.007.10 ( m , obscured by sm ) , 7.607.70 ( m , 3h ) . attempts to isolate and thoroughly characterize 12a were unsuccessful . a suspension of 8a ( 0.050 g , 0.21 mmol , 1.0 equiv ) and tio2 ( p25 , 0.025 g , 0.32 mmol , 1.5 equiv ) in mecn ( 20 ml ) was reacted according to the general procedure for 9 h to yield 10a as a colorless powder ( 20% by nmr ) . h nmr ( 400 mhz , cdcl3 , 294 k ) : 2.52 ( dd , j = 9.5 , 16.5 hz , 1h ) , 2.73 ( dd , j = 5.3 , 16.5 hz , 1h ) , 3.72 ( s , 3h ) , 3.843.91 ( m , 1h ) , 4.18 ( dd , j = 6.3 , 9.2 hz , 1h ) , 4.68 ( t , j = 9.0 hz , 1h ) , 6.80 ( d , j = 7.7 hz , 1h ) , 6.86 ( t , j = 8.3 hz , 1h ) , 7.087.17 ( m , 2h ) . c nmr ( 75 mhz , cdcl3 , 295 k ) : 38.7 , 39.7 , 52.3 , 110.2 , 114.1 , 121.0 , 124.6 , 129.1 , 129.5 , 160.2 , 172.7 . hr - esims : m / z 215.0674 ( calcd for c11h12o3na , 215.0679 ) . photoisomerization of 8a to its z isomer was observed during the course of this experiment . h nmr ( 400 mhz , cd3cn , 298 k ) : 7.48 ( dd , j = 7.6 , 1.8 hz , 1h ) , 7.31 ( t , j = 7.8 hz , 1h ) , 7.19 ( d , j = 12.5 hz , 1h ) , 6.97 ( t , j = 7.6 hz , 1h ) , 6.88 ( d , j = 8.3 hz , 1h ) , 6.01 ( d , j = 12.3 hz , 1h ) , 4.69 ( s , 2h ) , 3.63 ( s , 3h ) . a solution of 8b ( 0.050 g , 0.246 mmol ) in mecn ( 15 ml ) in a tio2 sol gel - coated tube the solvent was removed under reduced pressure , and the crude residue was purified by column chromatography ( ch2cl2 ) to yield 10b as a colorless powder ( 0.007 g , 28% ) . h nmr ( 400 mhz , cdcl3 , 296 k ) : 2.582.71 ( m , 2h ) , 3.743.82 ( m , 1h ) , 4.34 ( dd , j = 4.8 , 9.6 hz , 1h ) , 4.69 ( dd , j = 8.7 , 9.6 hz , 1h ) . 6.84 ( d , j = 8.1 hz , 1h ) , 6.93 ( td , j = 1.0 , 7.5 hz , 1h ) , 7.22 ( m , 1h ) , 7.32 ( d , j = 7.5 hz , 1h ) . c nmr ( 75 mhz , cdcl3 , 295 k ) : 22.8 , 38.8 , 75.4 , 110.3 , 117.7 , 121.2 , 124.4 , 127.1 , 129.7 , 159.7 . hr - esims : m / z 159.0676 ( calcd for c10h9no , 159.0679 ) . photoisomerization of 8b to its z isomer was observed during the course of this experiment . h nmr ( 400 mhz , cd3cn , 298 k ) : 8.01 ( dd , j = 7.6 , 1.6 hz , 1h ) , 7.66 ( d , j = 12.5 hz , 1h ) , 7.44 ( t , j = 8.0 hz , 1h ) , 7.11 ( t , j = 7.5 hz , 1h ) , 6.97 ( d , j = 8.3 hz , 1h ) , 5.64 ( d , j = 12.3 hz , 1h ) , 4.74 ( s , 2h ) . a suspension of 11c ( 59 mg , 0.20 mmol , 1.0 equiv ) and tio2 ( p25 , 0.024 g , 0.30 mmol , 1.5 equiv ) in mecn ( 20 ml ) was reacted in accordance with the general procedure for 18 h. following removal of the solvent under reduced pressure , h nmr analysis of the reaction mixture showed mainly unreacted acid ( 79% ) together with a small amount of the cyclized product 12c ( 0.01 mmol , 5% wrt ch2br2 ) . h nmr ( 400 mhz , cdcl3 , 296 k ) : 5.07 ( s , 2h ) , 6.936.96 ( m , 2h ) , 6.987.04 ( m ; obscured by sm ) , 7.727.65 ( m , 3h ) . gc ms analysis of the reaction mixture showed 12c as the main product : tr = 11.76 min ; m / z ( % ) 250 ( 58 ) , 249 ( 100 ) , 231 ( 6 ) , 201 ( 10 ) , 181 ( 10 ) , 152 ( 16 ) . also observed was a trace of what was probably the direct reduction product 2-methoxy-4-(trifluoromethyl)-1,1-biphenyl : tr = 11.84 min ; m / z ( % ) 252 ( 82 ) , 251 ( 48 ) , 250 ( 61 ) , 249 ( 100 ) , 237 ( 20 ) , 201 ( 16 ) , 183 ( 26 ) , 152 ( 32 ) . attempts to isolate and thoroughly characterize 12c were unsuccessful . a suspension of 13a ( 51 mg , 0.24 mmol , 1.0 equiv ) and tio2 ( p25 , 0.029 g , 0.36 mmol , 1.5 equiv ) in mecn ( 29 ml ) was reacted in accordance with the general procedure for 24 h. following removal of the solvent under reduced pressure , h nmr analysis of the reaction mixture showed that 13a had been entirely consumed and that a complex mixture of products had been formed . ms analysis of the mixture revealed that 14a had been formed : tr = 8.15 min ; m / z ( % ) 165 ( 29 ) , 133 ( 12 ) , 119 ( 100 ) , 91 ( 77 ) 77 ( 26 ) . also observed were benzofuran-3(2h)-one o - methyl oxime [ tr = 7.93 min ; m / z ( % ) 163 ( 100 ) , 118 ( 91 ) , 91 ( 52 ) ] , 2,3-dihydrobenzofuran [ tr = 7.87 min ; m / z ( % ) 120 ( 27 ) , 119 ( 100 ) , 91 ( 57 ) ] , 2,3-dihydrobenzofuran-3-amine [ tr = 5.06 min ; m / z ( % ) 135 ( 7 ) , 134 ( 74 ) , 119 ( 100 ) , 117 ( 11 ) ] , and benzofuran [ tr = 3.43 min ; m / z ( % ) 118 ( 100 ) , 90 ( 22 ) , 84 ( 30 ) ] . attempts to isolate and thoroughly characterize 14a , or any other products , were unsuccessful . a suspension of 13b ( 62 mg , 0.22 mmol , 1.0 equiv ) and tio2 ( p25 , 0.026 g , 0.33 mmol , 1.5 equiv ) in mecn ( 26 ml ) was reacted in accordance with the general procedure for 24 h. following removal of solvent under reduced pressure , h nmr analysis of the reaction mixture showed that 13b had been entirely consumed and that a complex mixture of products had been formed . gc ms analysis of the mixture revealed that 14b had been formed : tr = 15.53 min ; m / z ( % ) 241 ( 9 ) , 210 ( 9 ) , 133 ( 6 ) , 91 ( 100 ) , 77 ( 14 ) . also observed were benzofuran-3(2h)-one o - benzyl oxime [ tr = 15.37 min ; m / z ( % ) 239 ( 37 ) , 222 ( 4 ) , 148 ( 3 ) , 119 ( 7 ) , 91 ( 100 ) , 77 ( 12 ) ] , 2,3-dihydrobenzofuran-3-amine [ tr = 5.04 min ; m / z ( % ) 135 ( 7 ) , 134 ( 74 ) , 119 ( 100 ) , 117 ( 11 ) ] , benzyl alcohol [ tr = 3.77 min ; m / z ( % ) 108 ( 100 ) , 107 ( 73 ) , 79 ( 76 ) ] , benzofuran [ tr = 3.41 min ; m / z ( % ) 118 ( 100 ) , 90 ( 22 ) , 84 ( 30 ) ] , and benzaldehyde [ tr = 2.90 min ; m / z ( % ) 106 ( 87 ) , 105 ( 100 ) , 77 ( 66 ) ] . tio2 ( 12 mg , 0.15 mmol ) was dried overnight at 150 c under a continuous vacuum in an oven - dried schlenk tube . phenoxyacetic acid ( 15.2 mg , 0.1 mmol ) and acrylamide ( 14.2 mg , 0.2 mmol ) in mecn ( 12 ml ) were added via syringe , and the resultant mixture was irradiated for 18 h. h nmr analysis of the resultant mixture revealed 19h ( 39% by nmr ) . phenoxyacetic acid ( 15.2 mg , 0.1 mmol ) , acrylamide ( 14.2 mg , 0.2 mmol ) , and tio2 ( 12 mg , 0.15 mmol ) in d3-mecn ( 12 ml ) were irradiated for 18 h. h nmr analysis of the resultant mixture revealed 19h ( 55% by nmr ) . d1-phenoxyacetic acid ( 15.3 mg , 0.1 mmol ) , acrylamide ( 14.2 mg , 0.2 mmol ) , and tio2 ( 12 mg , 0.15 mmol ) in mecn ( 12 ml ) were irradiated for 19 h. h nmr analysis ( wrt ch2br2 standard ) of the resultant mixture revealed 19h ( 49% by nmr ) . an oven - dried schlenk tube , complete with magnetic stirrer , was connected to a vacuum line , evacuated , and then back - filled with argon while still hot from the oven . in a glovebox , tio2 ( p25 , 0.15 mmol , 12 mg ) was added , and the tube was then heated at 150 c for 3 h under vacuum . in a separate oven - dried vessel , 10 ml of d2o ( freshly opened ) was degassed by three freeze pump thaw cycles before being transferred by cannula to the tube containing the tio2 once it had cooled to ambient temperature . the resultant mixture was allowed to stir overnight before the d2o was removed under reduced pressure . at this point the tio2 was again dried for 3 h at 150 c under vacuum . in a separate oven - dried vessel , phenoxyacetic acid ( 0.1 mmol , 15.2 mg ) and acrylamide ( 0.2 mmol , 14.2 mg ) were dissolved in mecn ( 12 ml ) that had been freshly collected after distillation over calcium hydride . the resultant mixture was degassed for 15 min by bubbling with argon and then transferred via cannula to the tube containing the tio2 once it had cooled to ambient temperature . the resultant mixture was then irradiated in accordance with the general procedure for 19 h. h nmr analysis of the resultant mixture revealed 19h ( 58% by nmr ) . this reaction was performed as above using d1-phenoxyacetic acid ( 15.3 mg , 0.1 mmol ) for 19 h. h nmr analysis of the resultant mixture revealed no clear signals indicating the formation of 19h or 19d .

photochemical reactions employing tio2 and carboxylic acids under dry anaerobic conditions led to several types of c c bond - forming processes with electron - deficient alkenes . the efficiency of alkylation varied appreciably with substituents in the carboxylic acids . the reactions of aryloxyacetic acids with maleimides resulted in a cascade process in which a pyrrolochromene derivative accompanied the alkylated succinimide . the selectivity for one or other of these products could be tuned to some extent by employing the photoredox catalyst under different conditions . aryloxyacetic acids adapted for intramolecular ring closures by inclusion of 2-alkenyl , 2-aryl , or 2-oximinyl functionality reacted rather poorly . profiles of reactant consumption and product formation for these systems were obtained by an in situ nmr monitoring technique . an array of different catalyst forms were tested for efficiency and ease of use . the proposed mechanism , involving hole capture at the tio2 surface by the carboxylates followed by co2 loss , was supported by epr spectroscopic evidence of the intermediates . deuterium labeling indicated that the titania likely donates protons from surface hydroxyl groups as well as supplying electrons and holes , thus acting as both a catalyst and a reaction partner .

Introduction Results and Discussion Conclusions Experimental Section

carboxylic acids are pervasive in nature and are produced industrially on a large scale . conventionally , decarboxylative alkylations have relied on preparations of unappealing precursors such as peroxides , barton esters , or hunsdieker salts . decarboxylative copper - catalyzed reactions of carboxylic acids with carbonyls and with aldimines , and of amino acids with alkynes have been reported . under anhydrous , anaerobic conditions and with certain substrates , tio2 scpc can lead to the generation of specific radicals adapted for molecular assembly applications . notably , scpc additions of enol ethers to various acceptors have been investigated , and successful additions of tertiary amines to electron - deficient alkenes , some bearing chiral auxiliaries , have been described . we recently discovered that under dry , anaerobic conditions , tio2 mediation with carboxylic acid precursors could result in carbon alkylations and annulations were achieved when photolyses of certain acid precursors were carried out in the presence of suitable electron - deficient alkenes . this paper reports advances in crucial aspects of this process in the following areas : ( 1 ) structural features in the carboxylic acids that are necessary and sufficient ; ( 2 ) the range of functionality in the alkene acceptors that can be tolerated ; ( 3 ) how the reactions respond to modifications to the catalyst form and the catalyst support ; and ( 4 ) the mechanism followed by the reaction . during initial investigations , n - phenylmaleimide ( 2 ) was found to be an ideal radical acceptor in this system , as it is symmetrical and photostable , its photodimerization is negligible ( under our experimental conditions ) , and its electron - withdrawing character favors addition by weakly nucleophilic radicals . this alkene was therefore chosen to test the applicabliity of our process with simple aliphatic carboxylic acids 1 . in all instances , 0.1 mmol of 1 and 0.2 mmol of 2 were photolyzed in a suspension of the p25 catalyst ( 12 mg , 0.15 mmol ) in ch3cn ( 12 ml ) for 18 h. the reactions did indeed lead to the formation of adducts 3 incorporating the r moiety of the carboxylic acid and an additional h atom , together with significant amounts of succinimide 4 ( scheme 1 ) . the efficiency of the process evidently increased as the stabilization energy of the released radical increased ( see the supporting information for a plot of radical stabilization energy vs yield ) . we next examined a set of addition reactions with n - phenylmaleimide involving more diverse carboxylic acids ( scheme 2 ) . methoxy- and tert - butoxyacetic acids gave the corresponding adducts ( 3 g and 3h ) in reasonable yields of 54% and 57% , respectively , while 2-tetrahydrofuroic acid furnished a 1:1 mixture of the two diastereomers of 3i in a very pleasing yield of 75% . moderate and poor yields were obtained with benzyl ( 57% ) and 2-thienylmethyl acids ( 22% ) , but these adducts ( 3k and 3l ) were accompanied by the dimers bibenzyl ( 18% ) and 1,2-bis(thiophen-2-yl)ethane ( 27% ) , respectively . while boc - proline was transformed into adduct 3n in a fine yield of 75% , boc - alanine and the unnatural -amino acid boc - piperidinecarboxylic acid gave much more modest yields of 38% and 29% , respectively . no retention of chirality was observed when optically pure amino acids were employed because the released radicals have planar , or close to planar , configurations at their reactive centers . to compare the efficiencies of different forms of the titania catalyst , the reaction of phenoxyacetic acid with acrylamide , which was clean and efficient ( 82% adduct isolated ) was chosen . because of their buoyancy , they tended to accumulate at the top , and this was compounded by the fact that the high rate of magnetic stirring used in attempts to overcome this tended to result in their becoming damaged . an isolated yield of 68% was achieved upon scale - up of the reaction in the coated tube ( entry 3 ) . incorporation of 4-tert - butyl and 4-trifluoromethyl substituents in the aryl ring of the acid component furnished the corresponding adducts in similarly pleasing yields of 68% and 66% , respectively . we therefore concluded that for ease of handling with conventional organic techniques , scale - up , and efficient product formation , the orthodox p25 catalyst was the best compromise . the reaction of aryloxyacetic acids ( 5 ) with n - substituted maleimides ( 2 ) led to n - substituted-3,4-dihydrochromenopyrrole-1,3-dione derivatives 6 , each accompanied by a significant amount of the expected adduct 7 ( scheme 3 ) . tricyclics 6 presumably resulted from cyclization of the initial adduct radical onto the aryl ring followed by rearomatization . table 3 shows that the process worked well with both electron - releasing and electron - withdrawing substituents in the aromatic ring and for methyl- and phenyl - substituted maleimides . the reactions with just p25 generally gave a slight excess of the chromenopyrrole 6 ( entries 1 , 4 , 6 , 8 , and 10 ) , except in the case of the cf3 substituent ( entry 12 ) , for which an excess of adduct 7 was observed . the selectivity for chromenopyrroles 6 was improved by using a more dense dispersion of p25 ( entries 2 , 7 , and 11 ) . the possibility to tune h coupling constants between the two protons at the junction of the pyran and dihydropyrrole rings within the range of 9.29.3 hz . three types of acid framework with different acceptor groups were prepared to test the applicability of p25-mediated ring closures . 2-(2-vinylphenoxy)acetic acids having alkene acceptors with ester ( 8a ) , nitrile ( 8b ) , and ketone ( 8c ) substituents ( scheme 4 ) were obtained by treatment of 2-formylphenoxyacetic acid with the appropriate phosphorane ( see the experimental section ) . the tube was held at a slight angle with respect to horizontal in the chuck of the overhead stirrer so that , when rotated , it described a small ( ca . in conventional p25-mediated reactions , 8a c all gave rather complex product mixtures containing unreacted alkene as a mixture of e and z isomers ( 8/9 ) as well as the 5-exo ring - closed dihydrobenzofurans 10 . nmr and gc ms analyses showed complex product mixtures containing possible trace amounts of the cyclized 6h - benzo[c]chromenes 12a c . the reaction profile from irradiation of acid 8a in a sol gel - coated tube obtained by nmr monitoring ( figure 1 ) shows the sharp decrease in 8a ( the e isomer ) , which was all consumed in < 150 min . analogous reaction profiles were obtained for the sol gel tube reactions of 8a c , 11b , and 13a . a significant yield ( 28% ) of the cyclized product n-(2,3-dihydrobenzofuranyl)-o - methylhydroxylamine ( 14a ) was found in the sol gel tube reaction of oxime ether - functionalized acid 13a ( scheme 4 ) . reaction profile for acid 8a obtained by nmr monitoring . hole capture at the tio2 surface by a carboxylate creates the corresponding acyloxyl radical . transient t - bu radicals and phoch2 radicals were observed during uv irradiation of the acids t - buco2h and phoch2co2h , respectively , with pc-500 in fluid phh at 300 k. in similar epr experiments with vinylacetic acid and 2,2,2-triphenylacetic acid , we were also able to detect and characterize allyl and triphenylmethyl radicals , respectively ( see the supporting information ) . the isotropic character of the solution epr spectra of all the rxch2 radicals established that in the main they were freely tumbling and not attached to the tio2 surface ( scheme 5 ) . literature precedents imply that weakly nucleophilic rxch2 radicals should add rapidly to the electron - deficient double bonds of maleimides ( and similar acceptors ) . , addition produces electrophilic imidoalkyl radicals 15 , for which a competition exists between reduction to adducts 7 or homolytic closure ( 6-endo - trig ) onto the aryl ring . this aromatization could result from hole capture from tio2 by the radicals 17 and subsequent proton loss as shown . protonation of the resulting maleimide radical anions , followed by further electron capture and protonation steps , would explain the significant yields of succinimides obtained in our reactions . however , as greater amounts of succinimides than cyclized products 6 were generally formed , it is thought that direct reduction of the maleimide acceptor by the conduction band of the p25 also initiates this process . from the results described in schemes 1 and 2 and table 1 , it can be deduced that the stability of the initial radicals is an important factor in this system . carboxylic acids dissociate on the tio2 surface to give the corresponding surface - bound carboxylate and a proton . the tio2 surface is extensively hydroxylated , and it is thought that these species act as surface trapping sites for the valence - band holes . electron transfer from the system of the carboxylate to a hole trap site furnishes the surface - bound rxch2co2 radical . for acid precursors that generate stabilized rxch2 radicals , such as those depicted in scheme 2 , the decarboxylation step is more favorable , thus explaining the improved yields and conversions recorded . it should be noted that while we believe radical stability to be a key aspect in this system , it is clear that there are other factors at play as well and that these take precedence in deciding the reaction outcome in a few instances . while we did observe the allyl radical in our epr experiments , no products at all were observed in the preparative photolysis involving vinylacetic acid and 2 . in this case , other as yet unknown factors at the tio2 surface must come into play . a crucial feature of the process is that in the formation of adducts 3 and 7 a hydrogen atom is gained from some source within the dispersion . however , it is well - established that h2o molecules and ho groups are attached to the surface of the tio2 particles . drying p25 in a vacuum at 150 c removes a significant amount of the surface h2o while leaving the chemically attached ho groups . when p25 dried in this way was used , the yield of 18 was scarcely reduced , suggesting that the oh groups were likely the proton donors . attempts were made to deuterate the h2o and oh groups on the p25 surface by refluxing in degassed d2o in a glovebox . in view of the partially deuterated nature of the p25d and the ease with which it reverts to the protiated form , making handling difficult , our tentative conclusion is still that the p25 is the source of the h atoms . we have found that photochemical reactions employing p25 and carboxylic acids under dry anaerobic conditions generate c - centered radicals that can be deployed in several types of c c bond - forming processes . acids that yielded alkoxyalkyl , alkylthioalkyl , and benzyl radicals alkylated electron - deficient alkenes in moderate to good yields . the reactions of aryloxyacetic acids with maleimides resulted in a cascade process in which a pyrrolochromene derivative accompanied the alkylated succinimide . selectivity for one or other of these products could be tuned to some extent by employing the tio2 catalyst under different conditions . aryloxyacetic acids adapted for intramolecular ring closures by the inclusion of 2-alkenyl , 2-aryl , or 2-oximinyl functionality reacted rather poorly , especially the 2-aryl acceptor types . the mechanism ( scheme 5 ) is supported by epr spectroscopic evidence of the intermediates . experiments with deuterium - labeled solvent and reactants implied that this was the hydroxyl groups on the surface of the p25 . it appears that the titania supplies electrons and holes upon photostimulation but also donates protons from surface hydroxyl groups , thus acting as both a catalyst and a reaction partner . a photoreactor consisting of two lots of six 8 w blb uv lamps ( emission max = 365 nm ) , each arranged in a semicylinder with an aluminum reflector , was used to irradiate the contents of the nmr tube . the tube was held at a slight angle with respect to horizontal in the chuck of the overhead stirrer so that , when rotated , it described a small ( ca . these experiments were carried out in a similar fashion to the photoredox experiments outlined above . further details of these experiments are provided in the supporting information . prepared in the same manner as 8a . prepared in the same manner as 11a . a suspension of 11a ( 0.052 g , 0.23 mmol , 1.0 equiv ) and tio2 ( p25 , 0.027 g , 0.34 mmol , 1.5 equiv ) in mecn ( 20 ml ) was reacted in accordance with the general procedure for 18 h. following removal of the solvent under reduced pressure , h nmr analysis of the reaction mixture showed mainly unreacted 11a together with a small amount of the cyclized product 12a ( 0.01 mmol , 5% wrt ch2br2 ) . a suspension of 11c ( 59 mg , 0.20 mmol , 1.0 equiv ) and tio2 ( p25 , 0.024 g , 0.30 mmol , 1.5 equiv ) in mecn ( 20 ml ) was reacted in accordance with the general procedure for 18 h. following removal of the solvent under reduced pressure , h nmr analysis of the reaction mixture showed mainly unreacted acid ( 79% ) together with a small amount of the cyclized product 12c ( 0.01 mmol , 5% wrt ch2br2 ) . ms analysis of the mixture revealed that 14a had been formed : tr = 8.15 min ; m / z ( % ) 165 ( 29 ) , 133 ( 12 ) , 119 ( 100 ) , 91 ( 77 ) 77 ( 26 ) . a suspension of 13b ( 62 mg , 0.22 mmol , 1.0 equiv ) and tio2 ( p25 , 0.026 g , 0.33 mmol , 1.5 equiv ) in mecn ( 26 ml ) was reacted in accordance with the general procedure for 24 h. following removal of solvent under reduced pressure , h nmr analysis of the reaction mixture showed that 13b had been entirely consumed and that a complex mixture of products had been formed . phenoxyacetic acid ( 15.2 mg , 0.1 mmol ) and acrylamide ( 14.2 mg , 0.2 mmol ) in mecn ( 12 ml ) were added via syringe , and the resultant mixture was irradiated for 18 h. h nmr analysis of the resultant mixture revealed 19h ( 39% by nmr ) . d1-phenoxyacetic acid ( 15.3 mg , 0.1 mmol ) , acrylamide ( 14.2 mg , 0.2 mmol ) , and tio2 ( 12 mg , 0.15 mmol ) in mecn ( 12 ml ) were irradiated for 19 h. h nmr analysis ( wrt ch2br2 standard ) of the resultant mixture revealed 19h ( 49% by nmr ) . an oven - dried schlenk tube , complete with magnetic stirrer , was connected to a vacuum line , evacuated , and then back - filled with argon while still hot from the oven . in a separate oven - dried vessel , 10 ml of d2o ( freshly opened ) was degassed by three freeze pump thaw cycles before being transferred by cannula to the tube containing the tio2 once it had cooled to ambient temperature . in a separate oven - dried vessel , phenoxyacetic acid ( 0.1 mmol , 15.2 mg ) and acrylamide ( 0.2 mmol , 14.2 mg ) were dissolved in mecn ( 12 ml ) that had been freshly collected after distillation over calcium hydride . the resultant mixture was degassed for 15 min by bubbling with argon and then transferred via cannula to the tube containing the tio2 once it had cooled to ambient temperature . this reaction was performed as above using d1-phenoxyacetic acid ( 15.3 mg , 0.1 mmol ) for 19 h. h nmr analysis of the resultant mixture revealed no clear signals indicating the formation of 19h or 19d .

although attention - deficit / hyperactivity disorder ( adhd ) has been conceptualized as a disorder of deficient self - regulation,1 the dsm - iv ( diagnostic and statistical manual of mental disorders , 4th edition , text revision)2 symptoms of the disorder have been limited to those of cognitive ( inattention ) and behavioral ( hyperactivity/ impulsivity ) deficits , leaving out deficient emotional self - regulation ( desr ) . desr refers to : deficits in self - regulating the physiological arousal caused by strong emotions ; difficulties inhibiting inappropriate behavior in response to either positive or negative emotions ; problems refocusing attention from strong emotions ; and disorganization of coordinated behavior in response to emotional activation.3 desr traits include low tolerance of frustration , impatience , quickness to anger , and being easily excited to emotional reactions . although these traits may be a source of significant morbidity , there has been a paucity of research on the subject.36 the symptoms of desr should not be confused with the emotional symptoms of mood disorders . desr differs from the persistent and severe irritability often seen in pediatric mood disorder.7 unlike desr , the abnormal mood of mood disorder is not defined by poor self - regulation and includes features that are not part of desr ( eg , the other mood criteria in dsm - iv ) . we recently examined8 whether a unique profile of the child behavior checklist ( cbcl ) would help identify desr in children with adhd . we defined desr if a child had a sum of t scores of > 180 but < 210 ( between one and two standard deviations [ sd ] above the mean ) on the anxiety/ depression , aggression , and attention scales of the cbcl ( cbcl - desr ) . this profile was selected because of its conceptual congruence with the clinical concept of desr and because its extreme ( > 210 , more than 2 sd above the mean ) form had been previously associated with severe forms of mood and behavioral dysregulation in children with adhd.9 we found that 44% of children with adhd had a positive cbcl - desr profile versus 2% of controls ( p < 0.001 ) . the cbcl - desr profile was associated with elevated rates of anxiety and disruptive behavior disorders , as well as significantly more impairment in emotional and interpersonal functioning . these findings suggested that the cbcl - desr profile helped identify a subgroup of adhd children with a psychopathological and functional profile consistent with the clinical concept of desr . desr remained associated with poorer psychosocial functioning even after controlling for comorbid disorders , suggesting that desr may represent a burdensome source of morbidity in children with adhd that is partially independent of comorbid disorders such as oppositional defiant disorder . others have examined this cbcl - desr profile but not using the cutoffs defined by our group . ayer et al10 found that the profile was associated with lower functioning and child suicidal behavior . althoff et al11 found cross - informant agreement of the profile and describe its association with suicidal thoughts and behaviors . new insights into the role that desr plays in the clinical picture of adhd would have important clinical and scientific implications . clinically , desr could account for the frequent social interaction problems and impairments in several domains of major life activities that are commonly seen with adhd and other disorders . if so , the assessment of desr could be an important feature of child psychiatric assessment . scientifically understanding the relationships between adhd and desr could provide new insight into the neural basis of emotional regulation separating cortical regulatory regions from limbic ones . it could also help identify genes associated with emotional dysregulation apart from those associated with other behavioral and cognitive features of psychopathology . the main aim of this study was to assess the longitudinal course of desr in adhd children . to this end , we used longitudinal follow - up data from two identically designed large controlled studies of well characterized boys and girls with and without adhd . we hypothesized that a positive cbcl - desr profile will predict more psychopathology and poorer psychosocial functioning in children with adhd at follow - up . to the best of our knowledge , the methodology used in this study has been previously described.12,13 briefly , subjects were derived from two identically designed longitudinal case - control family studies of adhd . these studies recruited male and female subjects aged 618 years with ( n = 140 boys , n = 140 girls ) and without ( n = 120 boys , n = 122 girls ) dsm - iii - r adhd ascertained from pediatric and psychiatric clinics . male subjects were assessed at baseline and at four - year follow - up while female subjects were assessed at baseline and at five - year follow - up . potential subjects were excluded if they had been adopted or if their nuclear family was not available for study . we also excluded potential subjects if they had major sensorimotor handicaps ( paralysis , deafness , blindness ) , psychosis , autism , inadequate command of the english language , or a full scale iq less than 80 . parents and adult offspring provided written informed consent to participate , and parents also provided consent for offspring under the age of 18 years . psychiatric assessments of probands younger than 18 years relied on the kiddie schedule for affective disorders and schizophrenia - epidemiological version ( ksads - e).14,15 subjects 18 years of age and older were assessed with the structured clinical interview for dsm16,17 ( supplemented with modules from the k - sads - e to assess childhood diagnoses ) . diagnoses for this analysis were considered positive if full criteria were met within the past year of the assessment . we interviewed the mothers for all subjects and directly interviewed subjects older than 12 years . we combined data from direct and indirect interviews by considering a diagnostic criterion positive if it was endorsed in either interview . interviewers blind to all previous information about the child and family assessed mothers and children . based on 500 assessments from interviews of children and adults , the median kappa coefficient for diagnoses was 0.98 . kappa coefficients for individual diagnoses included adhd ( 0.88 ) , cognitive disorder ( 1.0 ) , major depression ( 1.0 ) , mania ( 0.95 ) , separation anxiety ( 1.0 ) , agoraphobia ( 1.0 ) , panic ( 0.95 ) , and substance use disorder ( 1.0 ) . a committee of board - certified child and adult psychiatrists who were blind to the subject s adhd status , referral source , and all other data resolved diagnostic uncertainties . diagnoses presented for review were considered positive only when the committee determined that diagnostic criteria were met to a clinically meaningful degree . we estimated the reliability of the diagnostic review process by computing kappa coefficients of agreement for clinician reviewers . for these diagnoses , the median reliability between individual clinicians and the review committee assigned diagnoses was 0.87 . kappa coefficients for individual diagnoses included adhd ( 1.0 ) , conduct disorder ( 1.0 ) , major depression ( 1.0 ) , mania ( 0.78 ) , separation anxiety ( 0.89 ) , agoraphobia ( 0.80 ) , panic ( 0.77 ) , and substance use disorder ( 1.0 ) . all assessment personnel were blind to proband diagnosis ( adhd or control ) and ascertainment site ( psychiatric or pediatric ) . the diagnosis of major depression was made only if the depressive episode was associated with severe impairment.18,19 since there are many anxiety disorders measured by our structured interviews , we aggregated them into a binary measure - coded positive if two or more anxiety disorders were endorsed , and negative otherwise . two or more anxiety disorders previously provided a reasonable trade - off between case identification and the false positive rate when compared against an independently defined anxiety standard in youth with adhd.20 psychoactive substance use disorder was defined as any alcohol abuse , alcohol dependence , substance abuse , or substance dependence . total comorbidities were calculated as the number of up to six psychiatric disorders / categories ( bipolar disorder , major depressive disorder , multiple anxiety disorders , oppositional defiant disorder , conduct / antisocial personality disorder , psychoactive substance use disorder ) present at any time in the year before the structured interview . global functioning was assessed using the global assessment of functioning scale.21 psychosocial functioning was assessed using the social adjustment inventory for children and adolescents ( saica).22 the saica provides an evaluation of children s functioning in school , in spare time activities , and with peers , siblings , and parents . a saica subscale is an average of 311 likert items ( eg , 1 = not a problem , 2 = mild problem , 3 = moderate problem , 4 = severe problem ) from an area of social functioning . family functioning was defined using the moos family environment scale,23 which uses standard scores with a mean of 50 and sd of 10 . socioeconomic status was measured using the five - point hollingshead scale.24 assessment of desr relied on the cbcl,25 completed at baseline and follow - up . a parent ( usually the mother ) of each participant completed the 1991 version of the child behavior checklist for children aged 418 years . the cbcl is an affordable pencil and paper test completed by the child s caregiver , requiring no administration by a physician or rater . the cbcl queries the parent about the child s behavior in the past 6 months and aggregates this data into behavioral problem t scores.25 a computer program calculates the t scores for each scale . raw scores are converted to genderand age - standardized scores ( t scores having a mean of 50 and a sd of 10 ) . a minimum t score of 50 is assigned to scores that fall at midpoint percentiles of 50 on the syndrome scales to permit comparison of standardized scores across scales . desr was defined as positive if the sum of the cbcl attention problems , aggressive behavior , and anxious - depressed t scores was 180 and < 210 . these cutoff scores represent , on average , 1 sd ( t score = 60 ) to 2 sd ( t score = 70 ) on each of the three selected cbcl subscales . subjects with a sum of > 210 on these cbcl subscales were excluded , because we previously showed that these subjects were at risk for developing bipolar disorder and associated impairments.9 we compared adhd probands with and without desr on baseline demographic characteristics using t - tests , pearson s chi - square , or wilcoxon rank - sum tests . the one - year prevalence rates of baseline and follow - up psychiatric disorders were compared using logistic regression , and controlling for demographic confounders . linear regression was used to test continuous variables ( eg , global assessment of functioning scale ) and negative binomial regression was used to test count variables ( eg , total number of comorbidities ) . the methodology used in this study has been previously described.12,13 briefly , subjects were derived from two identically designed longitudinal case - control family studies of adhd . these studies recruited male and female subjects aged 618 years with ( n = 140 boys , n = 140 girls ) and without ( n = 120 boys , n = 122 girls ) dsm - iii - r adhd ascertained from pediatric and psychiatric clinics . male subjects were assessed at baseline and at four - year follow - up while female subjects were assessed at baseline and at five - year follow - up . potential subjects were excluded if they had been adopted or if their nuclear family was not available for study . we also excluded potential subjects if they had major sensorimotor handicaps ( paralysis , deafness , blindness ) , psychosis , autism , inadequate command of the english language , or a full scale iq less than 80 . parents and adult offspring provided written informed consent to participate , and parents also provided consent for offspring under the age of 18 years . psychiatric assessments of probands younger than 18 years relied on the kiddie schedule for affective disorders and schizophrenia - epidemiological version ( ksads - e).14,15 subjects 18 years of age and older were assessed with the structured clinical interview for dsm16,17 ( supplemented with modules from the k - sads - e to assess childhood diagnoses ) . diagnoses for this analysis were considered positive if full criteria were met within the past year of the assessment . we interviewed the mothers for all subjects and directly interviewed subjects older than 12 years . we combined data from direct and indirect interviews by considering a diagnostic criterion positive if it was endorsed in either interview . interviewers blind to all previous information about the child and family assessed mothers and children . based on 500 assessments from interviews of children and adults , the median kappa coefficient for diagnoses was 0.98 . kappa coefficients for individual diagnoses included adhd ( 0.88 ) , cognitive disorder ( 1.0 ) , major depression ( 1.0 ) , mania ( 0.95 ) , separation anxiety ( 1.0 ) , agoraphobia ( 1.0 ) , panic ( 0.95 ) , and substance use disorder ( 1.0 ) . a committee of board - certified child and adult psychiatrists who were blind to the subject s adhd status , referral source , and all other data resolved diagnostic uncertainties . diagnoses presented for review were considered positive only when the committee determined that diagnostic criteria were met to a clinically meaningful degree . we estimated the reliability of the diagnostic review process by computing kappa coefficients of agreement for clinician reviewers . for these diagnoses , the median reliability between individual clinicians and the review committee assigned diagnoses was 0.87 . kappa coefficients for individual diagnoses included adhd ( 1.0 ) , conduct disorder ( 1.0 ) , major depression ( 1.0 ) , mania ( 0.78 ) , separation anxiety ( 0.89 ) , agoraphobia ( 0.80 ) , panic ( 0.77 ) , and substance use disorder ( 1.0 ) . all assessment personnel were blind to proband diagnosis ( adhd or control ) and ascertainment site ( psychiatric or pediatric ) . the diagnosis of major depression was made only if the depressive episode was associated with severe impairment.18,19 since there are many anxiety disorders measured by our structured interviews , we aggregated them into a binary measure - coded positive if two or more anxiety disorders were endorsed , and negative otherwise . two or more anxiety disorders previously provided a reasonable trade - off between case identification and the false positive rate when compared against an independently defined anxiety standard in youth with adhd.20 psychoactive substance use disorder was defined as any alcohol abuse , alcohol dependence , substance abuse , or substance dependence . total comorbidities were calculated as the number of up to six psychiatric disorders / categories ( bipolar disorder , major depressive disorder , multiple anxiety disorders , oppositional defiant disorder , conduct / antisocial personality disorder , psychoactive substance use disorder ) present at any time in the year before the structured interview . global functioning was assessed using the global assessment of functioning scale.21 psychosocial functioning was assessed using the social adjustment inventory for children and adolescents ( saica).22 the saica provides an evaluation of children s functioning in school , in spare time activities , and with peers , siblings , and parents . a saica subscale is an average of 311 likert items ( eg , 1 = not a problem , 2 = mild problem , 3 = moderate problem , 4 = severe problem ) from an area of social functioning . family functioning was defined using the moos family environment scale,23 which uses standard scores with a mean of 50 and sd of 10 . assessment of desr relied on the cbcl,25 completed at baseline and follow - up . a parent ( usually the mother ) of each participant completed the 1991 version of the child behavior checklist for children aged 418 years . the cbcl is an affordable pencil and paper test completed by the child s caregiver , requiring no administration by a physician or rater . the cbcl queries the parent about the child s behavior in the past 6 months and aggregates this data into behavioral problem t scores.25 a computer program calculates the t scores for each scale . raw scores are converted to genderand age - standardized scores ( t scores having a mean of 50 and a sd of 10 ) . a minimum t score of 50 is assigned to scores that fall at midpoint percentiles of 50 on the syndrome scales to permit comparison of standardized scores across scales . desr was defined as positive if the sum of the cbcl attention problems , aggressive behavior , and anxious - depressed t scores was 180 and < 210 . these cutoff scores represent , on average , 1 sd ( t score = 60 ) to 2 sd ( t score = 70 ) on each of the three selected cbcl subscales . subjects with a sum of > 210 on these cbcl subscales were excluded , because we previously showed that these subjects were at risk for developing bipolar disorder and associated impairments.9 we compared adhd probands with and without desr on baseline demographic characteristics using t - tests , pearson s chi - square , or wilcoxon rank - sum tests . the one - year prevalence rates of baseline and follow - up psychiatric disorders were compared using logistic regression , and controlling for demographic confounders . linear regression was used to test continuous variables ( eg , global assessment of functioning scale ) and negative binomial regression was used to test count variables ( eg , total number of comorbidities ) . of 280 subjects with adhd and 242 subjects without adhd at baseline , 242 and 229 , respectively , had cbcl data ( figure 1 ) . of the 242 adhd subjects , 45 were dropped because they had an attention problems + aggressive behavior + an anxious - depressed ( aaa ) score of > 210 at baseline . of the 229 control subjects , five were dropped because they met criteria for desr . of the remaining 197 adhd subjects and 224 control subjects , 177 ( 100 boys , 77 girls ) and 204 ( 105 boys , 99 girls ) , respectively , had follow - up data . the mean follow - up time was 4.4 years ( sd 1.1 years , range 39 years ) . of the 177 adhd subjects with follow - up data , 119 subjects had cbcl data at the four - year follow - up . the kappa for desr between the baseline and four - year followup assessments was 0.29 ( p < 0.001 ) , 57% of subjects with desr at baseline also had desr at follow - up ( sensitivity , 33/58 ) , 72% of subjects without desr at baseline did not have desr at follow - up ( specificity , 44/61 ) , the positive predictive value was 66% ( 33/50 ) , and the negative predictive value was 64% ( 44/69 ) . the intraclass correlation of the continuous aaa score between baseline and follow - up was 0.47 ( p < 0.001 ) . persistent adhd ( full or subthreshold diagnosis in the last month ) was significantly associated with desr at follow - up ( 0/18 with remittent adhd had desr compared with 50/101 with persistent adhd had desr , ( 1 ) = 15.37 , p < 0.001 ) . comparisons were made between controls ( n = 204 ) versus adhd subjects with ( adhd + desr , n = 79 ) and without desr at baseline ( adhd , n = 98 ) . subjects used for this analysis ( n = 381 ) did not significantly differ from subjects lost to follow - up ( n = 40 ) on group status ( p = 0.64 ) , gender / study ( p = 0.65 ) , age ( p = 0.58 ) , socioeconomic status ( p = 0.16 ) , or ascertainment source ( p = 0.69 ) . there were no differences between groups on gender , socioeconomic status , family intactness , or ascertainment source ( table 1 ) . therefore , all analyses controlled for age . at the adolescent follow - up , both adhd groups had significantly higher 1-year prevalence rates of bipolar ( z = 3.89 , p < 0.001 ) , major depressive ( z = 4.59 , p < 0.001 ) , multiple anxiety ( z = 4.27 , p < 0.001 ) , and conduct / antisocial personality ( z = 3.34 , p = 0.001 ) disorders compared with controls ( figure 2 ) . in addition , the adhd + desr group continued to have significantly more total comorbidities at follow - up compared with the adhd and control groups ( 1.4 versus 0.9 , z = 2.55 , p = 0.01 and 1.4 versus 0.3 , z = 8.61 , p < 0.001 , respectively ) as well as a significantly higher one - year prevalence of oppositional defiant disorder ( z = 3.01 , p = 0.003 and z = 7.28 , p < 0.001 , respectively , figure 2d ) . desr was not significantly associated with mood disorders at baseline or follow - up ( all p > 0.05 ) . at follow - up , both adhd groups continued to have higher thought problems and delinquent behavior t scores at follow - up compared with the controls ( adhd + desr : t(227 ) = 5.38 , adhd : t(227 ) = 3.79 , both p < 0.001 ) . adhd + desr subjects continued to have a more impaired social problems t score compared with the adhd and control groups ( t(227 ) = 2.41 , p = 0.02 and t(227 ) = 7.68 , p < 0.001 , respectively , figure 3 ) . figure 4a shows that the adhd + desr group continued to have poorer global functioning than the adhd and control groups at follow - up ( t(377 ) = 3.71 , p < 0.001 and t(377 ) = 15.14 , p < 0.001 , respectively ) . while desr was associated with significantly poorer social adjustment at baseline ( t(365 ) = 4.38 , p < 0.001 ) , this no longer held true at follow - up due to the more impaired social adjustment inventory for children and adolescents ( saica ) score in the adhd group at follow - up ( t(166 ) = 0.75 , p = 0.45 , figure 4b ) . the adhd + desr group had poorer family environment scale scores at baseline ( expression : t(372 ) = 2.35 , p = 0.02 ; conflict : t(372 ) = 2.62 , p = 0.009 ; cohesion : t(372 ) = 2.05 , p = 0.04 ) and follow - up ( expression : t(241 ) = 1.33 , p = 0.18 ; conflict : t(241 ) = 0.96 , p = 0.34 ; cohesion : t(242 ) = 1.66 , p = 0.10 ) compared with the adhd group , although the differences attained statistical significance only at baseline ( figure 5 ) . desr was not associated with educational functioning ( extra tutoring , placement in special class , repeated grade ) at baseline or follow - up ( all p > 0.30 ) . there were no differences between groups on gender , socioeconomic status , family intactness , or ascertainment source ( table 1 ) . at the adolescent follow - up , both adhd groups had significantly higher 1-year prevalence rates of bipolar ( z = 3.89 , p < 0.001 ) , major depressive ( z = 4.59 , p < 0.001 ) , multiple anxiety ( z = 4.27 , p < 0.001 ) , and conduct / antisocial personality ( z = 3.34 , p = 0.001 ) disorders compared with controls ( figure 2 ) . in addition , the adhd + desr group continued to have significantly more total comorbidities at follow - up compared with the adhd and control groups ( 1.4 versus 0.9 , z = 2.55 , p = 0.01 and 1.4 versus 0.3 , z = 8.61 , p < 0.001 , respectively ) as well as a significantly higher one - year prevalence of oppositional defiant disorder ( z = 3.01 , p = 0.003 and z = 7.28 , p < 0.001 , respectively , figure 2d ) . desr was not significantly associated with mood disorders at baseline or follow - up ( all p > 0.05 ) . at follow - up , both adhd groups continued to have higher thought problems and delinquent behavior t scores at follow - up compared with the controls ( adhd + desr : t(227 ) = 5.38 , adhd : t(227 ) = 3.79 , both p < 0.001 ) . adhd + desr subjects continued to have a more impaired social problems t score compared with the adhd and control groups ( t(227 ) = 2.41 , p = 0.02 and t(227 ) = 7.68 , p < 0.001 , respectively , figure 3 ) . figure 4a shows that the adhd + desr group continued to have poorer global functioning than the adhd and control groups at follow - up ( t(377 ) = 3.71 , p < 0.001 and t(377 ) = 15.14 , p < 0.001 , respectively ) . while desr was associated with significantly poorer social adjustment at baseline ( t(365 ) = 4.38 , p < 0.001 ) , this no longer held true at follow - up due to the more impaired social adjustment inventory for children and adolescents ( saica ) score in the adhd group at follow - up ( t(166 ) = 0.75 , p = 0.45 , figure 4b ) . the adhd + desr group had poorer family environment scale scores at baseline ( expression : t(372 ) = 2.35 , p = 0.02 ; conflict : t(372 ) = 2.62 , p = 0.009 ; cohesion : t(372 ) = 2.05 , p = 0.04 ) and follow - up ( expression : t(241 ) = 1.33 , p = 0.18 ; conflict : t(241 ) = 0.96 , p = 0.34 ; cohesion : t(242 ) = 1.66 , p = 0.10 ) compared with the adhd group , although the differences attained statistical significance only at baseline ( figure 5 ) . desr was not associated with educational functioning ( extra tutoring , placement in special class , repeated grade ) at baseline or follow - up ( all p > 0.30 ) . our a priori definition of desr based on moderate ( > 1 and < 2 sd ) elevations of cbcl - desr identified a clinically meaningful subgroup of adhd children at high risk for long - term morbidity and dysfunction consistent with current conceptualizations of desr . the cbcl - desr profile was associated at the follow - up assessment into adolescent years with higher rates of persistence of adhd , more psychiatric comorbidity , more impaired scores on the social problems cbcl scale , as well as more impaired ratings of the global assessment of functioning scale . these findings suggest that the cbcl - desr profile can help identify a clinically meaningful subgroup of children with adhd with long - term compromised outcomes . the finding that 57% of subjects with desr at baseline also had desr at follow - up suggests that desr is a largely persistent trait ( intraclass correlation of the continuous aaa score between baseline and follow - up was 0.47 ) . also noteworthy is the finding that desr was predictive of a persistent course of adhd ; 50% of adhd youth with persistent adhd had desr versus 0% in those without . at the adolescent follow - up , adhd + desr subjects continued to have significantly more mean number of comorbidities compared with adhd and control subjects as well as a significantly higher one - year prevalence of oppositional defiant disorder . these findings are congruent with those derived from analysis of the follow - up cbcl data showing more social problems associated with desr in children with adhd growing up . although social functioning as measured by the saica was significantly associated with desr at baseline and not at follow - up , subjects with desr had the same high overall saica score at follow - up as they did at baseline ( figure 4b ) . also congruent with the psychopathological findings are those showing that children with adhd + desr continued to have poorer global functioning than the adhd and control groups at follow - up . these findings are consistent with those of whalen and henker,26 who found that emotional dysregulation in adhd children disrupted the smoothness , reciprocity , and cooperative activities involved in peer relationships and with the suggestion by barkley3 that desr might account for a significant portion of the social dysfunction documented in adhd . several diagnostic and functioning measures that were associated with desr at baseline were no longer significantly different at follow - up , including multiple anxiety disorders , conduct / antisocial personality disorder , several cbcl scales , saica , and family environment scale . therefore , the cbcl - desr profile appears better at identifying children with adhd with more extreme concurrent impairments than at identifying children at risk for future impairments . the fact that nearly half ( 43% ) of the children with desr at baseline no longer had desr at follow - up may explain the somewhat weaker findings at follow - up . desr was not associated with substance use disorders at baseline or follow - up . because a substantial proportion of this sample had not yet entered the age of risk for substance use disorders , future studies should seek to confirm this lack of association . our longitudinal findings that children with adhd with the cbcl - desr profile were at significantly increased risk for oppositional defiant disorder but not for depression and bipolar disorder stand in contrast with findings of increased risks for these mood disorders associated with the cbcl - pediatric bipolar profile.9 this latter profile requires very abnormal ( 2 sd ) scores on the same scales of anxiety/ depression , aggression , and attention of the cbcl used to define the cbcl - desr profile . these nonoverlapping profile findings between the high ( 2 sd ) and the intermediate ( 1 sd ) cbcl profiles ( used to define desr ) support the hypotheses that these are mutually exclusive profiles that identify different clinical entities , with the severe profile likely identifying the extreme mood dysregulation associated with mood disorder , while the intermediate one is limited to deficient emotional self - regulation . while both desr and mood disorders involve emotional dysregulation , these constructs are substantially different clinically . the cardinal feature of mood disorders is the experience of strong emotions , not their self - regulation , the hallmark of desr . children who fulfilled the cbcl - desr criteria were not more likely to have bipolar disorder or major depression or to have been hospitalized than other children with adhd . in this regard , our data support the utility of the cbcl in defining desr as a substantially different clinical entity than the cbcl - juvenile bipolar profile used to identify children at high risk for pediatric bipolar disorder . in fact , a comprehensive literature review2 documents numerous studies in which children and adults with adhd are highly likely to manifest desr characterized by low frustration tolerance , impatience , quickness to anger , and being easily excited to emotional reactions more generally . our findings need to be viewed in light of some methodological limitations . although our rate of retention was high ( the large age range of our sample makes it difficult to assess any developmentally sensitive outcomes . our definition of emotional dysregulation relied on elevations in three cbcl clinical scales ( a - a - a ) . others have used this scale without cutoffs to measure dysregulated mood , attention , and behavior pathology across dsm - iv diagnoses . 10 one limitation of our cbcl definition of desr is that , because it includes the attention problems scale , which is known to be associated with adhd , it may simply define a more severe form of adhd , rather than a categorically different form of adhd . in fact , our previous examination7 showed that desr was associated with greater adhd - associated impairment . on the other hand , our prior family studies of desr suggest that adhd + desr is distinct from a familial perspective.4,27 although we selected the cbcl - desr profile from ongoing conceptualizations of desr in the literature ( most fully articulated by barkley2 ) indexing deficits in self - regulating cognition and inhibiting behavior caused by strong emotions , we do not know whether our results would be replicated if other methods were used to define desr . investigating continuous measures or measures that do not have symptom overlap with other psychiatric diagnoses may help our understanding of desr . for example , gioia et al28 reported that children with adhd had higher ratings on the behavior rating inventory of executive function ( brief ) emotional control subscale compared with healthy children . however , since these authors did not report correlations of their brief - defined desr ( beyond impulsivity ) , it is unclear whether the brief - emotional control scale captures the same phenomenology as the cbcl - desr profile . on the other hand , the validity of our construct of cbcl - desr is supported by face validity , the consistency of the constituent symptoms with other definitions of desr , its expression across a wide range of comorbid disorders and elevations of clinical scales , and its association with significant impairments beyond those accounted for by adhd . nevertheless , further research is needed to evaluate other definitions of desr to determine its optimal definition . because our sample was clinically referred and primarily caucasian , our findings may not generalize to community samples and other ethnic groups . despite these considerations , these longitudinal findings indicate that intermediate ( > 1 and < 2 sd ) scores on the anxiety / depression , attention and aggression cbcl subscales can help identify a clinically meaningful subgroup of children with adhd at high risk for more psychopathology and poorer functioning than other children with adhd . these findings suggest that the cbcl - desr profile may aid in the identification of children with deficient emotional regulation within and without the context of adhd at high risk for compromised outcomes . systematic efforts should be undertaken to determine the pharmacological or psychological interventions to best prevent these poor outcomes in children with adhd and desr .

backgroundwhile symptoms of deficient emotional self - regulation ( desr ) have been long associated with attention - deficit / hyperactivity disorder ( adhd ) , there has been limited investigation of this aspect of the clinical picture of the disorder . the main aim of this study was to examine the predictive utility of desr in moderating the course of adhd children into adolescence.methodssubjects comprised 177 children with and 204 children without adhd followed for an average of 4 years ( aged 618 years at baseline , 54% male ) . subjects were assessed with structured diagnostic interviews and measures of psychosocial functioning . desr was defined by the presence ( n = 79 ) or absence ( n = 98 ) of child behavior checklist ( cbcl)-desr profile ( score 180 < 210 total of attention , aggression , and anxious / depressed subscales ) at the baseline assessment.resultsof subjects with desr at baseline , 57% had desr at follow - up . persistent adhd was significantly associated with desr at follow - up ( 2 ( 1 ) = 15.37 , p < 0.001 ) . at follow - up , adhd + desr subjects had significantly more comorbidities ( z = 2.55 , p = 0.01 ) , a higher prevalence of oppositional defiant disorder ( z = 3.01 , p = 0.003 ) , and more impaired cbcl social problems t - score ( t(227 ) = 2.41 , p = 0.02 ) versus adhd subjects.conclusionthis work suggests that a positive cbcl - desr profile predicts subsequent psychopathology and functional impairments in children with adhd suggesting that it has the potential to help identify children with adhd at high risk for compromised outcomes .

Introduction Materials and methods Subjects Measures Assessment of deficient emotional self-regulation Statistical analysis Results Sociodemographic characteristics Psychopathology at the 4-year follow-up Psychosocial functioning Discussion Conclusion

although attention - deficit / hyperactivity disorder ( adhd ) has been conceptualized as a disorder of deficient self - regulation,1 the dsm - iv ( diagnostic and statistical manual of mental disorders , 4th edition , text revision)2 symptoms of the disorder have been limited to those of cognitive ( inattention ) and behavioral ( hyperactivity/ impulsivity ) deficits , leaving out deficient emotional self - regulation ( desr ) . although these traits may be a source of significant morbidity , there has been a paucity of research on the subject.36 the symptoms of desr should not be confused with the emotional symptoms of mood disorders . desr differs from the persistent and severe irritability often seen in pediatric mood disorder.7 unlike desr , the abnormal mood of mood disorder is not defined by poor self - regulation and includes features that are not part of desr ( eg , the other mood criteria in dsm - iv ) . we recently examined8 whether a unique profile of the child behavior checklist ( cbcl ) would help identify desr in children with adhd . we defined desr if a child had a sum of t scores of > 180 but < 210 ( between one and two standard deviations [ sd ] above the mean ) on the anxiety/ depression , aggression , and attention scales of the cbcl ( cbcl - desr ) . this profile was selected because of its conceptual congruence with the clinical concept of desr and because its extreme ( > 210 , more than 2 sd above the mean ) form had been previously associated with severe forms of mood and behavioral dysregulation in children with adhd.9 we found that 44% of children with adhd had a positive cbcl - desr profile versus 2% of controls ( p < 0.001 ) . the cbcl - desr profile was associated with elevated rates of anxiety and disruptive behavior disorders , as well as significantly more impairment in emotional and interpersonal functioning . these findings suggested that the cbcl - desr profile helped identify a subgroup of adhd children with a psychopathological and functional profile consistent with the clinical concept of desr . desr remained associated with poorer psychosocial functioning even after controlling for comorbid disorders , suggesting that desr may represent a burdensome source of morbidity in children with adhd that is partially independent of comorbid disorders such as oppositional defiant disorder . others have examined this cbcl - desr profile but not using the cutoffs defined by our group . new insights into the role that desr plays in the clinical picture of adhd would have important clinical and scientific implications . the main aim of this study was to assess the longitudinal course of desr in adhd children . we hypothesized that a positive cbcl - desr profile will predict more psychopathology and poorer psychosocial functioning in children with adhd at follow - up . male subjects were assessed at baseline and at four - year follow - up while female subjects were assessed at baseline and at five - year follow - up . socioeconomic status was measured using the five - point hollingshead scale.24 assessment of desr relied on the cbcl,25 completed at baseline and follow - up . desr was defined as positive if the sum of the cbcl attention problems , aggressive behavior , and anxious - depressed t scores was 180 and < 210 . subjects with a sum of > 210 on these cbcl subscales were excluded , because we previously showed that these subjects were at risk for developing bipolar disorder and associated impairments.9 we compared adhd probands with and without desr on baseline demographic characteristics using t - tests , pearson s chi - square , or wilcoxon rank - sum tests . the methodology used in this study has been previously described.12,13 briefly , subjects were derived from two identically designed longitudinal case - control family studies of adhd . these studies recruited male and female subjects aged 618 years with ( n = 140 boys , n = 140 girls ) and without ( n = 120 boys , n = 122 girls ) dsm - iii - r adhd ascertained from pediatric and psychiatric clinics . male subjects were assessed at baseline and at four - year follow - up while female subjects were assessed at baseline and at five - year follow - up . kappa coefficients for individual diagnoses included adhd ( 1.0 ) , conduct disorder ( 1.0 ) , major depression ( 1.0 ) , mania ( 0.78 ) , separation anxiety ( 0.89 ) , agoraphobia ( 0.80 ) , panic ( 0.77 ) , and substance use disorder ( 1.0 ) . assessment of desr relied on the cbcl,25 completed at baseline and follow - up . a parent ( usually the mother ) of each participant completed the 1991 version of the child behavior checklist for children aged 418 years . desr was defined as positive if the sum of the cbcl attention problems , aggressive behavior , and anxious - depressed t scores was 180 and < 210 . subjects with a sum of > 210 on these cbcl subscales were excluded , because we previously showed that these subjects were at risk for developing bipolar disorder and associated impairments.9 we compared adhd probands with and without desr on baseline demographic characteristics using t - tests , pearson s chi - square , or wilcoxon rank - sum tests . of 280 subjects with adhd and 242 subjects without adhd at baseline , 242 and 229 , respectively , had cbcl data ( figure 1 ) . of the remaining 197 adhd subjects and 224 control subjects , 177 ( 100 boys , 77 girls ) and 204 ( 105 boys , 99 girls ) , respectively , had follow - up data . the mean follow - up time was 4.4 years ( sd 1.1 years , range 39 years ) . of the 177 adhd subjects with follow - up data , 119 subjects had cbcl data at the four - year follow - up . the kappa for desr between the baseline and four - year followup assessments was 0.29 ( p < 0.001 ) , 57% of subjects with desr at baseline also had desr at follow - up ( sensitivity , 33/58 ) , 72% of subjects without desr at baseline did not have desr at follow - up ( specificity , 44/61 ) , the positive predictive value was 66% ( 33/50 ) , and the negative predictive value was 64% ( 44/69 ) . the intraclass correlation of the continuous aaa score between baseline and follow - up was 0.47 ( p < 0.001 ) . persistent adhd ( full or subthreshold diagnosis in the last month ) was significantly associated with desr at follow - up ( 0/18 with remittent adhd had desr compared with 50/101 with persistent adhd had desr , ( 1 ) = 15.37 , p < 0.001 ) . comparisons were made between controls ( n = 204 ) versus adhd subjects with ( adhd + desr , n = 79 ) and without desr at baseline ( adhd , n = 98 ) . subjects used for this analysis ( n = 381 ) did not significantly differ from subjects lost to follow - up ( n = 40 ) on group status ( p = 0.64 ) , gender / study ( p = 0.65 ) , age ( p = 0.58 ) , socioeconomic status ( p = 0.16 ) , or ascertainment source ( p = 0.69 ) . at the adolescent follow - up , both adhd groups had significantly higher 1-year prevalence rates of bipolar ( z = 3.89 , p < 0.001 ) , major depressive ( z = 4.59 , p < 0.001 ) , multiple anxiety ( z = 4.27 , p < 0.001 ) , and conduct / antisocial personality ( z = 3.34 , p = 0.001 ) disorders compared with controls ( figure 2 ) . in addition , the adhd + desr group continued to have significantly more total comorbidities at follow - up compared with the adhd and control groups ( 1.4 versus 0.9 , z = 2.55 , p = 0.01 and 1.4 versus 0.3 , z = 8.61 , p < 0.001 , respectively ) as well as a significantly higher one - year prevalence of oppositional defiant disorder ( z = 3.01 , p = 0.003 and z = 7.28 , p < 0.001 , respectively , figure 2d ) . desr was not significantly associated with mood disorders at baseline or follow - up ( all p > 0.05 ) . at follow - up , both adhd groups continued to have higher thought problems and delinquent behavior t scores at follow - up compared with the controls ( adhd + desr : t(227 ) = 5.38 , adhd : t(227 ) = 3.79 , both p < 0.001 ) . adhd + desr subjects continued to have a more impaired social problems t score compared with the adhd and control groups ( t(227 ) = 2.41 , p = 0.02 and t(227 ) = 7.68 , p < 0.001 , respectively , figure 3 ) . figure 4a shows that the adhd + desr group continued to have poorer global functioning than the adhd and control groups at follow - up ( t(377 ) = 3.71 , p < 0.001 and t(377 ) = 15.14 , p < 0.001 , respectively ) . while desr was associated with significantly poorer social adjustment at baseline ( t(365 ) = 4.38 , p < 0.001 ) , this no longer held true at follow - up due to the more impaired social adjustment inventory for children and adolescents ( saica ) score in the adhd group at follow - up ( t(166 ) = 0.75 , p = 0.45 , figure 4b ) . the adhd + desr group had poorer family environment scale scores at baseline ( expression : t(372 ) = 2.35 , p = 0.02 ; conflict : t(372 ) = 2.62 , p = 0.009 ; cohesion : t(372 ) = 2.05 , p = 0.04 ) and follow - up ( expression : t(241 ) = 1.33 , p = 0.18 ; conflict : t(241 ) = 0.96 , p = 0.34 ; cohesion : t(242 ) = 1.66 , p = 0.10 ) compared with the adhd group , although the differences attained statistical significance only at baseline ( figure 5 ) . desr was not associated with educational functioning ( extra tutoring , placement in special class , repeated grade ) at baseline or follow - up ( all p > 0.30 ) . at the adolescent follow - up , both adhd groups had significantly higher 1-year prevalence rates of bipolar ( z = 3.89 , p < 0.001 ) , major depressive ( z = 4.59 , p < 0.001 ) , multiple anxiety ( z = 4.27 , p < 0.001 ) , and conduct / antisocial personality ( z = 3.34 , p = 0.001 ) disorders compared with controls ( figure 2 ) . in addition , the adhd + desr group continued to have significantly more total comorbidities at follow - up compared with the adhd and control groups ( 1.4 versus 0.9 , z = 2.55 , p = 0.01 and 1.4 versus 0.3 , z = 8.61 , p < 0.001 , respectively ) as well as a significantly higher one - year prevalence of oppositional defiant disorder ( z = 3.01 , p = 0.003 and z = 7.28 , p < 0.001 , respectively , figure 2d ) . desr was not significantly associated with mood disorders at baseline or follow - up ( all p > 0.05 ) . at follow - up , both adhd groups continued to have higher thought problems and delinquent behavior t scores at follow - up compared with the controls ( adhd + desr : t(227 ) = 5.38 , adhd : t(227 ) = 3.79 , both p < 0.001 ) . adhd + desr subjects continued to have a more impaired social problems t score compared with the adhd and control groups ( t(227 ) = 2.41 , p = 0.02 and t(227 ) = 7.68 , p < 0.001 , respectively , figure 3 ) . figure 4a shows that the adhd + desr group continued to have poorer global functioning than the adhd and control groups at follow - up ( t(377 ) = 3.71 , p < 0.001 and t(377 ) = 15.14 , p < 0.001 , respectively ) . while desr was associated with significantly poorer social adjustment at baseline ( t(365 ) = 4.38 , p < 0.001 ) , this no longer held true at follow - up due to the more impaired social adjustment inventory for children and adolescents ( saica ) score in the adhd group at follow - up ( t(166 ) = 0.75 , p = 0.45 , figure 4b ) . the adhd + desr group had poorer family environment scale scores at baseline ( expression : t(372 ) = 2.35 , p = 0.02 ; conflict : t(372 ) = 2.62 , p = 0.009 ; cohesion : t(372 ) = 2.05 , p = 0.04 ) and follow - up ( expression : t(241 ) = 1.33 , p = 0.18 ; conflict : t(241 ) = 0.96 , p = 0.34 ; cohesion : t(242 ) = 1.66 , p = 0.10 ) compared with the adhd group , although the differences attained statistical significance only at baseline ( figure 5 ) . desr was not associated with educational functioning ( extra tutoring , placement in special class , repeated grade ) at baseline or follow - up ( all p > 0.30 ) . our a priori definition of desr based on moderate ( > 1 and < 2 sd ) elevations of cbcl - desr identified a clinically meaningful subgroup of adhd children at high risk for long - term morbidity and dysfunction consistent with current conceptualizations of desr . the cbcl - desr profile was associated at the follow - up assessment into adolescent years with higher rates of persistence of adhd , more psychiatric comorbidity , more impaired scores on the social problems cbcl scale , as well as more impaired ratings of the global assessment of functioning scale . these findings suggest that the cbcl - desr profile can help identify a clinically meaningful subgroup of children with adhd with long - term compromised outcomes . the finding that 57% of subjects with desr at baseline also had desr at follow - up suggests that desr is a largely persistent trait ( intraclass correlation of the continuous aaa score between baseline and follow - up was 0.47 ) . also noteworthy is the finding that desr was predictive of a persistent course of adhd ; 50% of adhd youth with persistent adhd had desr versus 0% in those without . at the adolescent follow - up , adhd + desr subjects continued to have significantly more mean number of comorbidities compared with adhd and control subjects as well as a significantly higher one - year prevalence of oppositional defiant disorder . these findings are congruent with those derived from analysis of the follow - up cbcl data showing more social problems associated with desr in children with adhd growing up . although social functioning as measured by the saica was significantly associated with desr at baseline and not at follow - up , subjects with desr had the same high overall saica score at follow - up as they did at baseline ( figure 4b ) . also congruent with the psychopathological findings are those showing that children with adhd + desr continued to have poorer global functioning than the adhd and control groups at follow - up . several diagnostic and functioning measures that were associated with desr at baseline were no longer significantly different at follow - up , including multiple anxiety disorders , conduct / antisocial personality disorder , several cbcl scales , saica , and family environment scale . therefore , the cbcl - desr profile appears better at identifying children with adhd with more extreme concurrent impairments than at identifying children at risk for future impairments . the fact that nearly half ( 43% ) of the children with desr at baseline no longer had desr at follow - up may explain the somewhat weaker findings at follow - up . desr was not associated with substance use disorders at baseline or follow - up . our longitudinal findings that children with adhd with the cbcl - desr profile were at significantly increased risk for oppositional defiant disorder but not for depression and bipolar disorder stand in contrast with findings of increased risks for these mood disorders associated with the cbcl - pediatric bipolar profile.9 this latter profile requires very abnormal ( 2 sd ) scores on the same scales of anxiety/ depression , aggression , and attention of the cbcl used to define the cbcl - desr profile . these nonoverlapping profile findings between the high ( 2 sd ) and the intermediate ( 1 sd ) cbcl profiles ( used to define desr ) support the hypotheses that these are mutually exclusive profiles that identify different clinical entities , with the severe profile likely identifying the extreme mood dysregulation associated with mood disorder , while the intermediate one is limited to deficient emotional self - regulation . children who fulfilled the cbcl - desr criteria were not more likely to have bipolar disorder or major depression or to have been hospitalized than other children with adhd . in this regard , our data support the utility of the cbcl in defining desr as a substantially different clinical entity than the cbcl - juvenile bipolar profile used to identify children at high risk for pediatric bipolar disorder . 10 one limitation of our cbcl definition of desr is that , because it includes the attention problems scale , which is known to be associated with adhd , it may simply define a more severe form of adhd , rather than a categorically different form of adhd . on the other hand , our prior family studies of desr suggest that adhd + desr is distinct from a familial perspective.4,27 although we selected the cbcl - desr profile from ongoing conceptualizations of desr in the literature ( most fully articulated by barkley2 ) indexing deficits in self - regulating cognition and inhibiting behavior caused by strong emotions , we do not know whether our results would be replicated if other methods were used to define desr . however , since these authors did not report correlations of their brief - defined desr ( beyond impulsivity ) , it is unclear whether the brief - emotional control scale captures the same phenomenology as the cbcl - desr profile . on the other hand , the validity of our construct of cbcl - desr is supported by face validity , the consistency of the constituent symptoms with other definitions of desr , its expression across a wide range of comorbid disorders and elevations of clinical scales , and its association with significant impairments beyond those accounted for by adhd . despite these considerations , these longitudinal findings indicate that intermediate ( > 1 and < 2 sd ) scores on the anxiety / depression , attention and aggression cbcl subscales can help identify a clinically meaningful subgroup of children with adhd at high risk for more psychopathology and poorer functioning than other children with adhd . these findings suggest that the cbcl - desr profile may aid in the identification of children with deficient emotional regulation within and without the context of adhd at high risk for compromised outcomes .

hemodialysis ( hd ) patients usually experience relatively high mortality rates ; for example , approximately 23% per annum in the u.s . , 15% in europe and 9% in japan . as the most common treatment of advanced kidney failure , hd typically requires patients to follow a strict treatment schedule , which typically entails receiving dialysis on a thrice weekly schedule ; either monday - wednesday - friday ( mwf ) or tuesday - thursday - saturday ( tts ) . during the intervals between dialysis sessions , electrolytes , fluid and various uremic toxins accumulate and , as a result , therefore , the intermittent dialysis schedule , mwf or tts , may put patients at higher risk of death on certain days . in particular , patients on a mwf schedule may have higher risk of death on monday , whereas those on a tts schedule may experience an elevated risk on tuesday ; since these days follow the longest intervals without the benefit of dialysis . various studies have assessed the association between day - of - week - specific mortality risk and dialysis schedule . for example , bleyer , russell , and satko ( 1 ) found a significantly higher risk of sudden death and cardiac - related death on monday for mwf schedule patients , and on tuesday for tts schedule patients . ( 2 ) identified modifiable risk factors for cardiac arrest in dialysis units , including age , diabetes , using a catheter for vascular access , and being hospitalized within the past 30 days . in addition , results from karnik et al . ( 2 ) revealed that there is a higher risk of cardiac arrest on monday for mwf schedule patients . bleyer et al . ( 3 ) applied a strict definition of sudden death and investigated the association between the timing of hd and occurrences of sudden death among hd patients . the authors found an increased risk of sudden death in the 12-hour period after starting dialysis , and also in the 12-hour period at the end of the weekend interval ( i.e. , before starting hd , namely monday and tuesday ) . for several reasons , most prior studies were based on relatively small sample sizes ; most were confined to u.s . additionally , for the most part , these analyses did not examine interactions between weekday and patient characteristics . for example , it is possible that mortality elevations on given days may be accentuated for various patient subgroups ; e.g. , older patients , diabetics , and patients with various comorbid conditions . our study uses data from the dialysis outcomes and practice patterns study ( dopps ) , an international prospective observational study of hemodialysis treatment and patient outcomes . the dopps - i ( 1996 - 2001 ) comprised over 17,000 patients in seven countries ( france , germany , italy , japan , spain , united kingdom , and the u.s . ) from more than 300 dialysis facilities . the dopps - ii ( 2002 - 2004 ) population included over 12,000 patients in 12 countries ( dopps - i countries , plus australia / new zealand , belgium , canada , and sweden ) from more than 300 dialysis facilities . the sampled facilities have at least 20 - 25 hd patients and represent all geographic regions and all types of facilities in each country . the dopps selects a random sample of hd patients within each participating facility . in this study , we carry out a comprehensive investigation of the association between day - of - week - specific death rates and dialysis schedule , in the u.s . , japan , and several european countries ( belgium , france , germany , italy , spain , sweden , united kingdom ) . in addition to having a very large sample size and long follow - up period , the dopps database contains information on many demographic variables and comorbid conditions . since the endpoint is time - to - death , we used survival analysis ( cox regression ) with day - of - the - week serving as a time - dependent covariate . cox regression is well - suited for this purpose since it is designed to handle a data structure where time until death is potentially censored and accurately tracks which patients are at risk on a particular weekday . baseline characteristics of the prevalent cross - section of patients are presented in table 1 by region . patients , european and japanese patients were , on average , more likely to be male , to have lower bmi , less likely to have diabetes and less likely to have cardiovascular disease ( cvd ) . subsequent models were constructed from both prevalent ( cross - sectional ) and incident patients . in total , of the deaths , 2,663 were among u.s patients , 1,391 among european patients , and 341 among japanese patients . in addition , 1,744 out of 4,395 ( 40% ) death were caused by cvd . a total of 2,489 out of 4,395 ( 57% ) deaths were among patients on a mwf schedule , while 1,906 ( 43% ) were on a tts schedule . the distribution of deaths by day - of - week for each dialysis schedule group is shown in figure 1 . for mwf schedule patients , monday had a much higher percentage of deaths for u.s . , european and japanese patients ( 19.7% , 19.6% and 17.9% , respectively ) . for tts schedule patients , for mwf schedule patients , friday appears to have the highest risk of death in japan , with approximately 1/5 of deaths occurring on that day . for tts schedule patients ( 15.7% ) , while saturday appears to have a higher risk of death in europe and japan ( 18% for both ) . for each region , cox regression models were used to estimate the covariate - adjusted day - of - week effect on hazard of death , with day - of - week coded as a time - dependent covariate . the model was set up so that mortality hazard on each day was compared to the seven - day average . figure 2 contains three sets of covariate - adjusted hazard ratios ( hrs ) of all - cause mortality by day - of - week with mwf schedule for u.s . patients [ figure 2(a ) ] , for european patients [ figure 2(b ) ] , and for japanese patients [ figure 2(c ) ] . patients on a mwf dialysis schedule from the u.s . experienced a significant ( p<0.0001 ) 41% higher risk of all - cause death on mondays relative to the seven - day average . european patients experienced a significant 34% higher risk of all - cause death on mondays versus the seven - day average ( p=0.001 ) , while patients from japan experienced a 27% higher risk of all - cause death on monday ( p=0.154 ) as well as a 44% higher mortality risk on friday ( p= 0.04 ) . corresponding results for tts schedule patients are shown in figure 3 ; again , with each week day compared to the overall average . a significant 39% higher risk of all - cause mortality was observed on tuesdays for u.s . european patients experienced a significant 22% higher risk of all - cause death on tuesdays ( p=0.043 ) and a significant 31% higher risk of all - cause death on saturdays ( p=0.013 ) . japanese patients experienced a significant 43% higher risk of all - cause death on tuesday ( p=0.044 ) as well as a 43% higher risk on saturdays ( p=0.07 ) . figure 4 shows covariate - adjusted hrs of cvd and non - cvd mortality by day - of - week for mwf schedule patients . in the u.s . , cvd mortality risk was higher by 45% on mondays relative to the seven - day average ( p<0.0001 ) ; the corresponding value for non - cvd mortality was 38% ( p<0.0001 ) . in europe , patients experienced a significant 55% higher risk of cvd mortality on mondays ( p=0.006 ) , while also experiencing a significant 27% higher risk of non - cvd mortality on mondays ( p=0.023 ) . in japan , cvd mortality risk was higher by 62% on mondays ( p=0.098 ) ; the corresponding value for non - cvd mortality was 10% ( p=0.674 ) . japanese patients , however , experienced a significant 100% higher non - cvd mortality risk on fridays versus the seven - day average ( p=0.001 ) . in figure 5 , hrs of cvd and non - cvd mortality by day - of - week for tts schedule patients is displayed . in the u.s . , cvd mortality risk was higher by 56% on tuesdays relative to the seven - day average ( p<0.0001 ) ; patients also experienced a significant 26% higher non - cvd mortality risk on tuesdays ( p=0.016 ) . in europe , patients experienced 43% higher risk of cvd mortality on tuesdays ( p=0.058 ) , with the corresponding value for non - cvd mortality being 15% ( p=0.236 ) . however , european patients had an 88% higher risk of cvd death on saturdays ( p=0.001 ) . in japan , patients experienced a significant 75% higher risk of cvd mortality on tuesdays ( p=0.037 ) and also experienced a 93% higher cvd mortality risk on saturdays ( p=0.034 ) . however , in japan there was no evidence of higher non - cvd mortality on tuesdays compared to the seven - day average ( hr=1.26 ; p=0.300 ) . europe and japan ) to study which patient characteristics were associated with the day - of - week effect on all - cause mortality . for this part of the analysis , we focused specifically on the impact of sex , 14 comorbid conditions and vascular access type on each of two effects : the mortality elevation on mondays ( for mwf schedule patients ) , and that on tuesdays ( for tts patients ) . none of the above listed factors seemed to modulate or to account for the effects , with the possible exceptions of cancer ( other than skin ) being associated with a lower risk of all - cause mortality on mondays ( for mwf schedule patients ) and on tuesdays ( for tts patients ) ( hr = 1.18 for patients with cancer [ other than skin ] , hr = 1.40 for patients without cancer [ other than skin ] , p - value = 0.033 ) ; neurological disease being associated with a higher risk of all - cause mortality on mondays ( mwf schedule ) , and on tuesdays ( tts schedule ) ( hr = 1.59 for patients with neurologic disease , hr = 1.33 for patients without neurologic disease , p - value = 0.041 ) ; and sex , where the effect of tuesday was higher for males on a tts schedule . a final cox model was fitted which combined dopps - i patients from the u.s . , europe and japan to determine whether time - dependent measures of blood pressure , potassium , sodium , ultrafiltration rate , intradialytic weight loss , residual renal function ( rrf ) and the use of diuretics modify the monday and tuesday effects . table 4 shows that low ultrafiltration rate was associated with higher risk of all - cause mortality on mondays with mwf schedule and on tuesdays with tts schedule . diuretic use was associated with lower risk of all - cause mortality on mondays with mwf schedule and on tuesdays with tts schedules . despite its proven value as a life - saving therapy , hd remains an intermittent intervention , most typically administered thrice weekly . harmful waste products and fluid accumulated over the extended weekend interval may therefore put patients at a higher risk of death on certain days . our results from the dopps in u.s . , european , and japanese patients indicate that in all three regions , hd patients have a higher risk of all - cause death on mondays if they are on mwf schedule , or tuesdays if they are on tts schedule . thus findings from prior reports ( 1 , 3 ) are corroborated and expanded upon here . this day - of - week effect tends to be stronger for cvd than non - cvd death overall . the european and japanese data tend to confirm the effect , quite evident in the us data , of increased mortality on the day of the first dialysis session in the week . on the other hand , there are unique features to the results in europe and japan , such as high mortality on saturday in the tts schedule which may be due to practice patterns unique to those countries / regions . such practice patterns could , for example , include continued aggressive / usual ultrafiltration and dialysis on fridays or saturdays , ( the last dialysis session of the week ) in order to allow for the anticipated longer weekend gap in dialysis , despite the patient being closer to their target post dialysis weight at the time of the last dialysis session of the week . this could predispose to a greater degree of intradialytic hypotension and/or hypokalemia which in turn would increase the tendency for cvd events , including sudden death . however , this mechanism while plausible , remains speculative . non - dialysis days had lower rrs for mortality compared to dialysis days perhaps because dialysis itself increases the risk of mortality . for hd patients , very large amounts of fluids and toxins are removed in a relatively short time period , particularly if the time elapsed since the last dialysis is long . this increases the potential for occurrence of intradialytic hypotension , which has previously been found to be a risk factor for mortality among hd patients ( 4 , 5 ) . rapid reduction in post dialysis potassium may also be a contributory factor , as hypokalemia can enhance the risk for cardiac arrhythmia and sudden death , and this is more likely to occur on a dialysis day as opposed to the day preceding dialysis . this study presents some evidence that the day - of - week effect is modulated by cancer ( other than skin ) , neurological disease , sex , low ultrafiltration rate and use of diuretics ; but apparently not by other suspected factors including other comorbid conditions , blood pressure , serum potassium , serum sodium , intradialytic weight loss and rrf ( the hazard ratio for the interaction with day of the week in this instance was protective but statistically non significant ) . potential explanations toward the interpretation of some of these interactions are again speculative . for those with cancer , it could be speculated that the interaction with first day of the week was associated with lower mortality because the mechanism of death may predominantly be through non - cardiovascular mechanisms . those with neurologic disease may have an accentuated early in the week effect because of vascular diseases being the underlying cause for many types of neurologic disease ( e.g. , cerebrovascular disease ) . similarly males have a greater association with cardiovascular disease and hence may have an interaction with early in the week mortality . low ultrafiltration rate may at least in part reflect patients especially prone to dialysis - related hypotension which is itself a predictor of worse patient outcomes . in particular , the finding that modification of the early - in - the - week mortality risk in association with diuretic use has therapeutic implications , especially in patients with significant rrf who are likely to be responsive to diuretic therapy . this is consistent with previously published dopps findings showing a lower mortality among those receiving diuretics ( 6 ) . low ultrafiltration may represent patients with low blood pressure to begin with or those that are malnourished , thus being associated with higher early in the week mortality . dialysis schedule was inferred from the reported date of dose of dialysis reporting , which was collected once every four months . in addition , the dopps data do not include the exact time of death , nor the timing of dialysis sessions ; both of which would be useful additional data in further understanding the day - of - week effect . the secondary endpoint of cvd death is certainly of interest , but cvd death may not be ascertained completely or determined consistently across centers . both the smaller sample size and lower event rate , inferences pertaining to japanese hd patients are subject to substantially more uncertainty than that for u.s . however , these countries share similar genetic and environmental factors as well as practice patterns , at least to some extent . in the analyses reported here , europeans were considered as a single group and conclusions were made about average effects over the 5 to 7 countries from this entire region . dopps ii also includes data from canada , australia and new zealand , but the sample sizes in these countries were too small to support reliable conclusions in this study . non adherence with dialysis sessions ( shortening or skipping sessions ) has been associated with higher mortality as detailed in a previous dopps paper ( 7 ) , and would be expected to raise overall mortality rates . unfortunately , there is insufficient information to assess the rate or effect of noncompliance in the dopps data . often , patients who are hospitalized would no longer follow their regular dialysis schedule . in this case , one would expect the observed day of week effect in this study to be somewhat attenuated . our results imply that there may be an advantage to a more frequent dialysis schedule in europe , the u.s and potentially japan . this is supported by the relatively low rates of mortality for patients receiving daily dialysis based on various reports ( 8 , 9 , 10 , 11 ) . one could hypothesize that more frequent dialysis would reduce or eliminate the day - of - week effect and , therefore , lower mortality on hd . ( 8) found that for certain patients , daily hd might have advantages over thrice weekly hd . blagg et al . ( 9 ) observed that patients treated by short - daily hd had a better survival rate than those treated by conventional hd . ( 10 ) reported that the survival of united states renal data system patients on short daily hd was 2 - 3 times lower than that of matched thrice weekly hd patients . ( 11 ) examined survival and hospitalization between frequent hd patients and patients undergoing thrice - weekly conventional hd . their study found that there was a non - significant reduction in death risk for patients using short - daily hd , while hospitalization patterns did not differ significantly between daily hd patients and their matched counterparts . daily hd can minimize fluid volumes ( 10 ) , improve many abnormal physiological parameters and metabolic markers associated with a high mortality ( 12 , 13 , 14 , 15 , 16 ) . quality of life has been reported to improve for patients on daily hd ( 17 , 18 ) . it can mean 5 or 6 , 5 - 7 , or 4 - 7 times of dialysis per week . hence there is the possibility that death rates are still elevated on the first and/or last day of dialysis . therefore , our results do not imply directly that a more frequent dialysis could reduce mortality . we would need to analyze the data using a cohort that received daily dialysis in order to determine whether day - of - week effect really disappeared and , hence , whether mortality was reduced . ( 19 ) investigated the association of hd treatment shift with continued survival among elderly patients . their study found that the morning - shift hd patients survived significantly longer than the afternoon - shift patients . in the present study , we observed a higher risk of all - cause mortality among european patients on saturdays on a tts schedule and a higher risk of cvd mortality among european and japanese patients on saturdays on a tts schedule . in summary , we have carried out a comprehensive analysis of the interaction between dialysis schedule and day - of - week specific mortality among hd patients in the u.s . , europe and japan . in particular , future studies aimed at evaluating the benefit of additional weekly dialysis sessions or alternate - day dialysis should also assess the impact of schedule on day - of - week - specific mortality . utilization of diuretics among those with significant rrf is another potential area fertile for further study , in addition to examination of other practice patterns that can reduce early - in - the - week mortality for those on conventional thrice weekly hd schedule , as this is likely to remain the norm for the vast majority of hd patients , at least for the foreseeable future . further study of data with the exact time of death and the timing of dialysis sessions would be valuable in further understanding the day - of - week effect . data were obtained on u.s . , japanese , and european ( belgium , france , germany , italy , spain , sweden , united kingdom ) patients from the dopps phase i and phase ii . the overall design of the dopps has been described in some detail previously ( 20 , 21 ) . in total , the study population consisted of n=22,163 patients ; 9,227 were from the u.s . patients were then observed until the earliest of death , transplant , loss to follow - up , or the end of the observation period : december 31 , 2001 for phase i patients , and december 31 , 2004 for phase ii patients . the mean follow - up time is 569 days for phase i patients and 564 days for phase ii patients . about 5% of patients received transplantations , and 4% of patients withdrew from the study . the primary outcome of interest is death [ all - cause , cvd and non - cvd ] . cvd mortality was defined as the death with primary cause being any of acute myocardial infarction , hyperkalemia , pericarditis , atherosclerotic heart disease , cardiomyopathy , cardiac arrhythmia , cardiac arrest , valvular heart disease , pulmonary edema due to exogenous fluid or congestive heart failure . baseline data on demographics , laboratory values , medical history and medications were collected at the start of participation in the dopps . thereafter , longitudinal data were collected at four - month intervals , including updated hospitalization events , vascular access , laboratory values , medications , dialysis prescription , dialysis dose and rrf . detailed information is also reported for the final dialysis session in the four - month period , including the date dialysis was received . for purposes of analysis , if the reported date was a monday , wednesday or friday , the dialysis schedule was considered to be mwf in the corresponding four - month interval . if this date was missing or a sunday , the dialysis schedule was carried over from the preceding four - month reporting interval . about 79% of patients did not switch the dialysis schedule in this study , 13% patients switched once , either from mwf to tts , or from tts to mwf , and 8% patients switched more than once . baseline characteristics for prevalent patients ( at the start of dopps i and ii , n=13,820 ) were summarized , with simple means for continuous variables and proportions for categorical variables . the covariate of interest is the day - of - week ( sunday , monday , , saturday ) , which was coded as a time - dependent covariate in a cox model with time origin as the first ever hd day . since patients were not under observation until they entered the dopps study , using time scale of days since study entry can be subject to left - truncation bias , therefore , we used days since onset of dialysis ( dialysis vintage ) as the time scale . adjustment covariates included sex , race , 14 comorbid conditions ( coronary heart disease , cancer other than skin , other cardiovascular disease , cerebrovascular disease , congestive heart failure , diabetes , gastrointestinal bleeding , hiv / aids , hypertension , lung disease , neurologic disease , psychiatric disorder , peripheral vascular disease , and recurrent cellulitis ) , body mass index ( grouped as < 20 , [ 20 - 25 ) , [ 25 - 30 ) , 30 kg / m ) , and vascular access ( catheter use ) . dialysis schedule , country , phase and age group ( 18 - 29 , 30 - 39 , 40 - 44 , 45 - 49 , 50 - 54 , 55 - 59 , 60 - 64 , 65 - 69 , 70 years ) were adjusted through stratification . since patients from the same facility may be correlated due to shared practice patterns , valid statistical procedures must account for intra - cluster dependence , which was done using a robust ( sandwich ) estimator of the covariance ( 22 ) . day of the week was coded such that each day was compared to the average of the seven days of the week . table 5 shows the coding of the time - dependent covariates related to day of the week . the average of the seven days of the week is constrained to be zero in the model ; therefore , each day was compared to this geometric average ( i.e. , zero ) . the comparison of each level of a categorical predictor to the average is as defensible statistically as the comparison of each level to a reference level . the former is used much less frequently than the latter , but is the best choice for our analysis . further analyses used cox models fitted to patients from all three regions combined , in order to study whether sex , 14 comorbid conditions and vascular access ( catheter use ) modified the day - of - week effect . models including interactions between region and day - of - week have been compared to those without such interactions . since only small differences in estimates are observed between these two models , only results from the latter models are reported in this article . also , in assessing the potential modifiers of the day of week effect , we found that the interactions between the modifiers and mondays ( for mwf schedule patients ) are similar to those between the modifiers and tuesdays ( for tts schedule patients ) ; thus , in the models reported , these interactions are assumed equal and combined . further , another cox model was fitted to all patients ' laboratory covariates ( blood pressure , potassium , sodium , ultrafiltration rate , weight loss , rrf , and use of diuretics ) . each of these variables , except rrf and the diuretic use , was categorized into three groups : low ( lowest quartile ) , normal ( middle two quartiles ) and high ( highest quartile ) . these time - dependent covariates were measured monthly in the unit and weight loss was measured with each treatment . however , the dopps updated such measures only every four months . in this last time - dependent analysis , results were based on dopps - i data because only baseline potassium and sodium values were available in dopps - ii . in this study , the covariate of interest is day of the week ( sunday , monday , , saturday ) . cox regression analyses employed the phreg procedure , and handled time - dependent covariates and left - truncation through the counting process style of input . facility clustering effects were accounted for through the robust sandwich covariance estimates . in sas phreg procedure , this is implemented using covs(aggregate ) option , with i d specified as facility [ sas 9.2 documentation , example 64.11 in the phreg procedure ( 23 ) ] .

the risk of death for hemodialysis patients is thought to be highest on the days following the longest interval without dialysis ( usually mondays and tuesdays ) ; however , existing results are inconclusive . to clarify this we analyzed dialysis outcomes and practice patterns study ( dopps ) data of 22,163 hemodialysis patients from the united states , europe and japan . our study focused on the association between dialysis schedule and day - of - week of all - cause , cardiovascular and non - cardiovascular mortality with day - of - week coding as a time - dependent covariate . the models were adjusted for dialysis schedule , age , country , dopps phase i or ii , and other demographic and clinical covariates comparing mortality on each day to the 7-day average . patients on a monday - wednesday - friday ( mfw ) schedule had elevated all - cause mortality on monday , and those on a tuesday - thursday - saturday ( tts ) schedule increased risk of mortality on tuesday in all 3 regions . the association between day - of - week mortality and schedule was generally stronger for cardiovascular than non - cardiovascular mortality , and most pronounced in the united states . unexpectedly , japanese patients on a mwf schedule had a higher risk of non - cardiovascular mortality on fridays , and european patients on a tts schedule experienced an elevated cardiovascular mortality on saturdays . thus , future studies are needed to evaluate the influence of practice patterns on schedule - specific mortality and factors that could modulate this effect .

Introduction Results Discussion Methods

as the most common treatment of advanced kidney failure , hd typically requires patients to follow a strict treatment schedule , which typically entails receiving dialysis on a thrice weekly schedule ; either monday - wednesday - friday ( mwf ) or tuesday - thursday - saturday ( tts ) . during the intervals between dialysis sessions , electrolytes , fluid and various uremic toxins accumulate and , as a result , therefore , the intermittent dialysis schedule , mwf or tts , may put patients at higher risk of death on certain days . in particular , patients on a mwf schedule may have higher risk of death on monday , whereas those on a tts schedule may experience an elevated risk on tuesday ; since these days follow the longest intervals without the benefit of dialysis . various studies have assessed the association between day - of - week - specific mortality risk and dialysis schedule . for example , bleyer , russell , and satko ( 1 ) found a significantly higher risk of sudden death and cardiac - related death on monday for mwf schedule patients , and on tuesday for tts schedule patients . ( 2 ) identified modifiable risk factors for cardiac arrest in dialysis units , including age , diabetes , using a catheter for vascular access , and being hospitalized within the past 30 days . ( 2 ) revealed that there is a higher risk of cardiac arrest on monday for mwf schedule patients . ( 3 ) applied a strict definition of sudden death and investigated the association between the timing of hd and occurrences of sudden death among hd patients . the authors found an increased risk of sudden death in the 12-hour period after starting dialysis , and also in the 12-hour period at the end of the weekend interval ( i.e. our study uses data from the dialysis outcomes and practice patterns study ( dopps ) , an international prospective observational study of hemodialysis treatment and patient outcomes . the dopps - ii ( 2002 - 2004 ) population included over 12,000 patients in 12 countries ( dopps - i countries , plus australia / new zealand , belgium , canada , and sweden ) from more than 300 dialysis facilities . in this study , we carry out a comprehensive investigation of the association between day - of - week - specific death rates and dialysis schedule , in the u.s . since the endpoint is time - to - death , we used survival analysis ( cox regression ) with day - of - the - week serving as a time - dependent covariate . cox regression is well - suited for this purpose since it is designed to handle a data structure where time until death is potentially censored and accurately tracks which patients are at risk on a particular weekday . patients , european and japanese patients were , on average , more likely to be male , to have lower bmi , less likely to have diabetes and less likely to have cardiovascular disease ( cvd ) . in total , of the deaths , 2,663 were among u.s patients , 1,391 among european patients , and 341 among japanese patients . a total of 2,489 out of 4,395 ( 57% ) deaths were among patients on a mwf schedule , while 1,906 ( 43% ) were on a tts schedule . the distribution of deaths by day - of - week for each dialysis schedule group is shown in figure 1 . for mwf schedule patients , monday had a much higher percentage of deaths for u.s . for tts schedule patients , for mwf schedule patients , friday appears to have the highest risk of death in japan , with approximately 1/5 of deaths occurring on that day . for tts schedule patients ( 15.7% ) , while saturday appears to have a higher risk of death in europe and japan ( 18% for both ) . for each region , cox regression models were used to estimate the covariate - adjusted day - of - week effect on hazard of death , with day - of - week coded as a time - dependent covariate . the model was set up so that mortality hazard on each day was compared to the seven - day average . figure 2 contains three sets of covariate - adjusted hazard ratios ( hrs ) of all - cause mortality by day - of - week with mwf schedule for u.s . patients [ figure 2(a ) ] , for european patients [ figure 2(b ) ] , and for japanese patients [ figure 2(c ) ] . patients on a mwf dialysis schedule from the u.s . experienced a significant ( p<0.0001 ) 41% higher risk of all - cause death on mondays relative to the seven - day average . european patients experienced a significant 34% higher risk of all - cause death on mondays versus the seven - day average ( p=0.001 ) , while patients from japan experienced a 27% higher risk of all - cause death on monday ( p=0.154 ) as well as a 44% higher mortality risk on friday ( p= 0.04 ) . corresponding results for tts schedule patients are shown in figure 3 ; again , with each week day compared to the overall average . a significant 39% higher risk of all - cause mortality was observed on tuesdays for u.s . european patients experienced a significant 22% higher risk of all - cause death on tuesdays ( p=0.043 ) and a significant 31% higher risk of all - cause death on saturdays ( p=0.013 ) . japanese patients experienced a significant 43% higher risk of all - cause death on tuesday ( p=0.044 ) as well as a 43% higher risk on saturdays ( p=0.07 ) . figure 4 shows covariate - adjusted hrs of cvd and non - cvd mortality by day - of - week for mwf schedule patients . , cvd mortality risk was higher by 45% on mondays relative to the seven - day average ( p<0.0001 ) ; the corresponding value for non - cvd mortality was 38% ( p<0.0001 ) . in europe , patients experienced a significant 55% higher risk of cvd mortality on mondays ( p=0.006 ) , while also experiencing a significant 27% higher risk of non - cvd mortality on mondays ( p=0.023 ) . in japan , cvd mortality risk was higher by 62% on mondays ( p=0.098 ) ; the corresponding value for non - cvd mortality was 10% ( p=0.674 ) . japanese patients , however , experienced a significant 100% higher non - cvd mortality risk on fridays versus the seven - day average ( p=0.001 ) . in figure 5 , hrs of cvd and non - cvd mortality by day - of - week for tts schedule patients is displayed . , cvd mortality risk was higher by 56% on tuesdays relative to the seven - day average ( p<0.0001 ) ; patients also experienced a significant 26% higher non - cvd mortality risk on tuesdays ( p=0.016 ) . in europe , patients experienced 43% higher risk of cvd mortality on tuesdays ( p=0.058 ) , with the corresponding value for non - cvd mortality being 15% ( p=0.236 ) . however , european patients had an 88% higher risk of cvd death on saturdays ( p=0.001 ) . in japan , patients experienced a significant 75% higher risk of cvd mortality on tuesdays ( p=0.037 ) and also experienced a 93% higher cvd mortality risk on saturdays ( p=0.034 ) . however , in japan there was no evidence of higher non - cvd mortality on tuesdays compared to the seven - day average ( hr=1.26 ; p=0.300 ) . europe and japan ) to study which patient characteristics were associated with the day - of - week effect on all - cause mortality . for this part of the analysis , we focused specifically on the impact of sex , 14 comorbid conditions and vascular access type on each of two effects : the mortality elevation on mondays ( for mwf schedule patients ) , and that on tuesdays ( for tts patients ) . none of the above listed factors seemed to modulate or to account for the effects , with the possible exceptions of cancer ( other than skin ) being associated with a lower risk of all - cause mortality on mondays ( for mwf schedule patients ) and on tuesdays ( for tts patients ) ( hr = 1.18 for patients with cancer [ other than skin ] , hr = 1.40 for patients without cancer [ other than skin ] , p - value = 0.033 ) ; neurological disease being associated with a higher risk of all - cause mortality on mondays ( mwf schedule ) , and on tuesdays ( tts schedule ) ( hr = 1.59 for patients with neurologic disease , hr = 1.33 for patients without neurologic disease , p - value = 0.041 ) ; and sex , where the effect of tuesday was higher for males on a tts schedule . a final cox model was fitted which combined dopps - i patients from the u.s . , europe and japan to determine whether time - dependent measures of blood pressure , potassium , sodium , ultrafiltration rate , intradialytic weight loss , residual renal function ( rrf ) and the use of diuretics modify the monday and tuesday effects . table 4 shows that low ultrafiltration rate was associated with higher risk of all - cause mortality on mondays with mwf schedule and on tuesdays with tts schedule . diuretic use was associated with lower risk of all - cause mortality on mondays with mwf schedule and on tuesdays with tts schedules . harmful waste products and fluid accumulated over the extended weekend interval may therefore put patients at a higher risk of death on certain days . , european , and japanese patients indicate that in all three regions , hd patients have a higher risk of all - cause death on mondays if they are on mwf schedule , or tuesdays if they are on tts schedule . this day - of - week effect tends to be stronger for cvd than non - cvd death overall . the european and japanese data tend to confirm the effect , quite evident in the us data , of increased mortality on the day of the first dialysis session in the week . on the other hand , there are unique features to the results in europe and japan , such as high mortality on saturday in the tts schedule which may be due to practice patterns unique to those countries / regions . such practice patterns could , for example , include continued aggressive / usual ultrafiltration and dialysis on fridays or saturdays , ( the last dialysis session of the week ) in order to allow for the anticipated longer weekend gap in dialysis , despite the patient being closer to their target post dialysis weight at the time of the last dialysis session of the week . non - dialysis days had lower rrs for mortality compared to dialysis days perhaps because dialysis itself increases the risk of mortality . rapid reduction in post dialysis potassium may also be a contributory factor , as hypokalemia can enhance the risk for cardiac arrhythmia and sudden death , and this is more likely to occur on a dialysis day as opposed to the day preceding dialysis . this study presents some evidence that the day - of - week effect is modulated by cancer ( other than skin ) , neurological disease , sex , low ultrafiltration rate and use of diuretics ; but apparently not by other suspected factors including other comorbid conditions , blood pressure , serum potassium , serum sodium , intradialytic weight loss and rrf ( the hazard ratio for the interaction with day of the week in this instance was protective but statistically non significant ) . for those with cancer , it could be speculated that the interaction with first day of the week was associated with lower mortality because the mechanism of death may predominantly be through non - cardiovascular mechanisms . those with neurologic disease may have an accentuated early in the week effect because of vascular diseases being the underlying cause for many types of neurologic disease ( e.g. similarly males have a greater association with cardiovascular disease and hence may have an interaction with early in the week mortality . in particular , the finding that modification of the early - in - the - week mortality risk in association with diuretic use has therapeutic implications , especially in patients with significant rrf who are likely to be responsive to diuretic therapy . low ultrafiltration may represent patients with low blood pressure to begin with or those that are malnourished , thus being associated with higher early in the week mortality . dialysis schedule was inferred from the reported date of dose of dialysis reporting , which was collected once every four months . in addition , the dopps data do not include the exact time of death , nor the timing of dialysis sessions ; both of which would be useful additional data in further understanding the day - of - week effect . however , these countries share similar genetic and environmental factors as well as practice patterns , at least to some extent . in the analyses reported here , europeans were considered as a single group and conclusions were made about average effects over the 5 to 7 countries from this entire region . often , patients who are hospitalized would no longer follow their regular dialysis schedule . our results imply that there may be an advantage to a more frequent dialysis schedule in europe , the u.s and potentially japan . one could hypothesize that more frequent dialysis would reduce or eliminate the day - of - week effect and , therefore , lower mortality on hd . ( 9 ) observed that patients treated by short - daily hd had a better survival rate than those treated by conventional hd . ( 10 ) reported that the survival of united states renal data system patients on short daily hd was 2 - 3 times lower than that of matched thrice weekly hd patients . we would need to analyze the data using a cohort that received daily dialysis in order to determine whether day - of - week effect really disappeared and , hence , whether mortality was reduced . in the present study , we observed a higher risk of all - cause mortality among european patients on saturdays on a tts schedule and a higher risk of cvd mortality among european and japanese patients on saturdays on a tts schedule . in summary , we have carried out a comprehensive analysis of the interaction between dialysis schedule and day - of - week specific mortality among hd patients in the u.s . , europe and japan . in particular , future studies aimed at evaluating the benefit of additional weekly dialysis sessions or alternate - day dialysis should also assess the impact of schedule on day - of - week - specific mortality . utilization of diuretics among those with significant rrf is another potential area fertile for further study , in addition to examination of other practice patterns that can reduce early - in - the - week mortality for those on conventional thrice weekly hd schedule , as this is likely to remain the norm for the vast majority of hd patients , at least for the foreseeable future . further study of data with the exact time of death and the timing of dialysis sessions would be valuable in further understanding the day - of - week effect . , japanese , and european ( belgium , france , germany , italy , spain , sweden , united kingdom ) patients from the dopps phase i and phase ii . patients were then observed until the earliest of death , transplant , loss to follow - up , or the end of the observation period : december 31 , 2001 for phase i patients , and december 31 , 2004 for phase ii patients . the mean follow - up time is 569 days for phase i patients and 564 days for phase ii patients . about 5% of patients received transplantations , and 4% of patients withdrew from the study . the primary outcome of interest is death [ all - cause , cvd and non - cvd ] . baseline data on demographics , laboratory values , medical history and medications were collected at the start of participation in the dopps . detailed information is also reported for the final dialysis session in the four - month period , including the date dialysis was received . for purposes of analysis , if the reported date was a monday , wednesday or friday , the dialysis schedule was considered to be mwf in the corresponding four - month interval . if this date was missing or a sunday , the dialysis schedule was carried over from the preceding four - month reporting interval . about 79% of patients did not switch the dialysis schedule in this study , 13% patients switched once , either from mwf to tts , or from tts to mwf , and 8% patients switched more than once . the covariate of interest is the day - of - week ( sunday , monday , , saturday ) , which was coded as a time - dependent covariate in a cox model with time origin as the first ever hd day . dialysis schedule , country , phase and age group ( 18 - 29 , 30 - 39 , 40 - 44 , 45 - 49 , 50 - 54 , 55 - 59 , 60 - 64 , 65 - 69 , 70 years ) were adjusted through stratification . since patients from the same facility may be correlated due to shared practice patterns , valid statistical procedures must account for intra - cluster dependence , which was done using a robust ( sandwich ) estimator of the covariance ( 22 ) . day of the week was coded such that each day was compared to the average of the seven days of the week . table 5 shows the coding of the time - dependent covariates related to day of the week . the average of the seven days of the week is constrained to be zero in the model ; therefore , each day was compared to this geometric average ( i.e. further analyses used cox models fitted to patients from all three regions combined , in order to study whether sex , 14 comorbid conditions and vascular access ( catheter use ) modified the day - of - week effect . models including interactions between region and day - of - week have been compared to those without such interactions . since only small differences in estimates are observed between these two models , only results from the latter models are reported in this article . also , in assessing the potential modifiers of the day of week effect , we found that the interactions between the modifiers and mondays ( for mwf schedule patients ) are similar to those between the modifiers and tuesdays ( for tts schedule patients ) ; thus , in the models reported , these interactions are assumed equal and combined . further , another cox model was fitted to all patients ' laboratory covariates ( blood pressure , potassium , sodium , ultrafiltration rate , weight loss , rrf , and use of diuretics ) . these time - dependent covariates were measured monthly in the unit and weight loss was measured with each treatment . in this last time - dependent analysis , results were based on dopps - i data because only baseline potassium and sodium values were available in dopps - ii . cox regression analyses employed the phreg procedure , and handled time - dependent covariates and left - truncation through the counting process style of input .

as a part of the age - associated deterioration of the immune system [ 13 ] , there is a chronic proinflammatory state even in the absence of clinically - apparent disease . inflamm - aging is characterized by elevated circulating levels of proinflammatory factors ( il-1 , il-6 , tnf , and prostaglandin e2 ) and anti - inflammatory mediators , ( il-1 receptor antagonist , soluble tnf receptor , il-10 , transforming growth factor beta , acute phase proteins , c - reactive protein , and serum amyloid a ) and contributes to the decreased ability of the elderly to mount an appropriate immune response following an infectious challenge [ 47 ] . one of the most prominent aspects of inflamm - aging is in presence of elevated circulating levels of the proinflammatory cytokine , interleukin ( il)-6 [ 8 , 9 ] . in an attempt to better define the role of il-6 in the systemic inflammatory response in the aged , we gave young and aged wild type ( wt ) and il-6 knockout ( ko ) balb / c female mice lipopolysaccharide ( lps ) . following the inflammatory challenge , aged il-6 ko mice had improved survival when compared to aged wt mice . moreover , we previously reported that aged il-6 ko mice showed a decreased acute phase response when compared to that of aged wt mice . in addition , hepatic injury was drastically reduced in aged il-6 ko mice given lps as compared with lps - exposed aged wt mice . recently , starr et al . reported similar findings in male c57bl/6 il-6ko mice using a higher dose of lps ( 5 mg / kg ) and a slightly older group of mice ( 26 months old ) than our study . taken together , these observations have suggested a crucial role for il-6 as a component of the aberrant systemic innate immune responses of the aged after an inflammatory challenge or injury and that this role is not sex or strain specific . macrophages play critical roles in phagocytosis of antigens , microorganisms , and cellular debris ; killing of invading pathogens and tumors ; and wound healing . many of the macrophage functions are carried out by secreted cytokines , which in turn , regulate multiple immune functions , especially inflammatory responses . the effects of aging on cytokine production by macrophages have shown conflicting results with in vitro stimulation of purified macrophage populations with lps and in vivo systemic administration of lps . for example , our laboratory and others reported that macrophages from aged mice produce less tnf and il-6 after in vitro exposure to lps than comparably stimulated cells from young mice [ 1519 ] . these findings have been observed despite the presence of inflamm - aging and the elevated baseline inflammatory state seen in healthy aged individuals [ 20 , 21 ] . since macrophages are considered to be an important source of proinflammatory cytokines in vivo , many possible explanations for this inconsistency can be proposed . among the multiple possibilities , the effect of the local environment in vitro versus the effect of the aged microenvironment in vivo has been considered [ 2224 ] . we reported that serum from aged fisher 344 rats increased the levels of il-6 by macrophages obtained from young animals and cultured in vitro without stimulants . these observations suggest that the elevated production of inflammatory mediators in the aged results , in part , from the interaction of macrophages with a variety of factors in their environment in situ . to analyze the role that il-6 might play in regulating the age - related defects in macrophages through alteration of proinflammatory cytokines , we exposed splenic macrophages from young and aged wt and il-6 ko mice to lps . our findings suggest that il-6 regulates the age - related defects in macrophages through alteration of proinflammatory cytokines and that these alterations are not due to changes in surface expression of tlr4 or the il-6 receptor . young ( 2 months old ) and aged ( 18 months old ) wt female balb / c mice were purchased from the national institute of aging colony at harlan laboratories ( indianapolis , in ) . manfred kopf , molecular biomedicine , eth zurich , switzerland , have a disruption of il-6 in the second exon ( first coding exon ) by insertion of a neo cassette . il-6 ko mice were backcrossed onto the balb / c background , bred , maintained , and aged at the taconic laboratories ( germantown , ny ) . young ( 2 months old ) and aged ( 18 months old ) female balb / c il-6 ko mice were used in the experiments described here . they were free of potential endemic viral pathogens that could influence their inflammatory response . all animals where maintained in an environmentally controlled facility at loyola university medical center for at least one week prior to experimentation . at the time of sacrifice , all mice were dissected and the organs were screened for visible tumors and/or gross abnormalities . if found , these animals were removed from the study . the experimental protocols described here followed the guidelines established by the publication , principles of laboratory animal care ( nih publication no . 8623 , revised 1985 ) , and were approved by the loyola university chicago institutional animal care and use committee . mice were sacrificed by co2 inhalation and subsequent cervical dislocation . in order to avoid confounding factors related to circadian rhythms , all animal protects briefly , spleens were aseptically removed and disrupted to yield a cell suspension in rpmi 1640 medium , supplemented with 5% fbs , penicillin ( 100 u / ml ) , streptomycin ( 100 g / ml ) , and 2 mm glutamine ( culture medium ) ( gibco - brl , grand island , ny ) . following red blood cell lysis with ack lysis buffer ( invitrogen corp . , carlsbad , ca ) , white blood cells were counted in a hemocytometer and viability was determined by trypan blue exclusion . two 10 cells were seeded in 96-well plates in 200 l culture medium . after incubation for 2 hours at 37c and 5% co2 , nonadherent cells were discarded by medium aspiration and washed twice with warm phosphate buffer saline ( pbs ) . this method resulted in an adherent population characterized as > 98% positive for mac-3 and di - i - acetylated low - density lipoprotein uptake , as we previously reported . adherent cells were treated in 200 l culture medium alone or containing 100 ng / ml lps from escherichia coli 0111:b4 ( sigma biosciences , st . louis , mo ) . in the absence of stimulation , the concentrations of tnf , il-1 , il-6 , and il-12 in macrophage supernatants were measured by commercially available opteia elisa kits ( bd pharmingen , san diego , ca ) according to the manufacturer instructions . flow cytometry was performed as previously described [ 16 , 17 ] . briefly , after blocking nonspecific staining with anti - cd16/cd32 ( fciii / ii ; bd - pharmingen , san diego , ca ) , total splenocyte suspensions were stained with apc - conjugated anti - cd3 ( 0.25 g/1 10 cells , clone 145 - 2c11 , ebioscience , san diego , ca ) , pe - conjugated anti - cd4 ( 0.125 g/1 10 cells , clone gk1.5 , ebioscience , san diego , ca ) , fitc - conjugated anti - cd8 ( 0.5 g/1 10 cells , clone 536.7 , ebioscience , san diego , ca ) , fitc - conjugated anti - ly49c ( 0.5 g/1 10 cells , clone 5e6 , ebioscience , san diego , ca ) , alexa fluor 750-conjugated anti - f4/80 ( 0.1 g/1 10 cells , clone bm8 , invitrogen corp . , camarillo , ca ) , pe - conjugated anti - il-6ra ( 0.06 g/1 10 cells , clone d7715a7 , bd - pharmingen , san diego , ca ) , apc - conjugated anti - cd19 ( 0.5 g/1 10 cells , clone mb 191 , ebioscience , san diego , ca ) , and biotin - conjugated anti - tlr4 ( 0.25 g/1 10 cells , clone mts510 , ebioscience , san diego , ca ) , biotin - conjugated anti - gp130 ( 1 g/1 10 cells , clone 125623 , r&d systems inc . , minneapolis , mn ) . following biotin - conjugate incubation , cells were incubated with percp - conjugated streptavidin ( 0.125 g/1 10 cells , ebioscience , san diego , ca ) . flow cytometric determinations were made using becton dickinson facscanto flow cytometer ( bdis , san jose , ca ) and data was analyzed with flowjo software ( tree star inc . , anova and tukey - kramer multiple comparisons were used to determine statistical significance using graphpad prism version 2.0 statistical package ( san diego , ca , usa ) . when stimulated in vitro with lps , macrophages from aged mice produce less cytokines than comparably stimulated cells from young mice [ 1519 ] . to determine if this phenotype may be determined in part by il-6 , which is elevated in the circulation of healthy aged individuals [ 9 , 10 , 28 , 29 ] , we evaluated proinflammatory cytokine production in splenic macrophages obtained from young aged wt and il-6 ko mice . macrophages from aged and young mice produced proinflammatory cytokines upon lps stimulation ( figures 1(a)1(d ) ) . as previously reported [ 1519 ] , macrophages from aged wt mice had decreased production of tnf and il-6 ( 55% ) , as well as il-1 ( 80% ) and il-12 ( 35% ) relative to splenic macrophages obtained from young wt mice given lps . similar to macrophages from aged wt mice , the production of cytokines by macrophages from young il-6 ko mice was reduced for tnf ( 67% ) , il-1 ( 31% ) , and il-12 ( 71% ) relative to that of young wt mice ( figure 1 ) . in contrast , as compared to macrophages from young il-6 ko mice , lps exposure induced higher cytokine production by cells from aged il-6 ko mice ( 1.3 , 2.2 and 1.2-fold for tnf , il-1 and il-12 , resp . ) ( figure 1 ) . cytokine production by macrophages from aged il-6 ko mice was elevated when compared to cells from aged wt mice ( 1.8 , 3.7 and 1.9-fold for tnf , il-1 and il-12 , resp . ) . overall , these results show that il-6 plays a role in regulating the age - related defects in macrophages through alteration of the production of proinflammatory cytokines . interleukin-6 is a pleiotropic cytokine , which has an important role in supporting the growth of t and b lymphocytes in lymphoid tissues , mainly in the spleen . therefore , lack of expression of il-6 could modify the phenotype of splenocytes and their production of cytokines when cultured in vitro . to test this hypothesis , total spleen cell suspensions were examined by flow cytometry for specific splenocyte cell populations . the percentage of t cells was determined by analyzing splenocytes bearing the cell surface markers cd3 ( t cells ) , cd4 ( cd3/cd4 ; helper / inducer cells ) , and cd8 ( cd3/cd8 ; suppressor / cytotoxic cells ) . relative to young wt mice , the percentage of cd3 t cells in splenocytes from aged wt mice was reduced ( 13% , p < .05 ) , table 1 . interestingly , this difference was more pronounced in splenocytes from aged il-6 ko mice relative to cells from young il-6 ko mice ( 24% reduction in cd3 t cells , p < .001 ) . an analysis of splenic cd3/cd4 helper / inducer t cells revealed no effects of aging or il-6 on their percentage . however , cd3/cd8 suppressor / cytotoxic t cells were reduced in splenocytes from aged wt mice ( 25% , p < .05 ) , relative to young wt mice . in addition , a marked ( 47% ) decrease in cd3/cd8 suppressor / cytotoxic t cells was found in aged il-6 ko animals , relative to splenocytes from young il-6 ko mice . analysis of b cell percentages in the spleen was also assessed by measuring b220 splenocytes , table 1 . this percentage was slightly increased by aging and il-6 deficiency , however , these differences failed to reach statistical significance . similarly , analysis of the f4/80 macrophage population showed no effects of aging or il-6 deficiency on their percentage . tlr4 involvement in age - related defects in macrophage activation following lps activation has been documented extensively ( reviewed in [ 1 , 2 ] ) . however , tlr4 levels in the context of aging and il-6 deficiency are unknown . to analyze the expression of tlr4 in macrophages from aged il-6 ko mice , splenic macrophages were examined by flow cytometry . as we reported previously [ 16 , 17 ] , f4/80 macrophages from aged wt mice did not exhibit age - associated differences in surface tlr4 expression ( 23.0% in young wt and 24.7% in aged wt ) , table 2 . in f4/80 macrophages from il-6 ko mice , a similar percentage of tlr4 cells was found relative to those from wt mice , regardless of age or il-6 deficiency ( 24.6% in young il-6 ko versus 23.6% in aged il-6 ko ) . these results suggest that changes in tlr4 expression in macrophages are not responsible for the increase in proinflammatory cytokine production observed in macrophages from aged il-6 ko mice . in vivo as well as in vitro studies suggest that stimulation with il-6 affects the expression of the il-6 receptor [ 32 , 33 ] . to analyze the effects of aging and il-6 on the initial steps of il-6 signaling , we measured surface levels of the il-6 receptor in total spleen cell suspensions by flow cytometry . il-6 signaling is initiated by binding of the cytokine to an 80-kda glycoprotein chain ( il-6ra / cd126 ) . f4/80 macrophages from aged wt mice did not differ in the percentage of il-6ra expressing cells ( table 2 ) or the level of the surface expression as measured by mean fluorescent intensity ( mfi ) ( data not shown ) relative to those from young wt mice . in f4/80 macrophages from il-6 ko mice , similar levels of il-6ra were found relative to those from wt mice , regardless of age or il-6 deficiency . following binding of il-6 to il-6ra , dimerization of signal - transducing glycoprotein 130 ( gp130/cd130 ) leads to activation of the janus kinase ( jak)/signal transducer and activator of transcription ( stat ) 3 signal transduction pathway . as shown in table 2 , gp130 levels on f4/80 macrophages from young or aged wt mice were similar . no effects of aging or il-6 were found in surface expression of gp130 in cells from il-6 ko mice . in this report , we analyzed the effects of aging and il-6 on cytokine production by macrophages in vitro . our results add to published data [ 1519 ] showing decreased synthesis and release of the proinflammatory cytokines , tnf , il-1 , il-6 , and il-12 , in macrophages from aged wt mice compared to those from young wt animals after lps stimulation . in addition , we have found for the first time that knocking out il-6 restores proinflammatory cytokine production by macrophages from aged mice to the levels of macrophages from young wt mice . when compared to aged wt mice , splenocytes from aged il-6 ko mice had similar age - related decreases in the relative abundance of cd3 t cells , cd3/cd4 t cells , and ly49c nk cells , however , a more pronounced decrease was found in cd3/cd8 t cells . no effects of aging or il-6 deficiency were found on the percentage of f4/80 macrophages and cd19 b cell populations , neither in the surface expression of tlr4 , nor the components of the il-6 receptor , il-6ra , and gp130 . overall , our results indicate that il-6 plays a role in regulating the age - related defects in macrophages through alteration of proinflammatory cytokines . macrophages show the impact of advanced aged in many of their biological properties including cytokine production ( recently reviewed in [ 1 , 2 ] ) . in general , when cultured in vitro with lps , macrophages from aged mice produce lower levels of proinflammatory cytokines than comparably stimulated cells from young mice [ 1519 ] . as previously reported , compared to macrophages from young wt mice , macrophages from aged wt mice had decreased production of tnf and il-6 . il-1 and il-12 production after lps stimulation also was reduced in macrophages from aged wt mice . these results are coincident with findings from chelvarajan and collaborators and are independent of the source used to isolate the macrophages ( plastic adherence utilized in current study versus positive selection from unfractionated splenocytes using magnetic sorting with cd11b microbeads used by chelvarajan and collaborators ) . overall , our findings agree with published literature in demonstrating decreased cytokine production in macrophages from aged wt mice . interleukin-6 deficiency in aged mice restored the cytokine production profile of macrophages to that of young wt mice . age - related alterations in the function of macrophages result from the combination of intrinsic and extrinsic defects and possibly the consequence of the complex interactions with other cell types or the aged milieu . our results , added to the previous findings [ 24 , 25 , 3537 ] , suggest that altered production of cytokines in macrophages from aged mice results in part from changes in the expression of extrinsic factors . cells from young il-6 ko mice had significantly lower levels of il-1 , tnf , and il-12 when incubated with lps relative to their wt counterparts . we still do n't have an explanation for these results , however , it can be speculated that a modification in the steady - state of components of cellular pathways shared between the il-6 signaling cascade and proinflammatory cytokine expression ( i.e. , erk and ap-1 ) may be a suitable common link . interleukin-6 supports the growth of splenic t and b lymphocytes [ 30 , 31 ] . it was therefore our interest to study if the effects of aging and the lack of il-6 in cytokine production by macrophages could be attributed to changes in the numbers of these cells in the spleen . while some of the splenocyte populations we tested showed age - related decreases in cytokine release , regardless of the presence of il-6 , we failed to find changes for f4/80 macrophages between young wt mice and young il-6 ko mice . unexpectedly , a more pronounced decrease was found in cd3/cd8 t cells ( 25% in aged wt versus 47% in aged il-6 ko , p < .05 ) . our results agree with those from kopf and collaborators indicating no effects of il-6 deficiency on the b cell compartment in the bone marrow and spleen of young il-6 ko mice , with normal expression of b220 , igm , igd , and cd23 . in this same study the expression of the t cell receptor , , , chains , cd4 , cd8 , cd44 ( pgpl ) , and cd24 ( hsa ) in splenocytes from young il-6 ko mice was unchanged . kopf et al . also reported a 30% to 50% reduction in the total number of splenocytes in young il-6 ko mice compared to young controls . similarly , we observed a 40% reduction in the number of splenocytes in young il-6 ko mice as well as in their concanavalin - a induced proliferative response relative to young wt mice ( data not shown ) . overall , these results stress the involvement of il-6 in the expansion and function of specific lymphoid cell subsets . further investigation will help to clarify the effects of il-6 on some of the age - related t cell defects , such as lymphocyte activation . the impact of aging on cell signaling in macrophages has been analyzed by several groups ( reviewed in ) . renshaw and collaborators linked defective production of cytokines in macrophages from aged mice following lps stimulation to a decline in all subclasses of mrna for tlrs as well as decreased surface expression of tlr4 on cells from the aged mice when compared with those from young mice . however , two independent studies from separate groups failed to show age - dependent differences in cell - surface expression of tlr4 [ 16 , 18 ] . in our study , we showed similar surface expression of tlr4 in macrophages from aged wt mice [ 16 , 18 ] . tlr4 expression was not affected by either aging or lack of il-6 in macrophages from senescent il-6 ko mice . previously , we reported that aged il-6 ko mice had similar serum levels of lipopolysaccharide - binding protein ( lpb ) relative to aged wt mice . neither advanced age nor il-6 deficiency modified the ability to induce production of this protein in vivo after an inflammatory challenge . therefore , age or il-6 deficiency does not appear to affect surface expression of the initial molecules involved in lps signal transduction . this conclusion also seems to be valid for the components of the il-6 receptor whose levels remained unchanged in macrophages regardless of age or lack of il-6 . in humans , there was a significant increase of soluble il-6 receptor until around age 70 and a gradual decline in its levels after age 70 . in mice , administration of il-6 triggered an upregulation of the components of the il-6 receptor in several tissues in mice embryos early during their development . however , aged mrl / lpr mice , genetically predisposed to the development of autoimmune diseases and reported to have elevated levels of il-6 and sil-6r levels , had a marked downregulation of gp130 in splenic t cells . our findings differ from the published literature and suggest that up or downregulation in expression of the il-6 receptor seen in other models is unlikely to operate in macrophages when il-6 is chronically absent and maybe alternative compensatory mechanisms are operating in cells from aged il-6 ko mice . most likely in our aged il-6 ko mice , the mechanisms responsible for age - dependent effects of il-6 in lps - mediated cell activation observed in macrophages may originate at the intracellular level , such as that which has been observed in macrophages from aged wt mice . examples of this may include defects in intracellular activation cascades [ 1517 , 41 ] . there is only a handful of publications on the effects of il-6 and aging on specific cell types involved in inflammatory responses . most of the work has been performed in mouse models of systemic inflammatory responses following burn injury , during sepsis , or after lps administration . in one of these studies , we analyzed the effects of aging and il-6 on the hepatic inflammatory response in two models of systemic injury : dorsal scald ( burn ) injury versus intraperitoneal lps administration . evidence obtained from histological observation showed comparable numbers of polymorphonuclear cells ( pmns ) in the livers of burn - injured mice regardless of age or il-6 deficiency . however , increased hepatic neutrophils were seen in aged wild type ( wt ) mice given lps relative to young wt mice given lps . accumulation of hepatic pmns was drastically reduced in aged il-6 ko mice given lps as compared with lps - exposed aged wt mice . starr and collaborators examined the expression of il-6 in various tissues in aged wt mice during lps - induced systemic inflammation . among the different tissues tested , white adipose tissue from epididymal fat pad expressed the highest level of il-6 mrna in both young and aged mice with a 5.5-fold higher level in the aged . immunohistochemistry revealed that lps - induced il-6 expression was associated to both the adipocytes and stromal cells . aged il-6 ko mice exhibited reduced mortality to lps suggesting a deleterious effect of il-6 overexpression in the aged . these results suggest that increased vulnerability to systemic inflammation with age is due in part to augmented il-6 production by the adipose tissue . overall , a role for other cells , besides macrophages , in work documenting advanced age and il-6 in vivo is suggested by the two publications showed above [ 11 , 12 ] . this report is the first to analyze the effects of aging and il-6 on cytokine production in macrophages in vitro . additional research will clarify the cellular mechanisms involved in our findings as well as the effects of aging and il-6 on other cell types .

here , we studied in vitro cytokine production by splenic macrophages obtained from young and aged balb / c wild type ( wt ) and il-6 knockout ( il-6 ko ) mice . relative to macrophages obtained from young wt mice given lipopolysaccharide ( lps ) , those from aged wt mice had decreased production of proinflammatory cytokines . in contrast , when compared to macrophages from young il-6 ko mice , lps stimulation yielded higher levels of these cytokines by cells from aged il-6 ko mice . aging or il-6 deficiency did not affected the percentage of f4/80 + macrophages , or the surface expression of toll - like receptor 4 ( tlr4 ) and components of the il-6 receptor . overall , our results indicate that il-6 plays a role in regulating the age - related defects in macrophages through alteration of proinflammatory cytokines , adding to the complexity of il-6-mediated impairment of immune cell function with increasing age .

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

as a part of the age - associated deterioration of the immune system [ 13 ] , there is a chronic proinflammatory state even in the absence of clinically - apparent disease . inflamm - aging is characterized by elevated circulating levels of proinflammatory factors ( il-1 , il-6 , tnf , and prostaglandin e2 ) and anti - inflammatory mediators , ( il-1 receptor antagonist , soluble tnf receptor , il-10 , transforming growth factor beta , acute phase proteins , c - reactive protein , and serum amyloid a ) and contributes to the decreased ability of the elderly to mount an appropriate immune response following an infectious challenge [ 47 ] . one of the most prominent aspects of inflamm - aging is in presence of elevated circulating levels of the proinflammatory cytokine , interleukin ( il)-6 [ 8 , 9 ] . in an attempt to better define the role of il-6 in the systemic inflammatory response in the aged , we gave young and aged wild type ( wt ) and il-6 knockout ( ko ) balb / c female mice lipopolysaccharide ( lps ) . following the inflammatory challenge , aged il-6 ko mice had improved survival when compared to aged wt mice . moreover , we previously reported that aged il-6 ko mice showed a decreased acute phase response when compared to that of aged wt mice . in addition , hepatic injury was drastically reduced in aged il-6 ko mice given lps as compared with lps - exposed aged wt mice . many of the macrophage functions are carried out by secreted cytokines , which in turn , regulate multiple immune functions , especially inflammatory responses . the effects of aging on cytokine production by macrophages have shown conflicting results with in vitro stimulation of purified macrophage populations with lps and in vivo systemic administration of lps . for example , our laboratory and others reported that macrophages from aged mice produce less tnf and il-6 after in vitro exposure to lps than comparably stimulated cells from young mice [ 1519 ] . since macrophages are considered to be an important source of proinflammatory cytokines in vivo , many possible explanations for this inconsistency can be proposed . among the multiple possibilities , the effect of the local environment in vitro versus the effect of the aged microenvironment in vivo has been considered [ 2224 ] . we reported that serum from aged fisher 344 rats increased the levels of il-6 by macrophages obtained from young animals and cultured in vitro without stimulants . to analyze the role that il-6 might play in regulating the age - related defects in macrophages through alteration of proinflammatory cytokines , we exposed splenic macrophages from young and aged wt and il-6 ko mice to lps . our findings suggest that il-6 regulates the age - related defects in macrophages through alteration of proinflammatory cytokines and that these alterations are not due to changes in surface expression of tlr4 or the il-6 receptor . young ( 2 months old ) and aged ( 18 months old ) wt female balb / c mice were purchased from the national institute of aging colony at harlan laboratories ( indianapolis , in ) . il-6 ko mice were backcrossed onto the balb / c background , bred , maintained , and aged at the taconic laboratories ( germantown , ny ) . young ( 2 months old ) and aged ( 18 months old ) female balb / c il-6 ko mice were used in the experiments described here . in order to avoid confounding factors related to circadian rhythms , all animal protects briefly , spleens were aseptically removed and disrupted to yield a cell suspension in rpmi 1640 medium , supplemented with 5% fbs , penicillin ( 100 u / ml ) , streptomycin ( 100 g / ml ) , and 2 mm glutamine ( culture medium ) ( gibco - brl , grand island , ny ) . , carlsbad , ca ) , white blood cells were counted in a hemocytometer and viability was determined by trypan blue exclusion . in the absence of stimulation , the concentrations of tnf , il-1 , il-6 , and il-12 in macrophage supernatants were measured by commercially available opteia elisa kits ( bd pharmingen , san diego , ca ) according to the manufacturer instructions . when stimulated in vitro with lps , macrophages from aged mice produce less cytokines than comparably stimulated cells from young mice [ 1519 ] . to determine if this phenotype may be determined in part by il-6 , which is elevated in the circulation of healthy aged individuals [ 9 , 10 , 28 , 29 ] , we evaluated proinflammatory cytokine production in splenic macrophages obtained from young aged wt and il-6 ko mice . macrophages from aged and young mice produced proinflammatory cytokines upon lps stimulation ( figures 1(a)1(d ) ) . as previously reported [ 1519 ] , macrophages from aged wt mice had decreased production of tnf and il-6 ( 55% ) , as well as il-1 ( 80% ) and il-12 ( 35% ) relative to splenic macrophages obtained from young wt mice given lps . similar to macrophages from aged wt mice , the production of cytokines by macrophages from young il-6 ko mice was reduced for tnf ( 67% ) , il-1 ( 31% ) , and il-12 ( 71% ) relative to that of young wt mice ( figure 1 ) . in contrast , as compared to macrophages from young il-6 ko mice , lps exposure induced higher cytokine production by cells from aged il-6 ko mice ( 1.3 , 2.2 and 1.2-fold for tnf , il-1 and il-12 , resp . ) cytokine production by macrophages from aged il-6 ko mice was elevated when compared to cells from aged wt mice ( 1.8 , 3.7 and 1.9-fold for tnf , il-1 and il-12 , resp . ) overall , these results show that il-6 plays a role in regulating the age - related defects in macrophages through alteration of the production of proinflammatory cytokines . therefore , lack of expression of il-6 could modify the phenotype of splenocytes and their production of cytokines when cultured in vitro . the percentage of t cells was determined by analyzing splenocytes bearing the cell surface markers cd3 ( t cells ) , cd4 ( cd3/cd4 ; helper / inducer cells ) , and cd8 ( cd3/cd8 ; suppressor / cytotoxic cells ) . relative to young wt mice , the percentage of cd3 t cells in splenocytes from aged wt mice was reduced ( 13% , p < .05 ) , table 1 . interestingly , this difference was more pronounced in splenocytes from aged il-6 ko mice relative to cells from young il-6 ko mice ( 24% reduction in cd3 t cells , p < .001 ) . an analysis of splenic cd3/cd4 helper / inducer t cells revealed no effects of aging or il-6 on their percentage . however , cd3/cd8 suppressor / cytotoxic t cells were reduced in splenocytes from aged wt mice ( 25% , p < .05 ) , relative to young wt mice . in addition , a marked ( 47% ) decrease in cd3/cd8 suppressor / cytotoxic t cells was found in aged il-6 ko animals , relative to splenocytes from young il-6 ko mice . this percentage was slightly increased by aging and il-6 deficiency , however , these differences failed to reach statistical significance . similarly , analysis of the f4/80 macrophage population showed no effects of aging or il-6 deficiency on their percentage . tlr4 involvement in age - related defects in macrophage activation following lps activation has been documented extensively ( reviewed in [ 1 , 2 ] ) . however , tlr4 levels in the context of aging and il-6 deficiency are unknown . to analyze the expression of tlr4 in macrophages from aged il-6 ko mice , splenic macrophages were examined by flow cytometry . as we reported previously [ 16 , 17 ] , f4/80 macrophages from aged wt mice did not exhibit age - associated differences in surface tlr4 expression ( 23.0% in young wt and 24.7% in aged wt ) , table 2 . in f4/80 macrophages from il-6 ko mice , a similar percentage of tlr4 cells was found relative to those from wt mice , regardless of age or il-6 deficiency ( 24.6% in young il-6 ko versus 23.6% in aged il-6 ko ) . these results suggest that changes in tlr4 expression in macrophages are not responsible for the increase in proinflammatory cytokine production observed in macrophages from aged il-6 ko mice . in vivo as well as in vitro studies suggest that stimulation with il-6 affects the expression of the il-6 receptor [ 32 , 33 ] . to analyze the effects of aging and il-6 on the initial steps of il-6 signaling , we measured surface levels of the il-6 receptor in total spleen cell suspensions by flow cytometry . f4/80 macrophages from aged wt mice did not differ in the percentage of il-6ra expressing cells ( table 2 ) or the level of the surface expression as measured by mean fluorescent intensity ( mfi ) ( data not shown ) relative to those from young wt mice . in f4/80 macrophages from il-6 ko mice , similar levels of il-6ra were found relative to those from wt mice , regardless of age or il-6 deficiency . following binding of il-6 to il-6ra , dimerization of signal - transducing glycoprotein 130 ( gp130/cd130 ) leads to activation of the janus kinase ( jak)/signal transducer and activator of transcription ( stat ) 3 signal transduction pathway . as shown in table 2 , gp130 levels on f4/80 macrophages from young or aged wt mice were similar . no effects of aging or il-6 were found in surface expression of gp130 in cells from il-6 ko mice . in this report , we analyzed the effects of aging and il-6 on cytokine production by macrophages in vitro . our results add to published data [ 1519 ] showing decreased synthesis and release of the proinflammatory cytokines , tnf , il-1 , il-6 , and il-12 , in macrophages from aged wt mice compared to those from young wt animals after lps stimulation . in addition , we have found for the first time that knocking out il-6 restores proinflammatory cytokine production by macrophages from aged mice to the levels of macrophages from young wt mice . when compared to aged wt mice , splenocytes from aged il-6 ko mice had similar age - related decreases in the relative abundance of cd3 t cells , cd3/cd4 t cells , and ly49c nk cells , however , a more pronounced decrease was found in cd3/cd8 t cells . no effects of aging or il-6 deficiency were found on the percentage of f4/80 macrophages and cd19 b cell populations , neither in the surface expression of tlr4 , nor the components of the il-6 receptor , il-6ra , and gp130 . overall , our results indicate that il-6 plays a role in regulating the age - related defects in macrophages through alteration of proinflammatory cytokines . in general , when cultured in vitro with lps , macrophages from aged mice produce lower levels of proinflammatory cytokines than comparably stimulated cells from young mice [ 1519 ] . as previously reported , compared to macrophages from young wt mice , macrophages from aged wt mice had decreased production of tnf and il-6 . il-1 and il-12 production after lps stimulation also was reduced in macrophages from aged wt mice . overall , our findings agree with published literature in demonstrating decreased cytokine production in macrophages from aged wt mice . interleukin-6 deficiency in aged mice restored the cytokine production profile of macrophages to that of young wt mice . age - related alterations in the function of macrophages result from the combination of intrinsic and extrinsic defects and possibly the consequence of the complex interactions with other cell types or the aged milieu . our results , added to the previous findings [ 24 , 25 , 3537 ] , suggest that altered production of cytokines in macrophages from aged mice results in part from changes in the expression of extrinsic factors . cells from young il-6 ko mice had significantly lower levels of il-1 , tnf , and il-12 when incubated with lps relative to their wt counterparts . we still do n't have an explanation for these results , however , it can be speculated that a modification in the steady - state of components of cellular pathways shared between the il-6 signaling cascade and proinflammatory cytokine expression ( i.e. it was therefore our interest to study if the effects of aging and the lack of il-6 in cytokine production by macrophages could be attributed to changes in the numbers of these cells in the spleen . while some of the splenocyte populations we tested showed age - related decreases in cytokine release , regardless of the presence of il-6 , we failed to find changes for f4/80 macrophages between young wt mice and young il-6 ko mice . unexpectedly , a more pronounced decrease was found in cd3/cd8 t cells ( 25% in aged wt versus 47% in aged il-6 ko , p < .05 ) . our results agree with those from kopf and collaborators indicating no effects of il-6 deficiency on the b cell compartment in the bone marrow and spleen of young il-6 ko mice , with normal expression of b220 , igm , igd , and cd23 . in this same study the expression of the t cell receptor , , , chains , cd4 , cd8 , cd44 ( pgpl ) , and cd24 ( hsa ) in splenocytes from young il-6 ko mice was unchanged . also reported a 30% to 50% reduction in the total number of splenocytes in young il-6 ko mice compared to young controls . similarly , we observed a 40% reduction in the number of splenocytes in young il-6 ko mice as well as in their concanavalin - a induced proliferative response relative to young wt mice ( data not shown ) . overall , these results stress the involvement of il-6 in the expansion and function of specific lymphoid cell subsets . further investigation will help to clarify the effects of il-6 on some of the age - related t cell defects , such as lymphocyte activation . renshaw and collaborators linked defective production of cytokines in macrophages from aged mice following lps stimulation to a decline in all subclasses of mrna for tlrs as well as decreased surface expression of tlr4 on cells from the aged mice when compared with those from young mice . however , two independent studies from separate groups failed to show age - dependent differences in cell - surface expression of tlr4 [ 16 , 18 ] . in our study , we showed similar surface expression of tlr4 in macrophages from aged wt mice [ 16 , 18 ] . tlr4 expression was not affected by either aging or lack of il-6 in macrophages from senescent il-6 ko mice . previously , we reported that aged il-6 ko mice had similar serum levels of lipopolysaccharide - binding protein ( lpb ) relative to aged wt mice . neither advanced age nor il-6 deficiency modified the ability to induce production of this protein in vivo after an inflammatory challenge . therefore , age or il-6 deficiency does not appear to affect surface expression of the initial molecules involved in lps signal transduction . this conclusion also seems to be valid for the components of the il-6 receptor whose levels remained unchanged in macrophages regardless of age or lack of il-6 . in mice , administration of il-6 triggered an upregulation of the components of the il-6 receptor in several tissues in mice embryos early during their development . however , aged mrl / lpr mice , genetically predisposed to the development of autoimmune diseases and reported to have elevated levels of il-6 and sil-6r levels , had a marked downregulation of gp130 in splenic t cells . our findings differ from the published literature and suggest that up or downregulation in expression of the il-6 receptor seen in other models is unlikely to operate in macrophages when il-6 is chronically absent and maybe alternative compensatory mechanisms are operating in cells from aged il-6 ko mice . most likely in our aged il-6 ko mice , the mechanisms responsible for age - dependent effects of il-6 in lps - mediated cell activation observed in macrophages may originate at the intracellular level , such as that which has been observed in macrophages from aged wt mice . most of the work has been performed in mouse models of systemic inflammatory responses following burn injury , during sepsis , or after lps administration . in one of these studies , we analyzed the effects of aging and il-6 on the hepatic inflammatory response in two models of systemic injury : dorsal scald ( burn ) injury versus intraperitoneal lps administration . evidence obtained from histological observation showed comparable numbers of polymorphonuclear cells ( pmns ) in the livers of burn - injured mice regardless of age or il-6 deficiency . however , increased hepatic neutrophils were seen in aged wild type ( wt ) mice given lps relative to young wt mice given lps . accumulation of hepatic pmns was drastically reduced in aged il-6 ko mice given lps as compared with lps - exposed aged wt mice . starr and collaborators examined the expression of il-6 in various tissues in aged wt mice during lps - induced systemic inflammation . among the different tissues tested , white adipose tissue from epididymal fat pad expressed the highest level of il-6 mrna in both young and aged mice with a 5.5-fold higher level in the aged . aged il-6 ko mice exhibited reduced mortality to lps suggesting a deleterious effect of il-6 overexpression in the aged . overall , a role for other cells , besides macrophages , in work documenting advanced age and il-6 in vivo is suggested by the two publications showed above [ 11 , 12 ] . this report is the first to analyze the effects of aging and il-6 on cytokine production in macrophages in vitro .

mercury is a toxic metal that is widespread in the environment and harmful to human and mammalian . the different chemical forms ( organic and inorganic ) of mercurial compounds exhibit different degrees of toxicity with effects including hearing loss , vision disturbance , motor deficits , and retarded or abnormal walking ability [ 1 , 2 ] . cinnabar ( an inorganic mercurial compound ) contains more than 95% mercury sulfide ( hgs ) and has been used for many thousands of years in traditional chinese medicine ( tcm ) and in asian and middle eastern countries as a sedative and hypnotic [ 36 ] . although cinnabar is supposed to have many therapeutic properties and is generally disregarded to result in significant mercury absorption from the gastrointestinal ( g - i ) tract following oral administration , its high mercury content highlights the need for studies on its possible toxic effects . various reports have reported that cinnabar can be absorbed following oral administration at high doses and accumulated in the brain and other tissues , causing mercury intoxication [ 5 , 7 , 8 ] . many studies have reported that the total amount of mercury accumulated in tissues from methylmercuric chloride ( mehgcl ) was about 5000-fold higher than that from hgs , but that exposure to a high - dose of cinnabar or hgs ( 1.0 g / kg / day , for 7 or 14 consecutive days ) was able to cause neurotoxicity , including dysfunction of the vestibule - ocular reflex ( vor ) system , an abnormal of auditory brainstem response , learning memory deficits , impairment of spontaneous locomotor activity , and suppression of compound muscle action potentials ( cmaps ) , as has been reported for mehg [ 1012 ] . recently , huang et al . also reported that long - term exposure to low dose of cinnabar ( 10 mg / kg / day , for more than 77 consecutive days ) induced neurotoxicological effects , which were associated with significant hg accumulation in the brain . moreover , cinnabar - containing tcms , such as ba paul san , which is used as a sedative and for the management of external infections in infants and children , have been reported to contain excess amounts of cinnabar that cause occasional intoxication in the chinese population [ 3 , 9 , 14 ] . therefore , expectant infants and children exposed to cinnabar may cause toxic effects because of overdosage and long - term administration . nonetheless , available data on the toxicological effects and action mechanisms of cinnabar in children at the perinatal stage exposed to low doses are still insufficient . auditory brainstem response ( abr ) test , a method to provide a clear and reliable indicator of hearing function in the central auditory system , is a useful indicator of neurotoxicity in body burdens of toxic metals [ 15 , 16 ] . it has been demonstrated the significant correlation between the abnormal abr test ( the hearing loss and the latency of wave iii and v delayed ) and hg accumulation during prenatal , postnatal , development , adult stage exposed to mercury or existed in patients with mercury - contaminated area [ 2 , 1720 ] . in experimental animals , the irreversible loss of hearing after exposure to chemicals ( such as mehg or cisplatin ) has been reported to be associated with toxicity to the central auditory system , including auditory loss or damage to the cochlea [ 16 , 21 , 22 ] . however , possible ototoxic effects of low dose of cinnabar exposure remain unclear , especially during the perinatal or developmental periods . on the other hand , it has been shown that chronic mercury intoxication is characterized by inhibition of neuronal na / k - atpase , which is a key enzyme for neurological function [ 23 , 24 ] . na / k - atpase of the neuronal membranes , which is responsible for the active transport of sodium and potassium ions in the nervous system , plays a critical role in the maintenance of cellular ionic homeostasis and in the physiological function of the inner ear as well as the nervous system [ 25 , 26 ] . the inactivation of na / k - atpase leads to partial membrane depolarization , which allows excessive ca entry into neurons with resultant toxic events similar to excitotoxicity and has been implicated in pathological and physiological abnormalities and neurodegenerative diseases [ 2628 ] . recently , the accumulated evidence has revealed that mercurial compounds specifically bind to this enzyme and cause cell or organ dysfunction [ 10 , 21 , 22 , 29 , 30 ] . based on these findings , we attempted through this study to clarify the toxicological effects of low dose of cinnabar in offspring that were exposed after weaning , only during the perinatal and weaning stages , or across all these stages . subsequently , we verified the working hypothesis that long - term exposure to low dose of cinnabar induced neurobehavioral abnormalities and central auditory system dysfunction through interference with na / k - atpase activities . we , therefore , examined a low dosage of cinnabar ( 10 mg / kg / day ) and a longer duration ( 7 weeks ) of oral application in mice and then monitored various neurobehavioral effects ( including spontaneous locomotor activities , pentobarbital - induced sleeping time , and motor equilibrium performance ) and hearing function ( by abr test , a useful parameter for indicating mercuric compounds - induced oto - toxicity ) , followed by analyzing changes in the hg content and na / k - atpase activities of the brain tissues and nox levels of whole blood at the end of the treatment . randomly bred , male and female icr mice were obtained from the animal center of the college of medical , national taiwan university ( taipei , taiwan ) . the protocol was approved by the institutional animal care and use committee ( iacuc ) , and the care and use of laboratory animals were conducted in accordance with the guidelines of the animal research committee of college of medicine , national taiwan university . mice were housed seven per cage under standard laboratory conditions at a constant temperature ( 23 2c ) , 50 20% relative humidity , given a solid diet and tap water ad libitum and 12 hrs for light - dark cycles . mice were acclimatized to the laboratory conditions prior to the experiments , and all experiments were carried out between 8:00 am and 05:00 pm . the adult male and female icr mice ( 4~5 weeks old , 2225 g ) ( breeders of f0 and f1 generations ) were randomly assigned to four initial dosing groups and then were orally gavaged distilled water or 10 mg / kg / day cinnabar for 4 consecutive weeks before mating , and then two females were placed per cage with one male breeder for mating . gestational day 0 ( gd 0 ) was confirmed by the presence of a vaginal plug in the morning . at that time , the female mice with vaginal plugs ( dams ) were placed into individual cage form gd0 to postnatal day ( pnd ) 21 ( lactation period ) and then maintained exposure to cinnabar . thus , the exclusive route of offspring exposure to mercury was through maternal milk . at postnatal day ( pnd ) 0 , the offspring newborn mice ( pups ) were recorded the number in the litter and randomly selected from different litters ( three or four per litter ) and sacrificed after deep anesthesia by an intraperitoneal injection of pentobarbital ( 80 mg / kg ) , and whole blood samples of the mothers were collected to eppendorf from an eyehole vessel after light anesthesia by an intraperitoneal injection of pentobarbital ( 50 mg / kg ) . these samples were analyzed hg contents . at pnd 21 , offspring ( pups ) within the original dose group assignment were randomly separated into two groups ( seven per cage , total numbers = 1215/group ) and then orally gavaged with distilled water or 10 mg / kg / day cinnabar for 7 consecutive weeks , respectively . figure 1 illustrates the time course of administration of cinnabar ( 10 mg / kg / day ) to offspring and distilled water exposure during maternal gestation and weaning or following weaning . after the end of experiment , all experimental animals were sacrificed by decapitation under pentobarbital anesthesia ( 80 mg / kg , i.p . ) after administration with the vehicle control or cinnabar fed . these tissues were analysis of na / k - atpase activities ( cerebral cortex , cerebellar cortex and brainstem ) , nitric oxide levels of whole blood , and mercury content ( whole blood , cerebral cortex , cerebellar cortex , and brainstem ) . to determine the hg concentrations , various tissues ( 300 mg of whole blood , cerebral cortex , cerebellar cortex and brainstem or offspring newborn mice ) or cinnabar sample ( 100 mg ) were placed in a 15 ml polyethylene tube , and 0.4 ml of a 3 : 1 mixture of hydrochloric acid ( 35% ) and nitric acid ( 70% ) was added . the tubes were capped and allowed to stand overnight at 50 degree oven . after cooling , suitable dilution buffer ( 0.3% nitric acid and 0.1% triton x-100 in distilled water ) was added to the digested material , and the total mercury content was determined by inductively coupled plasma mass spectrometry ( icp - ms ) . the cinnabar sample used in this study was composed : 809.3 mg / g of mercury , 1.03 mg / g of sodium , 0.12 mg / g of magnesium , 0.06 mg / g of aluminum , 4.37 mg / g of potassium , 0.73 mg / g of calcium , 1.05 mg / g of barium , 1.05 mg / g of chromium , 1.62 mg / g of iron , 0.05 mg / g of zinc , 0.04 mg / g of selenium , 0.01 mg / g of lead , 0.01 mg / g of copper , and 0.02 mg / g nickel . the mice were orally administered cinnabar by gavage or distilled water once every day for 7 consecutive weeks , and the effects on the spontaneous locomotor activity were measured at end of 7 weeks treatment . in the spontaneous locomotor activity tests , the experiments were performed during the day ( 9:0018:00 ) . when the drugs were administered by consecutive oral route , the mice were individually placed in an open field . a large colorless rectangular box with a metallic grid floor was used ( 70 cm wide , 90 cm long and 60 cm high ) . the photobeam activity monitors ( tru scan coulbourn instruments ) were used as well as real - time for detecting track - type plots . typical application of x - y activity recording ( floor plane activity ) sensory ring drops over the cage and rests on ring support . movement was detected by 16 16 infrared photobeam detectors and transducers set 1.5 cm above the floor of the apparatus and measured by a pc . finally , the number of squares crossed and the plots of tracking were counted during a period of 30 min for all experiments , and quantification of data was by truscan 99 software . in order to investigate the neurotoxic effect of cinnabar - induced sleep disorder in offspring mice , prolongation of pentobarbital - induced sleeping time was performed [ 13 , 32 ] . briefly , the prolongation of sleeping time was induced by an intraperitoneal injection of pentobarbital ( 50 mg / kg ) and recorded the sleeping time from anesthesia to awakening at end of 7 weeks in the mice with or without cinnabar administration . the motor equilibrium performance on a rotating rod is a more complex motor skill task , which requires both fine motor coordination and precise postural control and is an useful marker for monitoring mercurial compounds - induced neurotoxicity [ 21 , 33 , 34 ] . the effect of cinnabar on motor coordination in the separate groups of the mice was tested using an accelerating rotating rod treadmill ( ugo basile ; stoelting co. , chicago , il ) . the rotating rod was set in motion at a constant speed ( 60 rpm ) , and the mice were placed into individual sections of rotating rod . each time an animal fell , it was noted whether the fall had occurred when it sat still or when it walked . the mice were administered with distilled water or cinnabar once every day for 7 consecutive weeks . briefly , experimental mice were deep anesthetized with an intraperitoneal injection of pentobarbital ( 50 mg / kg body weight ) , keeping the body temperature by an electric blanket and recording the brainstem evoked response in a sound attenuated room . subcutaneous needle electrodes with active electrodes placed in the vertex and ipsilateral retro - auricular region and a ground electrode on the neck of the animal recorded the click - evoked abr by an auditory evoked potential system ( nicolet , spirit , madison , wi , usa ) . mice were presented with a stimulus intensity series , which was initiated at 110 db sound pressure level ( spl ) and reached a minimum of 5 db spl . click stimuli were calibrated with a calibrated b & k precision sound level meter ( duration 100 s , stimulation rate 57.7/s , and frequency from 0 to 150 hz ) . abr threshold was defined as the lowest intensity capable of eliciting replicable and detectable waveforms . the absolute wave and interwave latencies of abr waveforms abr was evoked by clicks in this study because the click - elicited abr is a simplified and effective electrophysiological test to examine the hearing loss induced by mercurial compounds , and its hearing thresholds would correlate with the enzyme activities of the brainstem of the experimental mice . the quantitative nitric oxide ( nox ) assay was based on that described by huang et al . briefly , the whole blood samples were collected to eppendorf from an eyehole vessel of the experimental animals after light anesthesia by an intraperitoneal injection of pentobarbital ( 50 mg / kg ) . to avoid total protein denatured incompletely , we added 95% ethanol into the eppendorf at 4c overnight ( 1216 hrs ) . next day , all samples were centrifuged at 4c for 20 min at 12000 g . the supernatants of these samples were collected and assayed by the no / ozone chemiluminescence ( no analyzer 280a sievers ) for quantitative nox levels , which measured the oxidation products ( no2- and no3- ) of no using a reaction vessel containing a reducing system ( 0.1 m vanadium chloride , aldrich co. , germany ) . detection of nox is then completed by its reaction with ozone , which leads to the emission of red light ( no + o3no2 * + o2 ; no2 * no2 + hv ) . standard curves were made prior to concentration ( 1 , 5 , 10 , 15 , and 20 m no ) , which were prepared using freshly prepared solutions of nano2 in distilled water . the brain tissues ( cerebral cortex , cerebellar cortex and brainstem ) of the vehicle control and cinnabar treated mice were acquired and analyzed for na / k - atpase activity after 7 weeks subsequent cinnabar fed . membrane na / k - atpase activities were assayed as described previously ( huang et al . [ 21 , 34 ] ) . the method allowed for quantification of two distinct na / k - atpase and mg - atpase activities in the same sample . the enzymatic activities were measured in triplicate in covered 96-well microliter plates at 37 0.5c on a shaker . thirty microliters of assay buffer ( 118 mm nacl , 1.67 mm kcl , 1.2 mm mgcl2 , 12.3 mm nahco3 , 11 mm glucose , 0.5 mm egta , ph : 7.4 ) containing 2 g of membrane protein was added to each well . the na / k - atpase activity was determined by subtracting the ouabain ( 1.25 mm ) insensitive mg - atpase activity from the overall na / k / mg - atpase and the assay was started with the addition of 10 l of atp ( final concentration 5 mm ) making the final reaction volume of 100 l . the reaction was terminated after preincubation at 37 0.5c by the addition of 200 l of malachite green ( mg ) plus ammonium molybdate ( am ) ( 3 : 1 ) . the inorganic phosphate ( pi ) released from the substrate atp was colorimetrically assayed by a microplate elisa reader ( dynatech mr7000 , ashford , middesex , uk ) at 630 nm . the absorbance values obtained were converted to activity values by linear regression using a standard curve of sodium monobasic phosphate that included in the assay procedure . the specific atpase activities were expressed as pi mole ( micromoles inorganic phosphate ) released per mg protein per hr . the results in the text are given as mean standard errors ( se ) . when more than one group was compared with one control , significance was evaluated according to one - way analysis of variance ( anova ) was used for analysis , and the duncans 's post hoc test was applied to identify group differences . the p value less than 0.05 was considered to be significant . as shown in figure 2 , the mean of the number of offspring per litter whose dam exposed to low dose of cinnabar ( 10 mg / kg ) was significantly decreased ( 12.2 0.6 and 10.1 0.6 in f1 and f2 generation , resp . ) compared with f1-control group ( 14.4 0.4 ) . the body weight of the offspring in groups exposed to low dose of cinnabar ( 10 mg / kg ) ( f1 and f2 generations ) at pnd 1 was also significantly lower ( 1.44 0.02 and 1.45 0.02 g , resp . ) than in age - matched control ( 1.63 0.02 g ) moreover , the hg content of the cinnabar - exposed maternal ( dams ) whole blood was 8.1 1.2 and 6.3 0.4 ppb for f0 and f1 dams , respectively , which was significantly higher than that of the control ( 2.0 0.3 ppb ) ( table 1 ) . likewise , the hg content was markedly higher in the cinnabar - exposed pups at pnd 1 than in age - matched control ( table 1 ) . to investigate whether the mercury within cinnabar could be absorbed by the g - i tract , pass through the blood - brain - barrier ( bbb ) , and accumulate in the brain , we detected the hg content of the cerebral cortex , cerebellar cortex , and brainstem by icp - ms . as shown in table 2 , the hg contents in the cerebral cortex , cerebellar cortex , and brainstem were slightly increased in f1- and f2-cin - v groups ( * p < 0.05 as compared with f1-c - v group ( age - matched control ) ) and markedly increased in f1-c - cin group , f1- and f2-cin - cin groups , but only the brainstem of f1-c - cin group significantly accumulated hg than f1- and f2-cin - cin groups . moreover , the hg levels of liver and kidney , especially in the kidney , were also significantly accumulated in f1- and f2-cin - v groups ( * p < 0.05 as compared with f1-c - v group ) , and there were gradually and significantly increased hg levels in f1- and f2-cin - cin groups more than those in f1-c - cin group ( p < 0.05 as compared with f1-c - cin group ) . as shown in figure 3 , the growth of mice in both f1- and f2-cin - v groups ( as assessed by the gain in body weight ) was significantly decreased compared with f1-c - v group ( decreased by 12.9 1.6% in f1-cin - v ; 8.3 2.2% in f2-cin - v , resp . ) . however , further decrease in the continuing exposure to cinnabar for 7 consecutive weeks ( decreased by 12.8 2.2% in f1-cin - cin group ; 10.9 1.4% in f2-cin - cin group ) was not revealed . to investigate neurotoxicity induced by exposure to low dose of cinnabar in offspring , we examined spontaneous locomotor activities ( a useful method for detecting the central function of neurotransmission or motor dysfunction on mercury - induced neurotoxicity in cinnabar - treated offspring . as shown in figures 4(a ) and 4(b ) , f1-c - cin group was revealed to be hypoactive in terms of its quantitative ambulatory distances and stereotype-1 episodes following exposure to cinnabar ( * p < 0.05 as compared with f1-c - v group ) . however , those parameters were significantly increased ( hyperactive ) in f1- , f2-cin - v and f1- , f2-cin - cin groups ( * p < 0.05 as compared with f1-c - v group ; p < 0.05 as compared with f1-c - cin group ) , and only f1-cin - cin group showed more severely effects than f1-cin - v group ( p < 0.05 ) . in the parameter of jump ( figure 4(c ) ) , a marked decrease was observed in f1-c - cin group ( * p < 0.05 as compared with f1-c - v group ) . f1- and f2-cin - v groups were found to show slight declines in this parameter , which were higher as in f1- and f2-cin - cin groups ( p < 0.05 as compared with f1- or f2-cin - v group , resp . ) . meanwhile , the motor equilibrium performance test of f1-c - cin and f1- and f2-cin - cin groups showed remarkably decreased retention times on the rotating rod ( * p < 0.05 as compared with f1-c - v group ) , with the decrease being especially severe in f2-cin - cin group ( p < 0.05 as compared with f2-cin - v ; p < 0.05 as compared with f1-c - cin group ) ( figure 4(d ) ) . in addition , the administration of cinnabar for 7 consecutive weeks after weaning ( f1-c - cin group ) caused a definite prolongation of pentobarbital - induced sleeping time , which was 33.4 2.4 min compared with 24.4 1.0 min in f1-c - v group ( * p < 0.05 , figure 5 ) . however , no significant increase in the pentobarbital - induced sleeping time was observed after exposure to cinnabar during the perinatal and weaning stages ( f1- and f2-cin - c groups ) . a remarkable prolongation of sleeping time by pentobarbital - induced was , however , revealed in f1- and f2-cin - cin groups by continuing exposure to cinnabar for further 7 consecutive weeks ( * p < 0.05 as compared with f1-c - v group ; p < 0.05 as compared with f1- or f2-cin - v group , respectively ) , and this was even more significant than that in f1-c - cin group ( p < 0.05 ) . to further understand whether the low dose of cinnabar ( 10 mg / kg ) induced ototoxicity in offspring , the hearing thresholds were determined using the abr test . as shown in figure 6 , the means of hearing threshold was significantly elevated in f1-c - cin group ( 11.67 2.99 db spl in f1-c - cin group as compared with 5.00 1.60 db spl in f1-c - v group ; * p < 0.05 ) . both f1- and f2-cin - v groups also had hearing thresholds ( 17.25 2.52 and 30.00 3.27 db spl respectively , as compared with the f1-c - v group ; * p < 0.05 ) , and this effect was found to be more severe following a further 7 consecutive weeks of exposure ( 23.80 2.02 and 50.63 6.44 db spl in the f1-cin - cin and f2-cin - cin groups , respectively ; * p < 0.05 as compared with the f1-c - v group ; p < 0.05 as compared with f1- or f2-cin - v group , respectively ; p < 0.05 as compared with the f1-c - cin group ) . moreover , the extent of the elevation of the hearing thresholds was greater in f2-cin - cin group than in f1-cin - cin group ( p < 0.05 ; figure 6(a ) ) . the absolute wave and interwave latencies of abr were also observed to be significantly delayed by low dose of cinnabar exposed ( figures 6(b ) and 6(c ) ) . the mean values of the absolute latency of wave i and iii were unaffected as compared with the age - matched control group ( f1-c - v group ) , but those of wave v were increased in all exposed groups ( * p < 0.05 as compared with f1-c - v group ; p < 0.05 as compared with f1- or f2-cin - v group , respectively ) , especially in f2-cin - cin group ( p < 0.05 as compared with f1-c - cin group ) . in addition , the interwave latencies of i v and iii v , but not that of i iii , were significantly increased in all exposed groups . in particularly , the iii v interwave of f2-cin - cin group was more markedly increased than that in f1-cin - cin group ( p < 0.05 ; figure 6(c ) ) . to examine whether changes in nitric oxide levels and na / k - atpase activities were involved in low dose of cinnabar - induced neurotoxicity in offspring mice , we first analyzed the nitric oxide ( nox : nitrate plus nitrite ) levels of whole blood by the no / ozone chemiluminescence . as shown in figure 7 , nox levels in the whole blood were significantly increased in f1- and f2-cin - v groups ( 28.8 1.5 and 27.4 1.6 m , respectively ; * p < 0.05 ) as compared with f1-c - v group ( 19.9 0.8 m ) and even more markedly increased in f1-c - cin and f1-cin - cin groups ( 22.4 0.9 m and 37.5 0.6 m , resp . ; * p < 0.05 ) , particularly in f1-cin - cin group ( p < 0.05 as compared with f1-cin - v group ; p < 0.05 as compared with f1-c - cin group ) . conversely , there was a decrease the nox level in f2-cin - cin group , which was more intensively acted as compared with f1-cin - cin group ( 16.4 1.3 mm versus 19.9 0.8 mm ; p < 0.05 ; figure 7 ) . next , we detected na / k - atpase enzyme activities of the brain regions in offspring mice exposed to low dose of cinnabar for 7 consecutive weeks . as shown in figure 8 , na / k - atpase activities in f1-c - cin group were increased in the cerebral and cerebellar cortex ( * p < 0.05 as compared with f1-c - v group ) . in f1- and f2-cin - v groups , na / k - atpase activities were significantly enhanced in the cerebral and cerebellar cortex of f1-cin - v group , but unaffected that in f2-cin - v group . in f1- and f2-cin - cin groups , na / k - atpase activities were altered more intensively with an increase in the cerebral cortex of both groups and a decrease in the cerebellar cortex of the f1-cin - cin group and , most notably , an increase in the cerebellar cortex of the f2-cin - cin group ( p < 0.05 as compared with f1- or f2-cin - v group , resp . ; p < 0.05 as compared with f1-c - cin group ) . in the brainstem ( the main organ of central auditory system ) , na / k - atpase activities were increased in f1-c - cin group and also changed in the mice exposed during the perinatal and weaning stages ( decreased in f1-cin - v group ; increased in f2-cin - v group ) . in f1- and f2-cin - cin groups , there was a gradual but clear increase in na / k - atpase activities of the brainstem compared with f1-c - cin group . furthermore , the changes of na / k - atpase activities of the brain in f2-cin - cin group were more significant than those observed in f1-cin - cin group ( p < 0.05 ; figure 8) . cinnabar has been used as a tcm for the management of various diseases for more than 2000 years , especially as a tranquilizer for infants or adults , and is still used in asia countries [ 3 , 5 , 8 ] . the reputed insolubility of cinnabar , or its counterpart hgs , has led to the assumption or disregard that it is not significantly absorbed from the g - i tract following oral administration , and thus it is generally considered to have a low toxicity in vivo . however , many studies have found that mercury content of cinnabar , or hgs , can still be significantly absorbed from g - i tract , and transported and accumulated to various tissues after oral administration at the high dose or sufficient levels in the experimental animals to induce neurotoxicity [ 1012 , 22 ] . subsequently , it has been revealed that oral administration of cinnabar ( 0.01 g / kg / day ) for 11 consecutive weeks produced neurobehavioral abnormalities . in addition , many studies have documented that mercury could be transferred to the fetus through the placenta and to newborn offspring through maternal milk , which caused the high level mercury accumulation in brain and the severe deficit of neurobehavioral and learning disability in offspring [ 1 , 31 , 33 , 35 ] . based on these findings , we considered that expectant women , infants , or children might encounter overdoses of cinnabar through long - term therapy with tcm leading to mercury poison . therefore , it was necessary to clarify whether a low dose ( an actual clinical dose of 525 mg / kg / day ) of cinnabar could be significantly absorbed from g - i tract and exert its toxic effects by interfering with the integrity and functional performance of the central nervous system ( cns ) following exposure during the perinatal and/or developmental periods . to investigate the extent of hg absorbed from g - i tract and the subsequent neurobiological effects of low dose of cinnabar , we first investigated the effects of cinnabar ( 10 mg / kg / day ) in offspring exposed to cinnabar during differential developmental stages . our results showed that : ( 1 ) exposure to low dose of cinnabar from weaning for 7 consecutive weeks ( f1-c - cin group ) induced neurotoxic responses ( figures 4 , 5 , and 6 ) and ( 2 ) exposure to cinnabar during the perinatal and weaning stages , and then continued exposure for a further 7 consecutive weeks after weaning ( f1- and f2-cin - cin groups ) caused more significant abnormalities of spontaneous locomotor activities ( hyperactivities ) , disruption of motor equilibrium performance , prolonged pentobarbital - induced sleeping time , and dysfunction of the auditory system ( elevated hearing thresholds and delayed absolute latency of wave v and interwave latencies of i v , iii v ) , particularly in f2-cin - cin group more severe than f1-cin - cin group , which were accompanied by significant hg accumulation in the brain regains . if those regions were injuries by toxic insults , it would induce neurobehavioral abnormalities , such as hyperactivities in the ambulatory distances and stereotype-1 episodes of the spontaneous locomotor activities and the disruption of rotarod performance [ 21 , 36 ] . due to this , our results not only agree with previous findings that the exposure to low dose mercurial compounds ( mehgcl and/or mercuric chloride ( hgcl2 ) ) for 3 to 7 consecutive weeks caused hyperactivity and disruption of motor equilibrium performance and auditory function [ 21 , 34 ] , but also verify that offspring were much more severe and susceptible to mercurial compounds - induced neurotoxicological injuries during the perinatal and/or developmental periods exposed . furthermore , results of this study also found that offspring treated with low dose of cinnabar ( f1- and f2-cin - cin groups ) exhibited more abnormal prolonged wave ( v ) and interwave ( i v and iii v ) latencies in the abr , indicating an abnormality at a late phase of the abr at the higher center ( figure 6(b ) and figure 6(c ) ) , which was in accordance with the clinical effects of mercury upon the brainstem auditory pathway of children or the occupational workers in the mercury contaminated area with a discernible prolongation of interwave latency i iii and iii v and the higher levels of hg accumulation [ 18 , 19 , 21 , 22 ] . v interwave latencies relate to the central auditory pathway of the brainstem , and cinnabar ( as well as mehg ) could be absorbed and passed through the bbb , accumulate in the brainstem , and cause central neurotoxicity . moreover , our results also showed that offspring that were exposed to low dose of cinnabar only during the perinatal and weaning stages ( in f1- and f2-cin - v groups ) still revealed irreversible neurotoxicological damage ( figures 4 and 6 ) despite the finding that their brain hg levels ( about 30 ppb ) were lower than , or equal to , levels measured in brain tissue from victims of mercury - contaminated areas or experimental animals [ 3739 ] . these findings indicate that exposure to low dose of cinnabar can still be absorbed hg from the g - i tract , transported to brain regions where they can cause dysfunction of the neurobehavioral abnormalities and auditory system following a continuous long - term exposure regime of more than 7 consecutive weeks . in addition , exposure to cinnabar during perinatal and weaning stages can cause irreversible impairments . it was also observed through this work that the abr system may provide a sensitive and powerful tool for detecting subclinical central hearing impairment induced by cinnabar . . showed that short - term exposure of adult rat to mehg ( total doses of 10 and 30 mg / kg ) led to a marked sleep disorder ( an increase in both slow - wave sleep and paradoxical sleep in the dark phase , as well as long - lasting sleep - waking changes ) and that was accompanied by high levels of hg in the brain . in this study , the results showed that offspring exposed to low dose of cinnabar ( 10 mg / kg / day ) for 7 consecutive weeks beginning after weaning ( in f1-c - cin group ) had a significantly prolonged pentobarbital - induced sleeping time , and this was even more severe in all experimental stage exposed to low dose of cinnabar , particularly in f2-cin - cin group more than f1-cin - cin group ( p < 0.05 ; figure 5 ) . these changes were accompanied by notable hg accumulation in the brain ( table 2 ) . thus , these results suggest that offspring exposed to low dose of cinnabar during the prenatal and weaning stages and followed by continued exposure for further 7 consecutive weeks can suffer disturbances of the sleep - waking pattern ( a severe sleep disorder ) . the membrane bound na / k - atpase is essential for the generation or maintenance of basic cellular na and k ion homeostasis and the functioning of specialized tissues , such as the nervous system . the inhibition of this enzyme could result in membrane depolarization , leading to the suppression of neuronal and excitatory transmission [ 40 , 41 ] . it has been reported that na / k - atpase activities are very sensitive totoxic agents , which are the significant alteration ( increase or inhibition ) during mercurial compounds - induced neurological injuries in vivo or in vitro especially in brain regions and the cochlear lateral wall , which is accompanying with the significant increase in hearing loss [ 10 , 11 , 2123 , 4244 ] . furthermore , a recent study has indicated that low dose and long - term exposure to mehgcl and hgcl2 in offspring during the prenatal , neonatal , and/or postnatal periods cause neurotoxicological effects , which is accompanied with the marked increase na / k - atpase activities in the cerebral cortex , cerebellar cortex , and brainstem . nevertheless , an important role of na / k - atpase activities in the neurotoxic effects induced by long - term exposure to low dose of cinnabar in differential offspring remained unclear . moreover , nitric oxide ( nox ) is also an important signaling molecule that not only mediates several physiological functions , including the regulation of neurotransmission , but also regulates many pathological processes [ 45 , 46 ] . nox plays a crucial factor in the regulation of na / k - atpase activities in brain , and less or excess of nox production can result in neurotoxicity [ 47 , 48 ] . acute exposure to high - dose of toxic metals has been indicated to be capable of inhibiting nox levels in vivo and in vitro [ 22 , 49 , 50 ] . recently , accumulated evidence has reported that the chronic exposure to toxic insults induces significant alteration of na / k - atpase activities in brain accompanied with the whole blood and/or brain nox changes [ 12 , 51 , 52 ] and increased or decreased the whole blood nox levels , which closely correlates with the exposure to low dose of mercurial compounds - induced neurotoxicity , has been revealed [ 13 , 21 , 34 ] . here , our results demonstrated that na / k - atpase activities in the cerebral cortex , cerebellar cortex , and brainstem of offspring were significantly increased after the administration of a low dose of cinnabar for 7 consecutive weeks ( the f1-c - cin group ) , which were related to the increase of nox levels in the whole blood . furthermore , offspring that were only exposed during the perinatal and weaning stages ( f1- and f2-cin - v groups ) had markedly altered na / k - atpase activities in all of the three brain regions tested and elevated nox levels in the whole blood , which more severely changed following further exposure to cinnabar for further 7 consecutive weeks ( alterant effect : f2-cin - cin > f1-cin - cin group ; p < 0.05 ; figures 7 and 8) . these findings implicate that cinnabar can alter na / k - atpase activities of the brain and nox levels of the whole blood , which maybe responsible for inducing the dysfunctions of nervous ( abnormalities of locomotor activities and motor equilibrium performance ) and central auditory system ( elevation of hearing thresholds , and delay of absolute latency and interwave latency ) . in addition , recent studies have suggested that changes in na / k - atpase activities and/or nox levels could be the useful biochemical markers for chemical - induced neuronal injuries or subclinical disease , especially in mercuric compounds - induced neurotoxicity [ 21 , 53 , 54 ] . based on these suggestions and our findings , we suggest that na / k - atpase activities and nox levels appear to serve as an important and useful biochemical marker of lower dose of cinnabar - induced neurotoxicity . in conclusion , our results provide a toxicological basis for cinnabar - induced neurotoxic and ototoxic effects in offspring mice , which may be extrapolated to adults and children exposed to therapeutic dosage in tcm . changes in nox levels and na / k - atpase activities appear to be the underlying mechanism of the toxicological effects of cinnabar , which may supply an important and useful biomarker in offspring exposure to low dose and long - term mercuric compounds - induced neurotoxicity .

cinnabar , a naturally occurring mercuric sulfide ( hgs ) , has long been used in chinese mineral medicine for more than 2000 years . although mercury is well - known for its toxicity , whether cinnabar induces neurotoxicity , especially in infants and children , is unknown . the purpose of this study was to explore the neurotoxic effects of low - dose of cinnabar ( 10 mg / kg / day ) on developing mice . the results revealed neurobehavioral defects in f1-c - cin group , which were associated with hg accumulation , increased nox levels in whole blood , and na+/k+-atpase activities in brain tissues . f1- and f2-cin - v groups were found to increase brain hg contents and prominent neurobehavioral defects compared with f1-c - v group , suggesting that the fetal brain was more susceptible to irreversible effects for cinnabar - induced damage . moreover , f1- and f2-cin - cin groups had severely neurobehavioral dysfunctions , closely correlated with the further alteration of nox levels and na+/k+-atpase activities than f1- and f2-c - cin groups . effects in f2-cin - cin group were more significant than those in f1-cin - cin group . in conclusion , this study demonstrates that exposure to low - dose of cinnabar during the perinatal and developmental stages results in irreversible and severe injuries of the neurotoxicity in offspring , and nox and na+/k+-atpase activities may exist potential and useful biomarkers for neurotoxicity - induced by low - doses of mercuric compounds .

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

many studies have reported that the total amount of mercury accumulated in tissues from methylmercuric chloride ( mehgcl ) was about 5000-fold higher than that from hgs , but that exposure to a high - dose of cinnabar or hgs ( 1.0 g / kg / day , for 7 or 14 consecutive days ) was able to cause neurotoxicity , including dysfunction of the vestibule - ocular reflex ( vor ) system , an abnormal of auditory brainstem response , learning memory deficits , impairment of spontaneous locomotor activity , and suppression of compound muscle action potentials ( cmaps ) , as has been reported for mehg [ 1012 ] . also reported that long - term exposure to low dose of cinnabar ( 10 mg / kg / day , for more than 77 consecutive days ) induced neurotoxicological effects , which were associated with significant hg accumulation in the brain . moreover , cinnabar - containing tcms , such as ba paul san , which is used as a sedative and for the management of external infections in infants and children , have been reported to contain excess amounts of cinnabar that cause occasional intoxication in the chinese population [ 3 , 9 , 14 ] . however , possible ototoxic effects of low dose of cinnabar exposure remain unclear , especially during the perinatal or developmental periods . based on these findings , we attempted through this study to clarify the toxicological effects of low dose of cinnabar in offspring that were exposed after weaning , only during the perinatal and weaning stages , or across all these stages . we , therefore , examined a low dosage of cinnabar ( 10 mg / kg / day ) and a longer duration ( 7 weeks ) of oral application in mice and then monitored various neurobehavioral effects ( including spontaneous locomotor activities , pentobarbital - induced sleeping time , and motor equilibrium performance ) and hearing function ( by abr test , a useful parameter for indicating mercuric compounds - induced oto - toxicity ) , followed by analyzing changes in the hg content and na / k - atpase activities of the brain tissues and nox levels of whole blood at the end of the treatment . figure 1 illustrates the time course of administration of cinnabar ( 10 mg / kg / day ) to offspring and distilled water exposure during maternal gestation and weaning or following weaning . as shown in figure 2 , the mean of the number of offspring per litter whose dam exposed to low dose of cinnabar ( 10 mg / kg ) was significantly decreased ( 12.2 0.6 and 10.1 0.6 in f1 and f2 generation , resp . ) the body weight of the offspring in groups exposed to low dose of cinnabar ( 10 mg / kg ) ( f1 and f2 generations ) at pnd 1 was also significantly lower ( 1.44 0.02 and 1.45 0.02 g , resp . ) as shown in table 2 , the hg contents in the cerebral cortex , cerebellar cortex , and brainstem were slightly increased in f1- and f2-cin - v groups ( * p < 0.05 as compared with f1-c - v group ( age - matched control ) ) and markedly increased in f1-c - cin group , f1- and f2-cin - cin groups , but only the brainstem of f1-c - cin group significantly accumulated hg than f1- and f2-cin - cin groups . moreover , the hg levels of liver and kidney , especially in the kidney , were also significantly accumulated in f1- and f2-cin - v groups ( * p < 0.05 as compared with f1-c - v group ) , and there were gradually and significantly increased hg levels in f1- and f2-cin - cin groups more than those in f1-c - cin group ( p < 0.05 as compared with f1-c - cin group ) . as shown in figure 3 , the growth of mice in both f1- and f2-cin - v groups ( as assessed by the gain in body weight ) was significantly decreased compared with f1-c - v group ( decreased by 12.9 1.6% in f1-cin - v ; 8.3 2.2% in f2-cin - v , resp . ) however , further decrease in the continuing exposure to cinnabar for 7 consecutive weeks ( decreased by 12.8 2.2% in f1-cin - cin group ; 10.9 1.4% in f2-cin - cin group ) was not revealed . to investigate neurotoxicity induced by exposure to low dose of cinnabar in offspring , we examined spontaneous locomotor activities ( a useful method for detecting the central function of neurotransmission or motor dysfunction on mercury - induced neurotoxicity in cinnabar - treated offspring . as shown in figures 4(a ) and 4(b ) , f1-c - cin group was revealed to be hypoactive in terms of its quantitative ambulatory distances and stereotype-1 episodes following exposure to cinnabar ( * p < 0.05 as compared with f1-c - v group ) . however , those parameters were significantly increased ( hyperactive ) in f1- , f2-cin - v and f1- , f2-cin - cin groups ( * p < 0.05 as compared with f1-c - v group ; p < 0.05 as compared with f1-c - cin group ) , and only f1-cin - cin group showed more severely effects than f1-cin - v group ( p < 0.05 ) . in the parameter of jump ( figure 4(c ) ) , a marked decrease was observed in f1-c - cin group ( * p < 0.05 as compared with f1-c - v group ) . f1- and f2-cin - v groups were found to show slight declines in this parameter , which were higher as in f1- and f2-cin - cin groups ( p < 0.05 as compared with f1- or f2-cin - v group , resp . ) meanwhile , the motor equilibrium performance test of f1-c - cin and f1- and f2-cin - cin groups showed remarkably decreased retention times on the rotating rod ( * p < 0.05 as compared with f1-c - v group ) , with the decrease being especially severe in f2-cin - cin group ( p < 0.05 as compared with f2-cin - v ; p < 0.05 as compared with f1-c - cin group ) ( figure 4(d ) ) . in addition , the administration of cinnabar for 7 consecutive weeks after weaning ( f1-c - cin group ) caused a definite prolongation of pentobarbital - induced sleeping time , which was 33.4 2.4 min compared with 24.4 1.0 min in f1-c - v group ( * p < 0.05 , figure 5 ) . however , no significant increase in the pentobarbital - induced sleeping time was observed after exposure to cinnabar during the perinatal and weaning stages ( f1- and f2-cin - c groups ) . a remarkable prolongation of sleeping time by pentobarbital - induced was , however , revealed in f1- and f2-cin - cin groups by continuing exposure to cinnabar for further 7 consecutive weeks ( * p < 0.05 as compared with f1-c - v group ; p < 0.05 as compared with f1- or f2-cin - v group , respectively ) , and this was even more significant than that in f1-c - cin group ( p < 0.05 ) . to further understand whether the low dose of cinnabar ( 10 mg / kg ) induced ototoxicity in offspring , the hearing thresholds were determined using the abr test . both f1- and f2-cin - v groups also had hearing thresholds ( 17.25 2.52 and 30.00 3.27 db spl respectively , as compared with the f1-c - v group ; * p < 0.05 ) , and this effect was found to be more severe following a further 7 consecutive weeks of exposure ( 23.80 2.02 and 50.63 6.44 db spl in the f1-cin - cin and f2-cin - cin groups , respectively ; * p < 0.05 as compared with the f1-c - v group ; p < 0.05 as compared with f1- or f2-cin - v group , respectively ; p < 0.05 as compared with the f1-c - cin group ) . moreover , the extent of the elevation of the hearing thresholds was greater in f2-cin - cin group than in f1-cin - cin group ( p < 0.05 ; figure 6(a ) ) . the mean values of the absolute latency of wave i and iii were unaffected as compared with the age - matched control group ( f1-c - v group ) , but those of wave v were increased in all exposed groups ( * p < 0.05 as compared with f1-c - v group ; p < 0.05 as compared with f1- or f2-cin - v group , respectively ) , especially in f2-cin - cin group ( p < 0.05 as compared with f1-c - cin group ) . to examine whether changes in nitric oxide levels and na / k - atpase activities were involved in low dose of cinnabar - induced neurotoxicity in offspring mice , we first analyzed the nitric oxide ( nox : nitrate plus nitrite ) levels of whole blood by the no / ozone chemiluminescence . as shown in figure 7 , nox levels in the whole blood were significantly increased in f1- and f2-cin - v groups ( 28.8 1.5 and 27.4 1.6 m , respectively ; * p < 0.05 ) as compared with f1-c - v group ( 19.9 0.8 m ) and even more markedly increased in f1-c - cin and f1-cin - cin groups ( 22.4 0.9 m and 37.5 0.6 m , resp . ; * p < 0.05 ) , particularly in f1-cin - cin group ( p < 0.05 as compared with f1-cin - v group ; p < 0.05 as compared with f1-c - cin group ) . conversely , there was a decrease the nox level in f2-cin - cin group , which was more intensively acted as compared with f1-cin - cin group ( 16.4 1.3 mm versus 19.9 0.8 mm ; p < 0.05 ; figure 7 ) . as shown in figure 8 , na / k - atpase activities in f1-c - cin group were increased in the cerebral and cerebellar cortex ( * p < 0.05 as compared with f1-c - v group ) . in f1- and f2-cin - v groups , na / k - atpase activities were significantly enhanced in the cerebral and cerebellar cortex of f1-cin - v group , but unaffected that in f2-cin - v group . in f1- and f2-cin - cin groups , na / k - atpase activities were altered more intensively with an increase in the cerebral cortex of both groups and a decrease in the cerebellar cortex of the f1-cin - cin group and , most notably , an increase in the cerebellar cortex of the f2-cin - cin group ( p < 0.05 as compared with f1- or f2-cin - v group , resp . in the brainstem ( the main organ of central auditory system ) , na / k - atpase activities were increased in f1-c - cin group and also changed in the mice exposed during the perinatal and weaning stages ( decreased in f1-cin - v group ; increased in f2-cin - v group ) . in f1- and f2-cin - cin groups , there was a gradual but clear increase in na / k - atpase activities of the brainstem compared with f1-c - cin group . furthermore , the changes of na / k - atpase activities of the brain in f2-cin - cin group were more significant than those observed in f1-cin - cin group ( p < 0.05 ; figure 8) . cinnabar has been used as a tcm for the management of various diseases for more than 2000 years , especially as a tranquilizer for infants or adults , and is still used in asia countries [ 3 , 5 , 8 ] . therefore , it was necessary to clarify whether a low dose ( an actual clinical dose of 525 mg / kg / day ) of cinnabar could be significantly absorbed from g - i tract and exert its toxic effects by interfering with the integrity and functional performance of the central nervous system ( cns ) following exposure during the perinatal and/or developmental periods . to investigate the extent of hg absorbed from g - i tract and the subsequent neurobiological effects of low dose of cinnabar , we first investigated the effects of cinnabar ( 10 mg / kg / day ) in offspring exposed to cinnabar during differential developmental stages . our results showed that : ( 1 ) exposure to low dose of cinnabar from weaning for 7 consecutive weeks ( f1-c - cin group ) induced neurotoxic responses ( figures 4 , 5 , and 6 ) and ( 2 ) exposure to cinnabar during the perinatal and weaning stages , and then continued exposure for a further 7 consecutive weeks after weaning ( f1- and f2-cin - cin groups ) caused more significant abnormalities of spontaneous locomotor activities ( hyperactivities ) , disruption of motor equilibrium performance , prolonged pentobarbital - induced sleeping time , and dysfunction of the auditory system ( elevated hearing thresholds and delayed absolute latency of wave v and interwave latencies of i v , iii v ) , particularly in f2-cin - cin group more severe than f1-cin - cin group , which were accompanied by significant hg accumulation in the brain regains . furthermore , results of this study also found that offspring treated with low dose of cinnabar ( f1- and f2-cin - cin groups ) exhibited more abnormal prolonged wave ( v ) and interwave ( i v and iii v ) latencies in the abr , indicating an abnormality at a late phase of the abr at the higher center ( figure 6(b ) and figure 6(c ) ) , which was in accordance with the clinical effects of mercury upon the brainstem auditory pathway of children or the occupational workers in the mercury contaminated area with a discernible prolongation of interwave latency i iii and iii v and the higher levels of hg accumulation [ 18 , 19 , 21 , 22 ] . moreover , our results also showed that offspring that were exposed to low dose of cinnabar only during the perinatal and weaning stages ( in f1- and f2-cin - v groups ) still revealed irreversible neurotoxicological damage ( figures 4 and 6 ) despite the finding that their brain hg levels ( about 30 ppb ) were lower than , or equal to , levels measured in brain tissue from victims of mercury - contaminated areas or experimental animals [ 3739 ] . these findings indicate that exposure to low dose of cinnabar can still be absorbed hg from the g - i tract , transported to brain regions where they can cause dysfunction of the neurobehavioral abnormalities and auditory system following a continuous long - term exposure regime of more than 7 consecutive weeks . in this study , the results showed that offspring exposed to low dose of cinnabar ( 10 mg / kg / day ) for 7 consecutive weeks beginning after weaning ( in f1-c - cin group ) had a significantly prolonged pentobarbital - induced sleeping time , and this was even more severe in all experimental stage exposed to low dose of cinnabar , particularly in f2-cin - cin group more than f1-cin - cin group ( p < 0.05 ; figure 5 ) . furthermore , a recent study has indicated that low dose and long - term exposure to mehgcl and hgcl2 in offspring during the prenatal , neonatal , and/or postnatal periods cause neurotoxicological effects , which is accompanied with the marked increase na / k - atpase activities in the cerebral cortex , cerebellar cortex , and brainstem . nevertheless , an important role of na / k - atpase activities in the neurotoxic effects induced by long - term exposure to low dose of cinnabar in differential offspring remained unclear . recently , accumulated evidence has reported that the chronic exposure to toxic insults induces significant alteration of na / k - atpase activities in brain accompanied with the whole blood and/or brain nox changes [ 12 , 51 , 52 ] and increased or decreased the whole blood nox levels , which closely correlates with the exposure to low dose of mercurial compounds - induced neurotoxicity , has been revealed [ 13 , 21 , 34 ] . here , our results demonstrated that na / k - atpase activities in the cerebral cortex , cerebellar cortex , and brainstem of offspring were significantly increased after the administration of a low dose of cinnabar for 7 consecutive weeks ( the f1-c - cin group ) , which were related to the increase of nox levels in the whole blood . furthermore , offspring that were only exposed during the perinatal and weaning stages ( f1- and f2-cin - v groups ) had markedly altered na / k - atpase activities in all of the three brain regions tested and elevated nox levels in the whole blood , which more severely changed following further exposure to cinnabar for further 7 consecutive weeks ( alterant effect : f2-cin - cin > f1-cin - cin group ; p < 0.05 ; figures 7 and 8) . based on these suggestions and our findings , we suggest that na / k - atpase activities and nox levels appear to serve as an important and useful biochemical marker of lower dose of cinnabar - induced neurotoxicity . in conclusion , our results provide a toxicological basis for cinnabar - induced neurotoxic and ototoxic effects in offspring mice , which may be extrapolated to adults and children exposed to therapeutic dosage in tcm . changes in nox levels and na / k - atpase activities appear to be the underlying mechanism of the toxicological effects of cinnabar , which may supply an important and useful biomarker in offspring exposure to low dose and long - term mercuric compounds - induced neurotoxicity .

development of apraxia and other cognitive deficits requires help of others with activities of daily living . unfortunately , the person with dementia sometimes does not cooperate with family or professional caregivers but actually resists care or exhibits behaviors during care that may be labeled abusive . these behaviors may be difficult to handle by family caregivers and may lead to institutionalization . in an institution , rejection of care and behaviors directed towards others may lead to injuries of the resident or staff and to staff burnout . a survey of nursing home physicians showed that the most common problem that they treated in residents with dementia was rejection of care . some of them investigated the effectiveness of pain control , others used personalized psychosocial interventions based on the unmet needs theory for treatment of agitation and found positive results . however , the problem with these studies is that they consider all behavioral symptoms to be agitation and do not differentiate between agitation and rejection of care . this is due to using the cohen - mansfield agitation inventory or neuropsychiatric inventory in which the item agitation / aggression is actually measuring rejection of care . similar problems are also present in drug trials investigating effects of antipsychotics and other medications . therefore , we decided to study specifically rejection of care that is a highly relevant problem in clinical practice . we have previously described strong correlations between rejection of care and lack of understanding , depression and psychotic symptoms . behaviors directed towards others were strongly correlated with rejection of care , depression and delusions . however , these findings were based only on cross - sectional data . in this study , we performed longitudinal analyses to further explore the relationships of these modifiable factors with rejection of care and behaviors directed towards others of nursing home residents . our hypothesis was that longitudinal data would support causal relationships between these modifiable factors and rejection of care . we used minimum data set - resident assessment instrument ( mds - rai ) data collected by 8 dutch nursing homes and 10 residential homes . we included the data of residents within a 12-month time window for each facility separately , resulting in a range from april 4 , 2007 , to december 1 , 2008 . of the records of 2,705 residents available from the 18 facilities , we selected the last records of 1,101 residents aged over 65 with alzheimer or other dementia ( i1q or u ) , who were dependent in decision making ( b4 not equal 0 ) and who were not comatose ( b1 equal 0 ) . four assessments from the mds within a period of 15 months were used for the analyses . the terminology was changed by the introduction of mds 3.0 , which uses rejection of care instead of resist care. other new , but equivalent , items in mds 3.0 are physical behavioral symptoms directed towards others instead of physical abuse and verbal behavioral symptoms directed towards others instead of verbal abuse . presence of rejection of care and behavioral symptoms directed towards others was obtained from mds 2.0 section e with choices from not exhibited to daily. the variables physical behavioral symptoms directed towards others ( e4ca ) and verbal behavioral symptoms directed towards others ( e4ba ) were combined into one variable called behaviors directed towards others. ability to understand ( c6 ) was rated as sometimes and rarely / never. psychotic symptoms [ delusions ( j1e ) and hallucinations ( j1i ) ] were combined into one variable , psychosis , whose values are number of symptoms . clinical diagnosis of depression was obtained from mds item i1ee , and depression symptoms were assessed by the validated depression rating scale using other mds items : negative statements ( e1a ) , anger ( e1d ) , unrealistic fears ( e1f ) , repetitive health complaints ( e1i ) , sad expression ( e1i ) and crying ( e1 m ; score 0 - 14 ) . this scale correlated well with the cornell and hamilton depression scales using at least mild depression as a cutoff point , and with psychiatric diagnosis . it was also more sensitive and specific than the 15-item geriatric depression scale in detecting depression in the nursing home population . data about presence of pain were obtained from the mds item j2a with options of no , less than daily and daily. demographic data were also obtained from the mds . we investigated the longitudinal relationship between changes in rejection of care and behaviors directed towards others , and changes in lack of understanding , pain , depression and psychotic symptoms with the structural equation modeling approach . within this approach , we used latent growth models , bivariate latent growth models and longitudinal latent growth mediation models . since the studied variables were ordinal , we used latent growth models for categorical variables to study change within each variable . this model assumes the existence of latent or nonobserved trajectories , which are observed only indirectly through the repeated measures . the intercept represents the initial level at the beginning of the study , and the slope represents the rate of change . the factor loadings for the slope represent the measurement points , and they were set to the values 0 , 3 , 6 and 9 that represent the months at which the measurements were taken . second , we used bivariate latent growth curve models to study the relationships between rejection of care and understanding , and rejection of care and depression . these models combine two latent growth curve models that describe two separate longitudinal processes , and study the relationship between the initial level of the first process ( y , e.g. rejection of care ) with the initial level and rate of change in the second process ( x , e.g. lack of understanding ) , and the relationship between the rate of change in the first process with the initial level and rate of change in the second process ( fig . 1 , process y and x ) . bivariate latent growth curve models were also used to describe the relationship between behavioral symptoms directed toward others with rejection of care and behavioral symptoms directed towards others with understanding , and depression . frequency distributions of the variables psychotic symptoms and presence of pain remained stable through the whole measurement period , and the latent growth models showed no trend . therefore , we did not use bivariate latent growth models but latent growth models that included these variables as time - dependent covariates . in the model for psychosis symptoms , we included only the measurements of the first and the fourth psychosis symptom because of the high correlation between the first and the second measurement , and the third and the fourth measurement . finally , longitudinal mediation models were used to study whether rejection of care mediated the relationship between lack of understanding and behaviors directed towards others , and whether it mediated the relationship between depression and behaviors directed towards others ( fig . this means that first changes in lack of understanding ( depression ) occurred , and they promoted changes in rejection of care . next , changes in behaviors directed towards others occurred as a consequence of changes in rejection of care . three sets of latent growth factors were specified , one set for the independent variable , one set for the mediator , and , finally , another set for the dependent variable . the mediation model examined whether the growth in the independent variable affected the growth trajectory of the mediating variable which , in turn , affected the growth trajectory of the dependent variable . the value , the root mean square error of approximation ( rmsea ) , the comparative fit index ( cfi ) , and the tucker - lewis ( tli ) index were used to evaluate the model . a model achieves a good fit if the rmsea is lower than 0.05 , and fit is acceptable for values between 0.05 and 0.09 . we used minimum data set - resident assessment instrument ( mds - rai ) data collected by 8 dutch nursing homes and 10 residential homes . we included the data of residents within a 12-month time window for each facility separately , resulting in a range from april 4 , 2007 , to december 1 , 2008 . of the records of 2,705 residents available from the 18 facilities , we selected the last records of 1,101 residents aged over 65 with alzheimer or other dementia ( i1q or u ) , who were dependent in decision making ( b4 not equal 0 ) and who were not comatose ( b1 equal 0 ) . four assessments from the mds within a period of 15 months were used for the analyses . for rejection of care , we used the mds 2.0 item resist care ( e4ea ) . the terminology was changed by the introduction of mds 3.0 , which uses rejection of care instead of resist care. other new , but equivalent , items in mds 3.0 are physical behavioral symptoms directed towards others instead of physical abuse and verbal behavioral symptoms directed towards others instead of verbal abuse . presence of rejection of care and behavioral symptoms directed towards others was obtained from mds 2.0 section e with choices from not exhibited to daily. the variables physical behavioral symptoms directed towards others ( e4ca ) and verbal behavioral symptoms directed towards others ( e4ba ) were combined into one variable called behaviors directed towards others. ability to understand ( c6 ) was rated as sometimes and rarely / never. psychotic symptoms [ delusions ( j1e ) and hallucinations ( j1i ) ] were combined into one variable , . clinical diagnosis of depression was obtained from mds item i1ee , and depression symptoms were assessed by the validated depression rating scale using other mds items : negative statements ( e1a ) , anger ( e1d ) , unrealistic fears ( e1f ) , repetitive health complaints ( e1i ) , sad expression ( e1i ) and crying ( e1 m ; score 0 - 14 ) . this scale correlated well with the cornell and hamilton depression scales using at least mild depression as a cutoff point , and with psychiatric diagnosis . it was also more sensitive and specific than the 15-item geriatric depression scale in detecting depression in the nursing home population . data about presence of pain were obtained from the mds item j2a with options of no , less than daily and daily. demographic data were also obtained from the mds . we investigated the longitudinal relationship between changes in rejection of care and behaviors directed towards others , and changes in lack of understanding , pain , depression and psychotic symptoms with the structural equation modeling approach . within this approach , we used latent growth models , bivariate latent growth models and longitudinal latent growth mediation models . since the studied variables were ordinal , we used latent growth models for categorical variables to study change within each variable . this model assumes the existence of latent or nonobserved trajectories , which are observed only indirectly through the repeated measures . the intercept represents the initial level at the beginning of the study , and the slope represents the rate of change . the factor loadings for the slope represent the measurement points , and they were set to the values 0 , 3 , 6 and 9 that represent the months at which the measurements were taken . second , we used bivariate latent growth curve models to study the relationships between rejection of care and understanding , and rejection of care and depression . these models combine two latent growth curve models that describe two separate longitudinal processes , and study the relationship between the initial level of the first process ( y , e.g. rejection of care ) with the initial level and rate of change in the second process ( x , e.g. lack of understanding ) , and the relationship between the rate of change in the first process with the initial level and rate of change in the second process ( fig . 1 , process y and x ) . bivariate latent growth curve models were also used to describe the relationship between behavioral symptoms directed toward others with rejection of care and behavioral symptoms directed towards others with understanding , and depression . frequency distributions of the variables psychotic symptoms and presence of pain remained stable through the whole measurement period , and the latent growth models showed no trend . therefore , we did not use bivariate latent growth models but latent growth models that included these variables as time - dependent covariates . in the model for psychosis symptoms , we included only the measurements of the first and the fourth psychosis symptom because of the high correlation between the first and the second measurement , and the third and the fourth measurement . finally , longitudinal mediation models were used to study whether rejection of care mediated the relationship between lack of understanding and behaviors directed towards others , and whether it mediated the relationship between depression and behaviors directed towards others ( fig . this means that first changes in lack of understanding ( depression ) occurred , and they promoted changes in rejection of care . next , changes in behaviors directed towards others occurred as a consequence of changes in rejection of care . three sets of latent growth factors were specified , one set for the independent variable , one set for the mediator , and , finally , another set for the dependent variable . the mediation model examined whether the growth in the independent variable affected the growth trajectory of the mediating variable which , in turn , affected the growth trajectory of the dependent variable . the value , the root mean square error of approximation ( rmsea ) , the comparative fit index ( cfi ) , and the tucker - lewis ( tli ) index were used to evaluate the model . a model achieves a good fit if the rmsea is lower than 0.05 , and fit is acceptable for values between 0.05 and 0.09 . the residents were mostly female , and some had diagnoses of both alzheimer 's disease and other dementias ( table 1 ) . over one third of them exhibited rejection of care for at least some days , and almost one third exhibited verbal behavioral symptoms directed towards others , while about 15.0% exhibited physical behavioral symptoms directed towards others . about half of these residents ( 51.0% ) exhibited significant symptoms indicating depression , but only 12.4% had a clinical diagnosis of depression . psychotic symptoms were present in 15.1% of residents , while almost one half of them experienced some pain ( table 1 ) . most common medications used for treatment of residents with rejection of care and behaviors directed towards others were antipsychotics that were administered at the beginning of the study to 32.2% of subjects ( table 1 ) . antidepressants were used in 18.7% of subjects , and antianxiety medications were used in 13.7% of subjects . further , all three classes of psychotropic medications were used more often in residents with depressive symptoms than in residents without depression ( antipsychotics 27.5 vs. 44.0% , antidepressants 13.0 vs. 27.4% , antianxiety 10.2 vs. 19.6% ) . the prevalence of psychotropic drug administration did not change during the study for antipsychotics ( t = 0.22 , p = 0.826 ) and antidepressants ( t = 0.81 , p = 0.416 ) , while the use of antianxiety medications increased ( 13.7 vs. 17.3% , t = 2.51 , p = 0.012 ) . table 2 shows the longitudinal trajectories of all the variables analyzed with latent growth models to investigate the development of the symptoms over time . based on the rmsea criteria , the model fit was good for the lack of understanding , rejection of care and behaviors directed towards others , and acceptable for depression and pain . the cfi and tli coefficients were equal to one for all the models . however , the increase was not significant for pain . the latent growth model for psychosis symptoms could not be estimated because there were almost no cases with change in psychosis scores within the study period . next , bivariate linear latent growth models were used to study the relationships between evolution in rejection of care and evolution in symptoms related to rejection of care . for psychosis and pain , we fitted a model with these variables as time - dependent covariates because previously estimated latent growth models did not show evidence of change for these variables . the model fit columns in table 3 show low rmsea values indicating a good model fit . changes in rejection of care were associated with or can be predicted by changes in lack of understanding and also by changes in depression . finally , lower initial levels of pain were associated with a stronger increase in rejection of care ( c = 0.009 , se = 0.004 , p = 0.029 ) . the relationships between the intercepts or initial level of rejection of care and intercepts of the other variables are not reported in table 3 because intercept parameters depend on the measurement scale of the analyzed variables . the relationships between psychosis symptoms and pain , and initial levels of rejection of care are scale free and can be interpreted . we found a strong relationship between psychosis and initial levels of rejection of care , with higher initial levels of psychosis associated with higher initial levels of rejection of care ( c = 0.207 , se = 0.053 , p < 0.001 ) . we also found that high initial levels of pain were associated with high initial levels of rejection of care ( c = 0.116 , se = 0.045 , p = 0.009 ) . then , bivariate linear latent growth models were used to describe the relationships between evolution in behaviors directed towards others and evolution in symptoms related to those behaviors . the columns reporting relationships between slopes show significant positive relationships between changes in rejection of care and changes in behaviors directed towards others . changes in understanding and changes in depression were also associated with changes in behaviors directed towards others . the relationships between intercepts for behaviors directed towards others are not reported in table 3 because of the same reasons explained above for the rejection of care analyses . regarding the relationship between initial behaviors directed towards others and initial levels in pain or psychotic symptoms , we found a significant relationship between initial behaviors directed towards others and initial psychosis ( c = 0.205 , se = 0.051 , p < 0.001 ) , indicating that initial psychosis was related to more frequent behaviors directed toward others . 1 ) , one to investigate whether rejection of care mediated the relationship between lack of understanding and behaviors directed towards others , and the other to investigate whether rejection of care mediated the relationship between depression and behaviors directed towards others . consistent with the literature on statistical mediation , the parameters illustrating the relationships in figure 1 are called a , b and c. parameter a describes the relationship between the slope for the independent variable ( x ) and the slope for the mediator ( m ) . parameter b describes the relationship between the slope of the mediator ( m ) and the slope in the dependent variable ( y ) . parameter c represents the direct effect , which is the part of the effect of x on y that is independent of the mediator . the fit of the first model was good ( test = 88.6 , d.f . = 61 , p = 0.012 , rmsea = 0.021 , cfi = 1.000 , tli = 1.000 ) . the relationship between lack of understanding and rejection of care was significant ( a = 0.876 , se = 0.124 , p < 0.000 ) . there was a significant relationship between rejection of care and behaviors directed towards others ( b = 1.249 , se = 0.337 , p < 0.001 ) , while the relationship between lack of understanding and behaviors directed towards others after adjusting for rejection of care was not significant ( c = 0.645 , se = 0.465 , p = 0.166 ) . therefore , this model suggests that the relationship between lack of understanding and behaviors directed towards others is mediated by rejection of care . to quantify the indirect effect , the product of the parameters a and b was calculated and tested with the sobel test . we found a significant mediation effect ( ab = 0.979 , se = 0.440 , p = 0.026 , with ab being the change in the outcome per one unit lack of understanding that goes through the mediator rejection of care ; fig . the fit of the second model was also good ( test = 105.1 , d.f . = 58 , p = 0.0002 , rmsea = 0.027 , cfi = 1.000 , tli = 1.000 ) . the relationship between the slope for depression and the slope for rejection of care was significant ( a = 0.828 , se = 0.138 , p < 0.001 ) , but there was not significant evidence that the slope in the mediator rejection of care was associated with the slope in behaviors directed towards others ( b = 0.561 , se = 0.306 , p = 0.067 ) . the adjusted direct effect ( c = 0.427 , se = 0.343 , p = 0.214 ) and the indirect effect were also not significant ( ab = 0.465 , se = 0.277 , p = 0.094 ) . therefore , we can not conclude that the relationship between depression and behaviors directed towards others is mediated by rejection of care . since we did not observe changes in psychosis and pain , we did not consider mediation models for these processes . table 2 shows the longitudinal trajectories of all the variables analyzed with latent growth models to investigate the development of the symptoms over time . based on the rmsea criteria , the model fit was good for the lack of understanding , rejection of care and behaviors directed towards others , and acceptable for depression and pain . the cfi and tli coefficients were equal to one for all the models . in table 2 , positive slopes show that all these variables increased with time . however , the increase was not significant for pain . the latent growth model for psychosis symptoms could not be estimated because there were almost no cases with change in psychosis scores within the study period . next , bivariate linear latent growth models were used to study the relationships between evolution in rejection of care and evolution in symptoms related to rejection of care . for psychosis and pain , we fitted a model with these variables as time - dependent covariates because previously estimated latent growth models did not show evidence of change for these variables . the model fit columns in table 3 show low rmsea values indicating a good model fit . the cfi and tli coefficients were equal to one . changes in rejection of care were associated with or can be predicted by changes in lack of understanding and also by changes in depression . finally , lower initial levels of pain were associated with a stronger increase in rejection of care ( c = 0.009 , se = 0.004 , p = 0.029 ) . the relationships between the intercepts or initial level of rejection of care and intercepts of the other variables are not reported in table 3 because intercept parameters depend on the measurement scale of the analyzed variables . the relationships between psychosis symptoms and pain , and initial levels of rejection of care are scale free and can be interpreted . we found a strong relationship between psychosis and initial levels of rejection of care , with higher initial levels of psychosis associated with higher initial levels of rejection of care ( c = 0.207 , se = 0.053 , p < 0.001 ) . we also found that high initial levels of pain were associated with high initial levels of rejection of care ( c = 0.116 , se = 0.045 , p = 0.009 ) . then , bivariate linear latent growth models were used to describe the relationships between evolution in behaviors directed towards others and evolution in symptoms related to those behaviors . the columns reporting relationships between slopes show significant positive relationships between changes in rejection of care and changes in behaviors directed towards others . changes in understanding and changes in depression were also associated with changes in behaviors directed towards others . the relationships between intercepts for behaviors directed towards others are not reported in table 3 because of the same reasons explained above for the rejection of care analyses . regarding the relationship between initial behaviors directed towards others and initial levels in pain or psychotic symptoms , we found a significant relationship between initial behaviors directed towards others and initial psychosis ( c = 0.205 , se = 0.051 , p < 0.001 ) , indicating that initial psychosis was related to more frequent behaviors directed toward others . 1 ) , one to investigate whether rejection of care mediated the relationship between lack of understanding and behaviors directed towards others , and the other to investigate whether rejection of care mediated the relationship between depression and behaviors directed towards others . consistent with the literature on statistical mediation , the parameters illustrating the relationships in figure 1 are called a , b and c. parameter a describes the relationship between the slope for the independent variable ( x ) and the slope for the mediator ( m ) . parameter b describes the relationship between the slope of the mediator ( m ) and the slope in the dependent variable ( y ) . parameter c represents the direct effect , which is the part of the effect of x on y that is independent of the mediator . the fit of the first model was good ( test = 88.6 , d.f . = 61 , p = 0.012 , rmsea = 0.021 , cfi = 1.000 , tli = 1.000 ) . the relationship between lack of understanding and rejection of care was significant ( a = 0.876 , se = 0.124 , p there was a significant relationship between rejection of care and behaviors directed towards others ( b = 1.249 , se = 0.337 , p < 0.001 ) , while the relationship between lack of understanding and behaviors directed towards others after adjusting for rejection of care was not significant ( c = 0.645 , se = 0.465 , p = 0.166 ) . therefore , this model suggests that the relationship between lack of understanding and behaviors directed towards others is mediated by rejection of care . to quantify the indirect effect , the product of the parameters a and b was calculated and tested with the sobel test . we found a significant mediation effect ( ab = 0.979 , se = 0.440 , p = 0.026 , with ab being the change in the outcome per one unit lack of understanding that goes through the mediator rejection of care ; fig . the fit of the second model was also good ( test = 105.1 , d.f . = 58 , p = 0.0002 , rmsea = 0.027 , cfi = 1.000 , tli = 1.000 ) . the relationship between the slope for depression and the slope for rejection of care was significant ( a = 0.828 , se = 0.138 , p < 0.001 ) , but there was not significant evidence that the slope in the mediator rejection of care was associated with the slope in behaviors directed towards others ( b = 0.561 , se = 0.306 , p = 0.067 ) . the adjusted direct effect ( c = 0.427 , se = 0.343 , p = 0.214 ) and the indirect effect were also not significant ( ab = 0.465 , se = 0.277 , p = 0.094 ) . therefore , we can not conclude that the relationship between depression and behaviors directed towards others is mediated by rejection of care . since we did not observe changes in psychosis and pain , we did not consider mediation models for these processes . the results of this longitudinal study showed that changes in lack of understanding or depression commonly precede changes in rejection of care . changes in behaviors directed towards others are related to changes in lack of understanding , depression and rejection of care . furthermore , a mediation model suggested that the relationship between lack of understanding and behaviors directed towards others was mediated by rejection of care . therefore , our results support conclusions of our previous cross - sectional study , which found that lack of understanding and depression are the two main risk factors for development of rejection of care and behaviors directed towards others . however , our results are more informative because longitudinal models provide more information regarding temporal relationships between the variables , and the mediation model provides information about the order in which the symptoms develop . initial psychotic symptoms and pain were also related to increased rejection of care , but we did not observe changes in the number of psychotic symptoms and pain within our study period . regarding to the absence of observed changes in pain , hendriks et al . found a high proportion of patients with pain persistence . that precluded inclusion of psychotic symptoms and pain in the mediation model . a relationship of depression to resistive behavior ( rejection of care ) and aggression ( abusive symptoms ) was already reported by lyketsos et al . and by leonard et al . . they found , in agreement with our results , that psychotic symptoms play a minor role in the development of these behaviors . the lack of understanding is caused by the progression of dementia , and it increases the prevalence of rejection of care ; this increase in rejection of care contributes to the increasing behaviors directed towards others . it may be improved by communication skills training of professionals and family caregivers that includes verbal skills , nonverbal and emotional skills , behavioral management skills , usage of tools and theoretical knowledge . such a program would be useful also in a hospital where many dementia patients are transferred . the most important intervention is to teach the staff that most people with dementia are not aggressive ; they just defend themselves against unwanted attention from the caregivers and may consider the caregivers to be the aggressors . there is a clear - cut distinction between rejection of care and agitation , and the term agitation should be reserved for behaviors that occur when the resident is solitary , e.g. restlessness , repetitive movements , crying out . we identified depressive symptoms using the mds depression scale , which uses 7 mds items . this scale correlated well with the cornell and hamilton depression scales using at least mild depression as a cutoff point , and with psychiatric diagnosis . this is comparable to 47.4% detected in a large study of a long - term care facility . high prevalence of depression in individuals with alzheimer 's disease can be expected because alzheimer 's disease causes serotoninergic deficit , and some data indicate that this deficit may be related to aggressive behaviors . the relationship between serotoninergic function and aggressive behavior may not be unique to alzheimer 's disease because decreased serotoninergic activity is present also in frontotemporal dementia . our results suggest that depression contributes to both rejection of care and behaviors directed towards others and that the relation between depression and behaviors towards others is not mediated by rejection of care . depression in dementia could be treated with psychological interventions as shown in a recent review . antidepressants could be the first line of medication for individuals exhibiting these behaviors directed towards others if psychotic symptoms are not present . although some studies found improvement of behavioral symptoms after treatment with antidepressants , other studies were negative . this was documented by the depression in alzheimer disease study , which found that treatment with sertraline decreased behavioral disturbance and caregiver distress only in patients whose depression responded to antidepressant treatment . antidepressant treatment was found effective in decreasing behavioral symptoms of dementia in a cochrane data analysis and in a recent citalopram study , but antidepressants may sometimes require augmentation with atypical antipsychotics . the results of this study show that the psychotropic medications administered most frequently to the participants in this study were antipsychotics . it is possible that these drugs somewhat reduced rejection of care by their sedative effects . similar prevalences of antipsychotic use were reported from a survey of nursing home physicians in the us and a dutch survey . in that study , 32% of subjects received antipsychotics , which is comparable with reports from sweden ( 38% ) and from germany ( 32.5% ) . antipsychotics are used frequently for treatment of behavioral symptoms of dementia despite multiple reports that found serious side effects including increased mortality in residents treated with these medications . efforts are made in several countries to decrease the use of antipsychotics in dementia . in the us , the prevalence of antipsychotic use in nursing homes is about 25% , and the use of antipsychotics was recently reduced by 9.1% . in the uk , antipsychotic use in people with dementia decreased from 19.9% in 1995 to 7.4% in 2011 , concomitant with an increase of antidepressants from 10.7 to 25.3% , indicating that antidepressants may be substituted for antipsychotics . our data were derived from mds records that were completed mostly by nurses . however , these were experienced clinicians , and mds data correlated well with other scales . we have studied the relationship of only four factors in abusive behavior of nursing home residents with dementia . however , there are probably also other factors that may be involved , e.g. physical causes such as thirst , hunger or inappropriate environmental temperature . using a discomfort scale in future studies would provide information about the importance of these other factors . our data could not evaluate these causes . a model with all predictors controlling for the influence of each variable would provide more evidence for causality than our models . another limitation is that we did not have data about specific drugs and drug dosages that may have been modified in residents with rejection of care and behaviors directed towards others even when the prevalence of their use did not change . the results of this study together with the results of previous investigations indicate that lack of understanding and depression are important factors in development of rejection of care and behaviors directed towards others , and that rejection of care may escalate into behaviors directed towards others . furthermore , the relationship between lack of understanding and behaviors directed towards others may be mediated by rejection of care . therefore , improved communication between caregivers and persons with dementia and perhaps also effective treatment of depression may prevent or ameliorate rejection of care and behaviors directed towards others .

aimsthe aim of this study was to analyze factors related to rejection of care and behaviors directed towards others in nursing home residents with dementia.methodsthe relationship of lack of understanding , depression , psychosis and pain with rejection of care and behaviors directed towards others was explored using four assessments from the minimum data set ( mds ) within a period of 15 months on 1,101 residents with dementia in dutch nursing homes . presence of depressive symptoms was ascertained using a validated mds scale , and presence of lack of understanding , rejection of care , psychosis and pain through the individual mds items . a structural equation modeling approach and latent growth models were used to investigate the longitudinal relationship between changes in rejection of care and physical or verbal behaviors directed towards others , and changes in lack of understanding , pain , depression and psychotic symptoms.resultschanges in lack of understanding predicted changes in rejection of care , and there was also a relationship between changes in depression and rejection of care . changes of behaviors directed towards others were related to changes in lack of understanding and depression . pain and behaviors directed towards others were unrelated , and psychosis was rather stable throughout . a mediation model suggested that the relationship of lack of understanding with behaviors directed towards others was mediated by rejection of care.conclusionthese results indicate that lack of understanding and depression are important factors in development of rejection of care and behaviors directed towards others . the relationship between lack of understanding and behaviors directed towards others is mediated by rejection of care . improvement in communication between residents and caregivers , and perhaps also effective treatment of depression may prevent or ameliorate these behaviors directed towards others .

Introduction Material and Methods Subject Population Measurements Analyses Results Longitudinal Trajectories of Each Process Combined Longitudinal Relationships Mediated Effects Discussion Conclusions Disclosure Statement

in an institution , rejection of care and behaviors directed towards others may lead to injuries of the resident or staff and to staff burnout . a survey of nursing home physicians showed that the most common problem that they treated in residents with dementia was rejection of care . however , the problem with these studies is that they consider all behavioral symptoms to be agitation and do not differentiate between agitation and rejection of care . this is due to using the cohen - mansfield agitation inventory or neuropsychiatric inventory in which the item agitation / aggression is actually measuring rejection of care . we have previously described strong correlations between rejection of care and lack of understanding , depression and psychotic symptoms . behaviors directed towards others were strongly correlated with rejection of care , depression and delusions . in this study , we performed longitudinal analyses to further explore the relationships of these modifiable factors with rejection of care and behaviors directed towards others of nursing home residents . our hypothesis was that longitudinal data would support causal relationships between these modifiable factors and rejection of care . we used minimum data set - resident assessment instrument ( mds - rai ) data collected by 8 dutch nursing homes and 10 residential homes . four assessments from the mds within a period of 15 months were used for the analyses . the terminology was changed by the introduction of mds 3.0 , which uses rejection of care instead of resist care. other new , but equivalent , items in mds 3.0 are physical behavioral symptoms directed towards others instead of physical abuse and verbal behavioral symptoms directed towards others instead of verbal abuse . presence of rejection of care and behavioral symptoms directed towards others was obtained from mds 2.0 section e with choices from not exhibited to daily. the variables physical behavioral symptoms directed towards others ( e4ca ) and verbal behavioral symptoms directed towards others ( e4ba ) were combined into one variable called behaviors directed towards others. clinical diagnosis of depression was obtained from mds item i1ee , and depression symptoms were assessed by the validated depression rating scale using other mds items : negative statements ( e1a ) , anger ( e1d ) , unrealistic fears ( e1f ) , repetitive health complaints ( e1i ) , sad expression ( e1i ) and crying ( e1 m ; score 0 - 14 ) . it was also more sensitive and specific than the 15-item geriatric depression scale in detecting depression in the nursing home population . we investigated the longitudinal relationship between changes in rejection of care and behaviors directed towards others , and changes in lack of understanding , pain , depression and psychotic symptoms with the structural equation modeling approach . within this approach , we used latent growth models , bivariate latent growth models and longitudinal latent growth mediation models . second , we used bivariate latent growth curve models to study the relationships between rejection of care and understanding , and rejection of care and depression . these models combine two latent growth curve models that describe two separate longitudinal processes , and study the relationship between the initial level of the first process ( y , e.g. lack of understanding ) , and the relationship between the rate of change in the first process with the initial level and rate of change in the second process ( fig . bivariate latent growth curve models were also used to describe the relationship between behavioral symptoms directed toward others with rejection of care and behavioral symptoms directed towards others with understanding , and depression . frequency distributions of the variables psychotic symptoms and presence of pain remained stable through the whole measurement period , and the latent growth models showed no trend . finally , longitudinal mediation models were used to study whether rejection of care mediated the relationship between lack of understanding and behaviors directed towards others , and whether it mediated the relationship between depression and behaviors directed towards others ( fig . this means that first changes in lack of understanding ( depression ) occurred , and they promoted changes in rejection of care . next , changes in behaviors directed towards others occurred as a consequence of changes in rejection of care . three sets of latent growth factors were specified , one set for the independent variable , one set for the mediator , and , finally , another set for the dependent variable . the value , the root mean square error of approximation ( rmsea ) , the comparative fit index ( cfi ) , and the tucker - lewis ( tli ) index were used to evaluate the model . we used minimum data set - resident assessment instrument ( mds - rai ) data collected by 8 dutch nursing homes and 10 residential homes . four assessments from the mds within a period of 15 months were used for the analyses . for rejection of care , we used the mds 2.0 item resist care ( e4ea ) . the terminology was changed by the introduction of mds 3.0 , which uses rejection of care instead of resist care. presence of rejection of care and behavioral symptoms directed towards others was obtained from mds 2.0 section e with choices from not exhibited to daily. the variables physical behavioral symptoms directed towards others ( e4ca ) and verbal behavioral symptoms directed towards others ( e4ba ) were combined into one variable called behaviors directed towards others. clinical diagnosis of depression was obtained from mds item i1ee , and depression symptoms were assessed by the validated depression rating scale using other mds items : negative statements ( e1a ) , anger ( e1d ) , unrealistic fears ( e1f ) , repetitive health complaints ( e1i ) , sad expression ( e1i ) and crying ( e1 m ; score 0 - 14 ) . we investigated the longitudinal relationship between changes in rejection of care and behaviors directed towards others , and changes in lack of understanding , pain , depression and psychotic symptoms with the structural equation modeling approach . since the studied variables were ordinal , we used latent growth models for categorical variables to study change within each variable . second , we used bivariate latent growth curve models to study the relationships between rejection of care and understanding , and rejection of care and depression . these models combine two latent growth curve models that describe two separate longitudinal processes , and study the relationship between the initial level of the first process ( y , e.g. lack of understanding ) , and the relationship between the rate of change in the first process with the initial level and rate of change in the second process ( fig . bivariate latent growth curve models were also used to describe the relationship between behavioral symptoms directed toward others with rejection of care and behavioral symptoms directed towards others with understanding , and depression . frequency distributions of the variables psychotic symptoms and presence of pain remained stable through the whole measurement period , and the latent growth models showed no trend . finally , longitudinal mediation models were used to study whether rejection of care mediated the relationship between lack of understanding and behaviors directed towards others , and whether it mediated the relationship between depression and behaviors directed towards others ( fig . this means that first changes in lack of understanding ( depression ) occurred , and they promoted changes in rejection of care . next , changes in behaviors directed towards others occurred as a consequence of changes in rejection of care . the value , the root mean square error of approximation ( rmsea ) , the comparative fit index ( cfi ) , and the tucker - lewis ( tli ) index were used to evaluate the model . over one third of them exhibited rejection of care for at least some days , and almost one third exhibited verbal behavioral symptoms directed towards others , while about 15.0% exhibited physical behavioral symptoms directed towards others . about half of these residents ( 51.0% ) exhibited significant symptoms indicating depression , but only 12.4% had a clinical diagnosis of depression . most common medications used for treatment of residents with rejection of care and behaviors directed towards others were antipsychotics that were administered at the beginning of the study to 32.2% of subjects ( table 1 ) . further , all three classes of psychotropic medications were used more often in residents with depressive symptoms than in residents without depression ( antipsychotics 27.5 vs. 44.0% , antidepressants 13.0 vs. 27.4% , antianxiety 10.2 vs. 19.6% ) . table 2 shows the longitudinal trajectories of all the variables analyzed with latent growth models to investigate the development of the symptoms over time . based on the rmsea criteria , the model fit was good for the lack of understanding , rejection of care and behaviors directed towards others , and acceptable for depression and pain . next , bivariate linear latent growth models were used to study the relationships between evolution in rejection of care and evolution in symptoms related to rejection of care . for psychosis and pain , we fitted a model with these variables as time - dependent covariates because previously estimated latent growth models did not show evidence of change for these variables . changes in rejection of care were associated with or can be predicted by changes in lack of understanding and also by changes in depression . finally , lower initial levels of pain were associated with a stronger increase in rejection of care ( c = 0.009 , se = 0.004 , p = 0.029 ) . the relationships between the intercepts or initial level of rejection of care and intercepts of the other variables are not reported in table 3 because intercept parameters depend on the measurement scale of the analyzed variables . the relationships between psychosis symptoms and pain , and initial levels of rejection of care are scale free and can be interpreted . we found a strong relationship between psychosis and initial levels of rejection of care , with higher initial levels of psychosis associated with higher initial levels of rejection of care ( c = 0.207 , se = 0.053 , p < 0.001 ) . we also found that high initial levels of pain were associated with high initial levels of rejection of care ( c = 0.116 , se = 0.045 , p = 0.009 ) . then , bivariate linear latent growth models were used to describe the relationships between evolution in behaviors directed towards others and evolution in symptoms related to those behaviors . the columns reporting relationships between slopes show significant positive relationships between changes in rejection of care and changes in behaviors directed towards others . changes in understanding and changes in depression were also associated with changes in behaviors directed towards others . the relationships between intercepts for behaviors directed towards others are not reported in table 3 because of the same reasons explained above for the rejection of care analyses . regarding the relationship between initial behaviors directed towards others and initial levels in pain or psychotic symptoms , we found a significant relationship between initial behaviors directed towards others and initial psychosis ( c = 0.205 , se = 0.051 , p < 0.001 ) , indicating that initial psychosis was related to more frequent behaviors directed toward others . 1 ) , one to investigate whether rejection of care mediated the relationship between lack of understanding and behaviors directed towards others , and the other to investigate whether rejection of care mediated the relationship between depression and behaviors directed towards others . the relationship between lack of understanding and rejection of care was significant ( a = 0.876 , se = 0.124 , p < 0.000 ) . there was a significant relationship between rejection of care and behaviors directed towards others ( b = 1.249 , se = 0.337 , p < 0.001 ) , while the relationship between lack of understanding and behaviors directed towards others after adjusting for rejection of care was not significant ( c = 0.645 , se = 0.465 , p = 0.166 ) . therefore , this model suggests that the relationship between lack of understanding and behaviors directed towards others is mediated by rejection of care . we found a significant mediation effect ( ab = 0.979 , se = 0.440 , p = 0.026 , with ab being the change in the outcome per one unit lack of understanding that goes through the mediator rejection of care ; fig . the relationship between the slope for depression and the slope for rejection of care was significant ( a = 0.828 , se = 0.138 , p < 0.001 ) , but there was not significant evidence that the slope in the mediator rejection of care was associated with the slope in behaviors directed towards others ( b = 0.561 , se = 0.306 , p = 0.067 ) . therefore , we can not conclude that the relationship between depression and behaviors directed towards others is mediated by rejection of care . since we did not observe changes in psychosis and pain , we did not consider mediation models for these processes . table 2 shows the longitudinal trajectories of all the variables analyzed with latent growth models to investigate the development of the symptoms over time . based on the rmsea criteria , the model fit was good for the lack of understanding , rejection of care and behaviors directed towards others , and acceptable for depression and pain . next , bivariate linear latent growth models were used to study the relationships between evolution in rejection of care and evolution in symptoms related to rejection of care . for psychosis and pain , we fitted a model with these variables as time - dependent covariates because previously estimated latent growth models did not show evidence of change for these variables . changes in rejection of care were associated with or can be predicted by changes in lack of understanding and also by changes in depression . finally , lower initial levels of pain were associated with a stronger increase in rejection of care ( c = 0.009 , se = 0.004 , p = 0.029 ) . the relationships between the intercepts or initial level of rejection of care and intercepts of the other variables are not reported in table 3 because intercept parameters depend on the measurement scale of the analyzed variables . the relationships between psychosis symptoms and pain , and initial levels of rejection of care are scale free and can be interpreted . we found a strong relationship between psychosis and initial levels of rejection of care , with higher initial levels of psychosis associated with higher initial levels of rejection of care ( c = 0.207 , se = 0.053 , p < 0.001 ) . we also found that high initial levels of pain were associated with high initial levels of rejection of care ( c = 0.116 , se = 0.045 , p = 0.009 ) . then , bivariate linear latent growth models were used to describe the relationships between evolution in behaviors directed towards others and evolution in symptoms related to those behaviors . the columns reporting relationships between slopes show significant positive relationships between changes in rejection of care and changes in behaviors directed towards others . changes in understanding and changes in depression were also associated with changes in behaviors directed towards others . the relationships between intercepts for behaviors directed towards others are not reported in table 3 because of the same reasons explained above for the rejection of care analyses . regarding the relationship between initial behaviors directed towards others and initial levels in pain or psychotic symptoms , we found a significant relationship between initial behaviors directed towards others and initial psychosis ( c = 0.205 , se = 0.051 , p < 0.001 ) , indicating that initial psychosis was related to more frequent behaviors directed toward others . 1 ) , one to investigate whether rejection of care mediated the relationship between lack of understanding and behaviors directed towards others , and the other to investigate whether rejection of care mediated the relationship between depression and behaviors directed towards others . consistent with the literature on statistical mediation , the parameters illustrating the relationships in figure 1 are called a , b and c. parameter a describes the relationship between the slope for the independent variable ( x ) and the slope for the mediator ( m ) . the relationship between lack of understanding and rejection of care was significant ( a = 0.876 , se = 0.124 , p there was a significant relationship between rejection of care and behaviors directed towards others ( b = 1.249 , se = 0.337 , p < 0.001 ) , while the relationship between lack of understanding and behaviors directed towards others after adjusting for rejection of care was not significant ( c = 0.645 , se = 0.465 , p = 0.166 ) . therefore , this model suggests that the relationship between lack of understanding and behaviors directed towards others is mediated by rejection of care . we found a significant mediation effect ( ab = 0.979 , se = 0.440 , p = 0.026 , with ab being the change in the outcome per one unit lack of understanding that goes through the mediator rejection of care ; fig . the relationship between the slope for depression and the slope for rejection of care was significant ( a = 0.828 , se = 0.138 , p < 0.001 ) , but there was not significant evidence that the slope in the mediator rejection of care was associated with the slope in behaviors directed towards others ( b = 0.561 , se = 0.306 , p = 0.067 ) . therefore , we can not conclude that the relationship between depression and behaviors directed towards others is mediated by rejection of care . since we did not observe changes in psychosis and pain , we did not consider mediation models for these processes . the results of this longitudinal study showed that changes in lack of understanding or depression commonly precede changes in rejection of care . changes in behaviors directed towards others are related to changes in lack of understanding , depression and rejection of care . furthermore , a mediation model suggested that the relationship between lack of understanding and behaviors directed towards others was mediated by rejection of care . therefore , our results support conclusions of our previous cross - sectional study , which found that lack of understanding and depression are the two main risk factors for development of rejection of care and behaviors directed towards others . initial psychotic symptoms and pain were also related to increased rejection of care , but we did not observe changes in the number of psychotic symptoms and pain within our study period . that precluded inclusion of psychotic symptoms and pain in the mediation model . a relationship of depression to resistive behavior ( rejection of care ) and aggression ( abusive symptoms ) was already reported by lyketsos et al . they found , in agreement with our results , that psychotic symptoms play a minor role in the development of these behaviors . the lack of understanding is caused by the progression of dementia , and it increases the prevalence of rejection of care ; this increase in rejection of care contributes to the increasing behaviors directed towards others . there is a clear - cut distinction between rejection of care and agitation , and the term agitation should be reserved for behaviors that occur when the resident is solitary , e.g. we identified depressive symptoms using the mds depression scale , which uses 7 mds items . high prevalence of depression in individuals with alzheimer 's disease can be expected because alzheimer 's disease causes serotoninergic deficit , and some data indicate that this deficit may be related to aggressive behaviors . our results suggest that depression contributes to both rejection of care and behaviors directed towards others and that the relation between depression and behaviors towards others is not mediated by rejection of care . antidepressants could be the first line of medication for individuals exhibiting these behaviors directed towards others if psychotic symptoms are not present . the results of this study show that the psychotropic medications administered most frequently to the participants in this study were antipsychotics . it is possible that these drugs somewhat reduced rejection of care by their sedative effects . in the us , the prevalence of antipsychotic use in nursing homes is about 25% , and the use of antipsychotics was recently reduced by 9.1% . we have studied the relationship of only four factors in abusive behavior of nursing home residents with dementia . another limitation is that we did not have data about specific drugs and drug dosages that may have been modified in residents with rejection of care and behaviors directed towards others even when the prevalence of their use did not change . the results of this study together with the results of previous investigations indicate that lack of understanding and depression are important factors in development of rejection of care and behaviors directed towards others , and that rejection of care may escalate into behaviors directed towards others . furthermore , the relationship between lack of understanding and behaviors directed towards others may be mediated by rejection of care . therefore , improved communication between caregivers and persons with dementia and perhaps also effective treatment of depression may prevent or ameliorate rejection of care and behaviors directed towards others .

environmental noise has become a serious socio - economic problem in many industrialized countries , and a large portion of the population is exposed to urban noise ; noise - induced hearing loss ( nihl ) is one of the most prominent occupational diseases in europe . the main significant event in nihl regards the outer hair cells ( ohc ) within the organ of corti . it is well known that , after noise exposure , ohcs death occurs , which in turn leads to a loss of cochlear amplification and thus to a permanent threshold shift , as reported in previous work by our group using a guinea pig model exposed to noise [ 46 ] . in humans , the audiological characteristics of noise - induced hearing loss include hearing threshold shift at high frequencies , presence of recruitment , reduction in the level of evoked otoacoustic emission ( oaes ) , and impairment of speech intelligibility . particular focus is devoted to the analysis of audiograms and oae tests which , as single diagnostic tools or in combination , may fail to provide early diagnosis since scientific consensus is lacking regarding screening protocols and optimal audiological findings for the early detection of nihl [ 79 ] . nihl does not simultaneously affect all frequencies pure tone audiometry usually shows a gradual progression and the first impairment begins around the frequency of 4 khz , with a typical notch at 4 khz and , at times , 6 khz frequencies . oaes tests are non - invasive and do not require co - operation from the patient , as does behavioral audiometry ; still , 30 years after their discovery , clinical applications of oaes are largely limited to qualitative pass / fail testing . oaes are sounds generated in the inner ear by the activity of the cochlear amplifiers ; they can be recorded when the auditory periphery is solicited by an external acoustic stimuli , such as brief click ( i.e. , transient evoked oae or teoae ) or 1 or 2 simultaneous pure tones ( i.e. , stimulus - frequency oaes and distortion product oaes ) , and even in the absence of stimulation ( i.e. , spontaneous oaes ) . protocols based on teoaes are a widely used method for identifying hearing losses [ 1720 ] and , as dpoaes , may provide an early indication of cochlear damage before evidence for nihl appears in pure - tone audiometry [ 2128 ] . compared teoae parameters with pure tone audiograms to determine early indications of cochlear damage ; results showed that teoae spectral parameters can discriminate between non - exposed normal subjects and subjects with unilateral noise - induced hearing loss . however , the validity of teoae measurements is hampered by their subject - dependent variations . taken together , constraints to the early diagnosis are patient s co - operation in conventional audiometry and subject s variance in teoaes ; both issues can be controlled and reduced by mathematical procedures . in this preliminary report we propose a mathematical screening protocol able to detect and quantify the magnitude of hearing loss . a quantification parameter , called henceforth 2-dimensional radius ( rad2d ) , is extracted from the teoae waveform by means of a non - linear signal analysis process referred to as recurrence quantification analysis ( rqa ) along with principal component analysis ( pca ) . rqa is an efficient and relatively simple tool in the analysis of many physiological signals and accounts for the deterministic structure of teoae signals ; pca is a multivariate statistical analysis successfully applied to teoaes signals since it minimizes redundant information . the proposed rad2d parameter is a global parameter ( comparable to whole waveform reproducibility - wwr ) , but , as will be shown , the embedding procedure in rqa tools permits observation of the fine structure of the waveform . with respect to the typical frequency - specific bands analysis obtained by using the fourier transform , this extracted parameter , rad2d , is expressed by an easily comparable numeric value in the same way as the wwr parameter . furthermore , this mathematical quantification of teoae s fine structure reduces the individual variability and can be easily applied to detect hearing impairments in the early pathologic stage . in the present study , the mathematical procedure is applied to teoaes recorded in normal ears and in ears exhibiting a notch at 4 khz greater than 30 db hl ( hearing level ) , and the obtained parameter is compared with the conventional analyses ( audiometry and teoae whole waveform reproducibility ) . to optimize and validate the procedure , an electronic model of the ear , developed by gigure and woodland and applied in a previous work , is used to simulate teoae signals both in normal ears and in ears with different amounts of cochlear damage . taken together , the aims of this paper are : 1 ) to compare the measured teoae among normal and ih ears ; 2 ) to simulate normal and ih ears by using the electronic model of the ear with a selective damage corresponding to a 4 khz notch ; 3 ) to obtain simulated teoae and to compare them with the measured ones ; 4 ) to estimate the rad2d parameter in the electronic model and to compare the obtained rad2d values with those evaluated in the normal and ih recorded teoae . the present study was carried out on 29 ears from 21 subjects ( average age = 38.78.12 years ) . of these , 14 ears were from 11 impaired - hearing ( ih ) subjects , and the remaining 15 ears were from 10 subjects with normal hearing . ih subjects ( average age = 41.36.5 years ) were included in this study if pure - tone audiometry revealed a 4 khz notch > 30 db hl . the normal hearing subjects ( normal group , average age = 36.39.0 years ) had no significant noise exposure . none of the subjects had any background of hereditary hearing loss or ear infections , and were fully informed of the aim , design , and clinical applications of this study . the study was approved by the ethics committee of the catholic university , rome , italy , and the investigations were performed in accordance with the principles of the declaration of helsinki . all subjects underwent a baseline hearing evaluation : otoscopic examination and 226-hz probe tone tympanometry were used to exclude middle ear pathologies . conventional pure tone audiometric testing was carried out in a soundproof room and the pure - tone thresholds ( ptt ) of each ear were measured at 0.25 , 0.50 , 1 , 2 , 4 , and 8 khz with a clinical audiometer ( ac-40 , interacoustics co , assens , denmark ) . classification of patients was obtained by using a ptt value at 4 khz . the otodynamic analyzer ( ilo92 , otodynamics ltd , hatfield , u.k . ) was used to record the teoae signals by inserting a sgs - type general purpose teoae probe into the external ear canal . the teoae recordings were carried out in a standard hospital room , corresponding to the usual clinical setting for these measurements . the automated differential non - linear test paradigm was used : the stimulus was characterized by a train of 4 clicks , 3 with the same amplitude and polarity , followed by a fourth with a 3-fold amplitude and opposite polarity with respect to the preceding ones . the responses were obtained , evaluating an average among 260 stimuli trains ( 1040 clicks ) stored into 2 different buffers ( a and b ) for a total of 2080 clicks . the value of the automatically computed correlation or reproducibility between the 2 obtained waveforms ( a and b ) of an oae signal is termed repro or whole waveform reproducibility ( wwr ) ( pearson correlation coefficient * 100 ) . according to the clinical settings , when the wwr value is > 70% and the snr is higher than 3 db , the signal is considered as physiological ( ilo92 test is pass ) , whereas a wwr value < 70% indicates the presence of a possible hearing malfunction ( ilo92 test is failed ) . in the measured data , the mean snr given by the ilo92 was 13.04 db for the normal ears and 3.96 db for the ih ears . the examined ears were classified as normal hearing or ih ears according to their pure tone thresholds at 1 , 2 , 4 and 8 khz . in particular , the subjects with a pure - tone threshold value greater than 30 db hl at 4 khz were assigned to the ih group even though their wwr value was high ( wwr 70% and pass at ilo92 test ) . to optimize and validate the procedure , teoae have been numerically simulated using an electronic model of the whole ear , originally introduced by guigure and woodland ( 1994 ) and used in other teoaes analysis and other studies . the electronic model is based on the electro - acoustic analogy , where the acoustic mass of cochlea fluids is represented by inductors ; the acoustic resistance , mass and stiffness of the basilar membrane are represented by shunt resistors , inductors and capacitors , respectively ; and the ohc active processes are represented by non - linear voltage sources ( details of the circuit are in ) . in the electronic model of the ear , the cochlea is divided into several sections ( termed partitions ) from the base to the apex . sixty - four partitions are used to segment the cochlea , so that , being the whole uncoiled cochlea 3.5 cm long ( comprising a total of about 12000 cells ) , each partition corresponds to a cochlear portion of 0.0547 cm and about 188 cells . in order to simulate the noise - induced ohc damage corresponding to the experimental ih condition , the suppression of cochlear amplification is obtained by zeroing the gain of some of the generators within the cochlear partitions . in particular , to establish different damage levels , a localized suppression of an increasing number ( ranged from n=2 to n=10 ) of adjacent cochlear amplifiers is applied close to the characteristic frequency ( cf ) of 4 khz . to simulate teoae measurements , the input voltage was a rectangular pulse 80 s in duration , with an amplitude corresponding to a stimulus level of 80 pe db spl . finally , to eliminate the initial ringing , the first 2.8 ms of the simulated teoae signals were excluded from the successive analysis . the wwr value of teoaes in ih subjects with a 4 khz notch can be > 70% as in normal hearing subjects . consequently , the usual waveform descriptive parameters are of no assistance in detecting cochlear pathology . in the present study , teoae signals were analyzed by rqa and pca techniques to reduce inter - subject variability and the dynamical structure of signals were investigated rather than signal - level differences . rqa introduces few parameters ( named rqa descriptors ) descriptive of the global complexity of a signal and computed from the so called recurrence plot the time behavior of the original signal was represented by a series of 512 points equally spaced in time ( e.g. , { a1 a2 . a 512 } where ai represents the value of the signal corresponding to the i - th time position ) . then , the series was arranged in successive columns ( the columns number is defined by the embedding dimension parameter n ) , each - one obtained by applying a delay in time ( lag parameter ) to the original sequence , thus creating an embedding matrix , as in equation 1 . finally , the recurrence plot was built , drawing a black dot ( named recurrent point ) in the represented space if the distance between the corresponding rows ( the distance between the j - th and the ( jth+1 ) row is i=1512(aj , i - aj+1,i)2 ) of the embedding matrix was lower than a fixed value ( radius ) . in the obtained plot , the horizontal and vertical axes represented the relative position of the 512 points into the teoae waveform . rqa descriptors were then calculated on the basis of the number and the location of dots in the recurrence plot . in particular , percent of recurrence ( rec ) is the percentage of recurrence points in a recurrent plot ; percent of determinism ( det ) is the percentage of recurrence points which form diagonal lines and it indicates the degree of deterministic structure of the signal ; entropy ( ent ) is the shannon entropy of the probability distribution of the diagonal line lengths and is linked to the richness of deterministic structure . the presence of horizontal and vertical lines in the recurrence plot shows that part of the considered signal matches closely with a later sequence . based on experience gained in previous research , the delay in the embedding procedure ( lag ) was set to 1 ; the number of the embedding matrix columns ( embedding dimension , n ) was 10 ; and the cut - off distance ( radius ) was 15 . pca is a common statistical technique which , exploiting the presence of correlations among the original variables , provides the possibility to reduce the starting data set dimension without consistent loss of information and with a separation of the different and independent features characterizing the data set . as a consequence , pca describes the original data set with a lower number of new parameters , termed main components . as shown in a previous study , if a set of at least 70 representative teoae signals is chosen as input to the rqa - pca analysis , the first 2 main components ( pc1 , pc2 ) explain more than 90% of the total variability in the data set . moreover , having pc1 and pc2 , by construction , zero mean and standard deviation equal to 1 , if a set of teoae signals from normal ears are studied , 96% of them will fall within a circle centered in the origin of the pc1/pc2 plane , and with a radius equal to 2 ( reference circle ) . in the present study , the pc1/pc2 plane was defined starting from a representative data set made by 118 signals measured from normal hearing subjects , in agreement with results of a previous study . the explained variability of the representative data set used was 90.02% , 6.29% , and 3.68% for pc1 , pc2 , and pc3 , respectively . the correlation between rqa parameters and pcs is described by the factor score coefficients the representative data set was used to define the circle in the pc1/pc2 plane in which the majority of teoae signals recorded in normal hearing subjects will fall . in this study , simulated signals , as well as the measured ones , were projected and analyzed into the reference circle of the pc1/pc2 plane defined by the 118 signals . then , a reference parameter , termed the 2-dimensional radius ( rad2d ) was introduced . rad2d is defined in the pc1/pc2 plane as the euclidean distance of 1 point representing a teoae signal from the plane origin : to determine the amount of damage , the ear model was used to simulate different levels of cochlear damage by zeroing a growing number of cochlear partitions , and the relation between the rad2d value and the number of silenced partitions was evaluated . the obtained relation allowed correlating the rad2d value obtained for all the measured signals with the entity of cochlear damage . a statistical analysis was performed on 15 parameters : a ) the age of the participants , b ) the whole waveform reproducibility , c ) 6 audiometric levels ( at 250 hz , 500 hz , 1 khz , 2 khz , 4 khz and 8 khz ) , d ) 3 rqa parameters ( rec , det , ent ) , e ) 3 pca coordinates ( pc1 , pc2 and pc3 ) , and f ) the rad2d parameter calculated from the first 2 principal components pc1 and pc2 ( see eq . ( 2 ) ) . a 2-sample t - test comparison was made between normal and ih ears for each variable separately ; moreover , a multi - variable anova test was also conducted , comprising both wwr and the rqa parameters . all the statistical data were calculated using a commercial statistical package ( systat , v.10.2 , chicago , il ) . the present study was carried out on 29 ears from 21 subjects ( average age = 38.78.12 years ) . of these , 14 ears were from 11 impaired - hearing ( ih ) subjects , and the remaining 15 ears were from 10 subjects with normal hearing . ih subjects ( average age = 41.36.5 years ) were included in this study if pure - tone audiometry revealed a 4 khz notch > 30 db hl . the normal hearing subjects ( normal group , average age = 36.39.0 years ) had no significant noise exposure . none of the subjects had any background of hereditary hearing loss or ear infections , and were fully informed of the aim , design , and clinical applications of this study . the study was approved by the ethics committee of the catholic university , rome , italy , and the investigations were performed in accordance with the principles of the declaration of helsinki . all subjects underwent a baseline hearing evaluation : otoscopic examination and 226-hz probe tone tympanometry were used to exclude middle ear pathologies . conventional pure tone audiometric testing was carried out in a soundproof room and the pure - tone thresholds ( ptt ) of each ear were measured at 0.25 , 0.50 , 1 , 2 , 4 , and 8 khz with a clinical audiometer ( ac-40 , interacoustics co , assens , denmark ) . classification of patients was obtained by using a ptt value at 4 khz . the otodynamic analyzer ( ilo92 , otodynamics ltd , hatfield , u.k . ) was used to record the teoae signals by inserting a sgs - type general purpose teoae probe into the external ear canal . the teoae recordings were carried out in a standard hospital room , corresponding to the usual clinical setting for these measurements . the automated differential non - linear test paradigm was used : the stimulus was characterized by a train of 4 clicks , 3 with the same amplitude and polarity , followed by a fourth with a 3-fold amplitude and opposite polarity with respect to the preceding ones . the responses were obtained , evaluating an average among 260 stimuli trains ( 1040 clicks ) stored into 2 different buffers ( a and b ) for a total of 2080 clicks . the value of the automatically computed correlation or reproducibility between the 2 obtained waveforms ( a and b ) of an oae signal is termed repro or whole waveform reproducibility ( wwr ) ( pearson correlation coefficient * 100 ) . according to the clinical settings , when the wwr value is > 70% and the snr is higher than 3 db , the signal is considered as physiological ( ilo92 test is pass ) , whereas a wwr value < 70% indicates the presence of a possible hearing malfunction ( ilo92 test is failed ) . in the measured data , the mean snr given by the ilo92 was 13.04 db for the normal ears and 3.96 db for the ih ears . the examined ears were classified as normal hearing or ih ears according to their pure tone thresholds at 1 , 2 , 4 and 8 khz . in particular , the subjects with a pure - tone threshold value greater than 30 db hl at 4 khz were assigned to the ih group even though their wwr value was high ( wwr 70% and pass at ilo92 test ) . to optimize and validate the procedure , teoae have been numerically simulated using an electronic model of the whole ear , originally introduced by guigure and woodland ( 1994 ) and used in other teoaes analysis and other studies . the electronic model is based on the electro - acoustic analogy , where the acoustic mass of cochlea fluids is represented by inductors ; the acoustic resistance , mass and stiffness of the basilar membrane are represented by shunt resistors , inductors and capacitors , respectively ; and the ohc active processes are represented by non - linear voltage sources ( details of the circuit are in ) . in the electronic model of the ear , the cochlea is divided into several sections ( termed partitions ) from the base to the apex . sixty - four partitions are used to segment the cochlea , so that , being the whole uncoiled cochlea 3.5 cm long ( comprising a total of about 12000 cells ) , each partition corresponds to a cochlear portion of 0.0547 cm and about 188 cells . in order to simulate the noise - induced ohc damage corresponding to the experimental ih condition , the suppression of cochlear amplification is obtained by zeroing the gain of some of the generators within the cochlear partitions . in particular , to establish different damage levels , a localized suppression of an increasing number ( ranged from n=2 to n=10 ) of adjacent cochlear amplifiers is applied close to the characteristic frequency ( cf ) of 4 khz . to simulate teoae measurements , the input voltage was a rectangular pulse 80 s in duration , with an amplitude corresponding to a stimulus level of 80 pe db spl . finally , to eliminate the initial ringing , the first 2.8 ms of the simulated teoae signals were excluded from the successive analysis . the wwr value of teoaes in ih subjects with a 4 khz notch can be > 70% as in normal hearing subjects . consequently , the usual waveform descriptive parameters are of no assistance in detecting cochlear pathology . in the present study , teoae signals were analyzed by rqa and pca techniques to reduce inter - subject variability and the dynamical structure of signals were investigated rather than signal - level differences . rqa introduces few parameters ( named rqa descriptors ) descriptive of the global complexity of a signal and computed from the so called recurrence plot [ 3233 ] . to build the recurrence plot , the time behavior of the original signal was represented by a series of 512 points equally spaced in time ( e.g. , { a1 a2 . a 512 } where ai represents the value of the signal corresponding to the i - th time position ) . then , the series was arranged in successive columns ( the columns number is defined by the embedding dimension parameter n ) , each - one obtained by applying a delay in time ( lag parameter ) to the original sequence , thus creating an embedding matrix , as in equation 1 . finally , the recurrence plot was built , drawing a black dot ( named recurrent point ) in the represented space if the distance between the corresponding rows ( the distance between the j - th and the ( jth+1 ) row is i=1512(aj , i - aj+1,i)2 ) of the embedding matrix was lower than a fixed value ( radius ) . in the obtained plot , the horizontal and vertical axes represented the relative position of the 512 points into the teoae waveform . rqa descriptors were then calculated on the basis of the number and the location of dots in the recurrence plot . in particular , percent of recurrence ( rec ) is the percentage of recurrence points in a recurrent plot ; percent of determinism ( det ) is the percentage of recurrence points which form diagonal lines and it indicates the degree of deterministic structure of the signal ; entropy ( ent ) is the shannon entropy of the probability distribution of the diagonal line lengths and is linked to the richness of deterministic structure . the presence of horizontal and vertical lines in the recurrence plot shows that part of the considered signal matches closely with a later sequence . based on experience gained in previous research , the delay in the embedding procedure ( lag ) was set to 1 ; the number of the embedding matrix columns ( embedding dimension , n ) was 10 ; and the cut - off distance ( radius ) was 15 . pca is a common statistical technique which , exploiting the presence of correlations among the original variables , provides the possibility to reduce the starting data set dimension without consistent loss of information and with a separation of the different and independent features characterizing the data set . as a consequence , pca describes the original data set with a lower number of new parameters , termed main components . as shown in a previous study , if a set of at least 70 representative teoae signals is chosen as input to the rqa - pca analysis , the first 2 main components ( pc1 , pc2 ) explain more than 90% of the total variability in the data set . moreover , having pc1 and pc2 , by construction , zero mean and standard deviation equal to 1 , if a set of teoae signals from normal ears are studied , 96% of them will fall within a circle centered in the origin of the pc1/pc2 plane , and with a radius equal to 2 ( reference circle ) . in the present study , the pc1/pc2 plane was defined starting from a representative data set made by 118 signals measured from normal hearing subjects , in agreement with results of a previous study . the explained variability of the representative data set used was 90.02% , 6.29% , and 3.68% for pc1 , pc2 , and pc3 , respectively . the correlation between rqa parameters and pcs is described by the factor score coefficients ( data not shown ) . the representative data set was used to define the circle in the pc1/pc2 plane in which the majority of teoae signals recorded in normal hearing subjects will fall . in this study , simulated signals , as well as the measured ones , were projected and analyzed into the reference circle of the pc1/pc2 plane defined by the 118 signals . then , a reference parameter , termed the 2-dimensional radius ( rad2d ) was introduced . rad2d is defined in the pc1/pc2 plane as the euclidean distance of 1 point representing a teoae signal from the plane origin : to determine the amount of damage , the ear model was used to simulate different levels of cochlear damage by zeroing a growing number of cochlear partitions , and the relation between the rad2d value and the number of silenced partitions was evaluated . the obtained relation allowed correlating the rad2d value obtained for all the measured signals with the entity of cochlear damage . a statistical analysis was performed on 15 parameters : a ) the age of the participants , b ) the whole waveform reproducibility , c ) 6 audiometric levels ( at 250 hz , 500 hz , 1 khz , 2 khz , 4 khz and 8 khz ) , d ) 3 rqa parameters ( rec , det , ent ) , e ) 3 pca coordinates ( pc1 , pc2 and pc3 ) , and f ) the rad2d parameter calculated from the first 2 principal components pc1 and pc2 ( see eq . ( 2 ) ) . a 2-sample t - test comparison was made between normal and ih ears for each variable separately ; moreover , a multi - variable anova test was also conducted , comprising both wwr and the rqa parameters . all the statistical data were calculated using a commercial statistical package ( systat , v.10.2 , chicago , il ) . the subjects age distributions of the 2 groups was not statistically different ( t - test p=0.095 ) . the recorded teoaes in all ears of the normal group and in 50% ears ( 7/14 ) of the ih group had the wwr 70% . in figure 1 , the audiogram levels ( mean values and standard deviations ) are shown for the normal hearing ears ( bold line ) and for the ih ears ( thin line ) . at 0.5 , 1 , 2 khz no differences were found on the contrary , the audiometric thresholds belonging to ih ears were characterized by a deep and narrow notch close to 4 khz , that in all patients was higher than 30 db hl with a mean value of 59.617.4 db hl , as compared to the flat shape of the normal hearing audiograms . the standard deviations demonstrated a large variability in ih subjects at 4 khz and 8 khz . figure 2 shows the recurrence plots ( figure 2a , b ) of measured teoae signals ( figure 2c , d ) from a normal hearing subject ( left side of figure 2 ) and from an ih subject ( right side of figure 2 ) . comparing figure 2a and b , it is clear that recurrence plots distinguish between normal hearing and ih teoaes , especially in terms of a reduction in the deterministic structure . the profile of the traveling wave along the basilar membrane ( bm volume velocity ) simulated by the electronic model of the ear is shown in figure 3 for normal hearing ( bold line ) and ih ( shaded grey line ) ear . in this case , ih was simulated by zeroing the generators gain in 2 consecutive cochlear partitions working close to 4 khz . bm volume velocity was obtained by using a pure tone stimulus at 4 khz and by plotting the current values evaluated in each cochlear section at a fixed instant of time ( 8.9 ms in figure 3 ) . the behavior evaluated for the normal hearing ear ( bold line in figure 3 ) is the typical bm volume velocity reported by several authors . the black circle symbols placed upon the plot represent the value of the characteristic frequencies ( cf ) corresponding to the different positions along the cochlea . comparing the bm volume velocity obtained in normal and ih ears , it is possible to observe that the largest difference is around the frequency of 4 khz , as expected ( see arrow and grey circles ) . table 1 reports the mean and standard deviation of the most significant parameters of teoae signals and rqa / pca analysis . in particular , the first 4 rows of table 1 refer to measured teoae from normal ( row 1 ) and ih ears ( row 2 ) , showing the values of the considered parameters and the results of the statistical analyses between normal and ih data . moreover , the last 2 rows show the corresponding data referring to simulated teoaes . in this case , the ih simulated signal ( last row ) was obtained by zeroing the generation gain of 6 partitions around the 4 khz frequency . from table 1 it can be seen that , on average , the percent of determinism ( det ) , entropy ( ent ) , and wwr significantly distinguished normal from ih measured signals ( see p of t - test , table 1 ) . moreover , comparing rows 1 and 5 ( normal ) and rows 2 and 6 ( ih ) , it is clear that the rqa parameters of the simulated signals were close to the mean values calculated for real ears , validating the use of the ear model to study teoae characteristics both in normal and in ih ears . rqa parameters were calculated both for measured and simulated signals , whereas wwr and ptt values were available only for measured signals . figure 4 shows the rad2d values calculated for simulated signals with an increasing number of silenced partitions ( crosses ) . the dashed line in figure 4 corresponds to the value of rad2d ( 0.21 ) obtained by considering the mean values of the rqa parameters obtained from the 15 normal hearing ears , whereas the black bold line corresponds to the value of rad2d ( 1.78 ) obtained from the mean values of the rqa parameters of the 14 ih ears . figure 4 shows that rad2d grew approximately linearly with the number of zeroed partitions ( amount of damage ) , so that it is possible to represent the scaling of rad2d with the number of silenced partitions with a line having a slope of 0.199 and intercept at 0.204 ( r= 0.883 ) ( thin line ) . it is important to note that the intercept value ( 0.20 ) was very close to the rad2d value ( 0.21 ) evaluated from the mean values of the rqa parameters calculated on teoaes of normal ears . from the linear behavior extrapolated by the rad2d simulated data it is possible to calculate the amount of damage corresponding to each measured signal , as the number of hypothetical silenced partitions . in this way , since each partition corresponds to a specific portion of the uncoiled cochlea and to a specific number of ohc cells , a descriptor of ohc integrity ( rad2d value ) is obtained . in table 2 the ptt at 4 khz , wwr , and the newly proposed rad2d parameter the rad2d calculated for each signal of the 2 groups ( normal and ih ) was statistically different ( t - test p=0.004 , the statistical power is 89.1% for =5% and =50% , and the minimum difference between means that can be observed with these samples is 0.35 ) . table 2 demonstrates that 9 ih signals ( 64% ) had a rad2d value > 1.78 , which was the mean value of the ih group . by defining an ear as normal if it scored a rad2d < 1.78 , only 1 ear resulted in a false positive . from a clinical point of view , it is important to note that 2 ears ( t2 and t3 ) were considered as normal from the standard ilo92 analysis because they had a wwr value > 70% ( false negative ) , whereas they had a rad2d value > 1.78 , which corresponded to a damaged ear according to the newly proposed parameter . from pearson s correlation values obtained within the ih group , it is shown that : a ) subjects age did not correlate with either the audiometric levels or with rqa parameters ( ie , the results were not biased by an aging effect ) ; b ) audiometric parameters did not correlate with rqa values ( i.e. , audiometric and rqa parameters carry independent information ) ; and c ) the new parameter rad2d correlated negatively with wwr ( r=0.698 ) , confirming that at large distances from the origin of the reference plane pc1/pc2 , the auditory functionality was poorer . the subjects age distributions of the 2 groups was not statistically different ( t - test p=0.095 ) . the recorded teoaes in all ears of the normal group and in 50% ears ( 7/14 ) of the ih group had the wwr 70% . in figure 1 , the audiogram levels ( mean values and standard deviations ) are shown for the normal hearing ears ( bold line ) and for the ih ears ( thin line ) . at 0.5 , 1 , 2 khz no differences were found on the contrary , the audiometric thresholds belonging to ih ears were characterized by a deep and narrow notch close to 4 khz , that in all patients was higher than 30 db hl with a mean value of 59.617.4 db hl , as compared to the flat shape of the normal hearing audiograms . the standard deviations demonstrated a large variability in ih subjects at 4 khz and 8 khz . figure 2 shows the recurrence plots ( figure 2a , b ) of measured teoae signals ( figure 2c , d ) from a normal hearing subject ( left side of figure 2 ) and from an ih subject ( right side of figure 2 ) . comparing figure 2a and b , it is clear that recurrence plots distinguish between normal hearing and ih teoaes , especially in terms of a reduction in the deterministic structure . the profile of the traveling wave along the basilar membrane ( bm volume velocity ) simulated by the electronic model of the ear is shown in figure 3 for normal hearing ( bold line ) and ih ( shaded grey line ) ear . in this case , ih was simulated by zeroing the generators gain in 2 consecutive cochlear partitions working close to 4 khz . bm volume velocity was obtained by using a pure tone stimulus at 4 khz and by plotting the current values evaluated in each cochlear section at a fixed instant of time ( 8.9 ms in figure 3 ) . the behavior evaluated for the normal hearing ear ( bold line in figure 3 ) is the typical bm volume velocity reported by several authors . the black circle symbols placed upon the plot represent the value of the characteristic frequencies ( cf ) corresponding to the different positions along the cochlea . comparing the bm volume velocity obtained in normal and ih ears , it is possible to observe that the largest difference is around the frequency of 4 khz , as expected ( see arrow and grey circles ) . table 1 reports the mean and standard deviation of the most significant parameters of teoae signals and rqa / pca analysis . in particular , the first 4 rows of table 1 refer to measured teoae from normal ( row 1 ) and ih ears ( row 2 ) , showing the values of the considered parameters and the results of the statistical analyses between normal and ih data . moreover , the last 2 rows show the corresponding data referring to simulated teoaes . in this case , the ih simulated signal ( last row ) was obtained by zeroing the generation gain of 6 partitions around the 4 khz frequency . from table 1 it can be seen that , on average , the percent of determinism ( det ) , entropy ( ent ) , and wwr significantly distinguished normal from ih measured signals ( see p of t - test , table 1 ) . moreover , comparing rows 1 and 5 ( normal ) and rows 2 and 6 ( ih ) , it is clear that the rqa parameters of the simulated signals were close to the mean values calculated for real ears , validating the use of the ear model to study teoae characteristics both in normal and in ih ears . rqa parameters were calculated both for measured and simulated signals , whereas wwr and ptt values were available only for measured signals . figure 4 shows the rad2d values calculated for simulated signals with an increasing number of silenced partitions ( crosses ) . the dashed line in figure 4 corresponds to the value of rad2d ( 0.21 ) obtained by considering the mean values of the rqa parameters obtained from the 15 normal hearing ears , whereas the black bold line corresponds to the value of rad2d ( 1.78 ) obtained from the mean values of the rqa parameters of the 14 ih ears . figure 4 shows that rad2d grew approximately linearly with the number of zeroed partitions ( amount of damage ) , so that it is possible to represent the scaling of rad2d with the number of silenced partitions with a line having a slope of 0.199 and intercept at 0.204 ( r= 0.883 ) ( thin line ) . it is important to note that the intercept value ( 0.20 ) was very close to the rad2d value ( 0.21 ) evaluated from the mean values of the rqa parameters calculated on teoaes of normal ears . from the linear behavior extrapolated by the rad2d simulated data it is possible to calculate the amount of damage corresponding to each measured signal , as the number of hypothetical silenced partitions . in this way , since each partition corresponds to a specific portion of the uncoiled cochlea and to a specific number of ohc cells , a descriptor of ohc integrity ( rad2d value ) is obtained . in table 2 the ptt at 4 khz , wwr , and the newly proposed rad2d parameter the rad2d calculated for each signal of the 2 groups ( normal and ih ) was statistically different ( t - test p=0.004 , the statistical power is 89.1% for =5% and =50% , and the minimum difference between means that can be observed with these samples is 0.35 ) . table 2 demonstrates that 9 ih signals ( 64% ) had a rad2d value > 1.78 , which was the mean value of the ih group . by defining an ear as normal if it scored a rad2d < 1.78 , only 1 ear resulted in a false positive . from a clinical point of view , it is important to note that 2 ears ( t2 and t3 ) were considered as normal from the standard ilo92 analysis because they had a wwr value > 70% ( false negative ) , whereas they had a rad2d value > 1.78 , which corresponded to a damaged ear according to the newly proposed parameter . from pearson s correlation values obtained within the ih group , it is shown that : a ) subjects age did not correlate with either the audiometric levels or with rqa parameters ( ie , the results were not biased by an aging effect ) ; b ) audiometric parameters did not correlate with rqa values ( i.e. , audiometric and rqa parameters carry independent information ) ; and c ) the new parameter rad2d correlated negatively with wwr ( r=0.698 ) , confirming that at large distances from the origin of the reference plane pc1/pc2 , the auditory functionality was poorer . screening protocols are fundamental for the early diagnosis of hearing impairment , and neither audiogram tests nor the different teoae analyses provide sufficiently reliable diagnostic tools . the primary aim of this preliminary study was to propose an additional global parameter , rad2d , useful in clinical applications to improve the early detection of cochlear damage in ih subjects with notches by way of a rqa / pca procedure . specifically , our results can be summarized as follows : a ) the combined use of the rqa and pca methods discriminated ih teoae from normal ones ; b ) the rad2d was validated by adopting an ear model where the cochlea was assumed to be an active electronic circuit that includes ohc active processes ; and c ) the new parameter , rad2d , by correlating the waveform dynamic structure of teoaes with the amount of damage , defined the topology and quantified the hearing impairment . in this study rqa was applied without any previous mathematical assumptions and data manipulation ( as in fourier transform ) to both the simulated and recorded teoae . rqa method is particularly suited for signals which exhibit clear frequency dispersion over time , as well as signals characterized by high sensitivity and low specificity such as teoaes . rqa parameters obtained from simulated signals were close to the mean values calculated for real ears , validating the use of the ear model to study teoae characteristics both in normal and in ih ears . in particular , the specific selection of ih ears with a notch at 4 khz larger than 30 db hl , evidenced a decrease of determinism in the considered signals ( i.e. , det and ent values obtained for the ih signals were lower than those obtained for normal signals ) . this loss of determinism can be considered a loss of deterministic structure , because the number of recurrence points was invariant ( see statistic tests in table 1 ) . in this context , it is important to observe that the loss of structure of the signals depends on the number of active resonators which have lost their normal function , and that the same loss occurs both in simulated ih and measured ih signals . in fact , the amount of damage assessment is necessarily based on surviving signal frequency components rather than on missing ones . the pca technique was then applied on the obtained rqa descriptors . on the basis of the above considerations , the combination of the rqa / pca techniques , focusing on the dynamic regime of teoae , reduced individual variability which , masking the relevant features of the signals , is the most relevant problem occurring in standard teoae technique , thus confirming previous results . the embedding procedure allowed expansion of the monodimensional signal into multidimensional space and produced few global parameters directly deriving from signal fine structure . the possibility to realize the rqa analysis on time windowing will in the future allow direct comparison with the results of time - frequency analysis . the proposed parameter , rad2d , may have a remarkable clinical interest in fact , rad2d not only discriminated between normal and ih teoaes , but it scaled on the basis of the integrity of cochlear amplification in ih ears . this result is of importance because ih ears are often characterized by a limited loss at a specific frequency ( e.g. , a deep notch at 4 khz ) and ih teoaes , often exhibiting high wwr , can be confused with ones from normal ears , as was the case in some of the patients examined in the present study ( e.g. , t2 , t3 in table 2 ) . a first validation of the proposed parameter was obtained by studying simulated signals . the evidence that rad2d value grew approximately linearly with the number of silenced amplifiers in the ear model confirmed that rad2d quantitatively described the level of damage of ih . a second validation was obtained by comparing measured and simulated signals . by taking the rad2d value of 1.78 as a threshold value ( the intercept of the bold horizontal line of figure 4 ) the rad2d was able to identify 2 more cases with respect to wwr data of ih subjects ; the mean normal rad2d value ( the intercept of the dashed horizontal line of figure 4 ) was very close to the value predicted by the model simulating a normal ear . in this study we used as wwr threshold value 70% , which is usually indicated in the clinical practice and some researchers used a different threshold value ( i.e. wwr 80% ) when performing teoae measurements in an unavoidable low frequency noisy environment ( nursery environment ) . if such a value was used to evaluate the ih ears of the present study , 9 ears out of 14 would have been recognized as ih ears by the wwr value . however , the rad2d procedure would still be able to identify 2 more ih ears , thus confirming that the use of the 2 global parameters , wwr and rad2d , can improve the early detection of hearing impairments through teoae analysis . with reference to the value chosen as threshold for the new rad2d parameter , the 1.78 value was obtained as the mean value from the teoae recorded in the ih ears . this is a preliminary study , and further research with a higher number of ih ears should be conducted . other investigators studied hearing loss and compared teoae with dpoae , finding a greater sensibility of teoaes with respect to dpoaes , especially using frequency - specific bands parameters . in general , however , a detailed assessment on the acoustic deficit can not be achieved by a single approach . thus , the use of the parameter obtained in the present research in conjunction with the teoae standard procedure can improve the percentage of successfully detected early stages hearing impairments that is fundamental for preventive measures to be applied . further implications concern the possibility of using teoae techniques reliably to monitor hearing function in noise - exposed and elderly populations . these implications are fundamental for prevention ( e.g. , early use of antioxidant agents ) , for design of new hearing protector devices , and hearing - aid fitting strategies . although preliminary , the findings of this study illustrate a global parameter to be used for the clinical screening of hearing impairments . being a global parameter , the rad2d value could be easily used in conjunction with the reproducibility parameter , an improvement over using the many other parameters such as teoae reproducibility in frequency bands . the analysis of the teoaes of 4 khz notch subjects , as chosen in this study , establishes the validity of the rad2d parameter , as evidenced by : a ) combining wwr value with rad2d unrevealed 2 hearing impaired cases ; b ) the ear model , used to simulate the 4 khz damage and to reproduce the corresponding teoaes , has validated the measured human signals ; and c ) the entity and location of the damage has been obtained . finally , the benefit of this additional parameter could consist in the introduction into the clinical practice of a numeric value similar to a pass and fail criterion to assess cochlear function in different ear pathologies such as nihl , ototoxicity , age - related hearing loss and other sensorineural hearing deficits .

summarybackgroundto identify a parameter to distinguish normal hearing from hearing impairment in the early stages . the parameter was obtained from transient - evoked otoacoustic emissions ( teoaes ) , overcoming the limitations of the usually adopted waveform descriptive parameters which may fail in standard clinical screenings.material/methodsaudiometric examinations and teoae analysis were conducted on 15 normal ears and on 14 hearing - impaired ears that exhibited an audiometric notch around 4 khz . teoae signals were analyzed through a multivariate technique to filter out the individual variability and to highlight the dynamic structure of the signals . the new parameter ( named radius 2-dimension rad2d ) was defined and evaluated for simulated teoae signals modeling a different amount of hearing impairment.resultsaudiometric examinations indicated 14 ears as impaired - hearing ( ih ) , while the teoae ilo92 whole reproducibility parameter ( wwr ) indicated as ih 7 signals out of 14 ( 50% ) . the proposed new parameter indicated as ih 9 signals out of 14 ( 64% ) , reducing the number of false negative cases of wwr.conclusionsin this preliminary study there is evidence that the new parameter rad2d defines the topology and the quantification of the damage in the inner ear . the proposed protocol can be useful in hearing screenings to identify hearing impairments much earlier than conventional pure tone audiometry and teoae pass / fail test .

Background Material and Methods Signal detection and clinical evaluation The ear model RQA-PCA of TEOAE signals Statistical analysis of TEOAE waveform parameters Results Comparison between TEOAEs recorded in normal and IH ears TEOAE simulation in the electronic model and comparison with measured TEOAEs RAD2D parameter evaluated in simulated and measured TEOAEs Discussion Conclusions

nihl does not simultaneously affect all frequencies pure tone audiometry usually shows a gradual progression and the first impairment begins around the frequency of 4 khz , with a typical notch at 4 khz and , at times , 6 khz frequencies . oaes are sounds generated in the inner ear by the activity of the cochlear amplifiers ; they can be recorded when the auditory periphery is solicited by an external acoustic stimuli , such as brief click ( i.e. taken together , constraints to the early diagnosis are patient s co - operation in conventional audiometry and subject s variance in teoaes ; both issues can be controlled and reduced by mathematical procedures . a quantification parameter , called henceforth 2-dimensional radius ( rad2d ) , is extracted from the teoae waveform by means of a non - linear signal analysis process referred to as recurrence quantification analysis ( rqa ) along with principal component analysis ( pca ) . rqa is an efficient and relatively simple tool in the analysis of many physiological signals and accounts for the deterministic structure of teoae signals ; pca is a multivariate statistical analysis successfully applied to teoaes signals since it minimizes redundant information . the proposed rad2d parameter is a global parameter ( comparable to whole waveform reproducibility - wwr ) , but , as will be shown , the embedding procedure in rqa tools permits observation of the fine structure of the waveform . furthermore , this mathematical quantification of teoae s fine structure reduces the individual variability and can be easily applied to detect hearing impairments in the early pathologic stage . in the present study , the mathematical procedure is applied to teoaes recorded in normal ears and in ears exhibiting a notch at 4 khz greater than 30 db hl ( hearing level ) , and the obtained parameter is compared with the conventional analyses ( audiometry and teoae whole waveform reproducibility ) . to optimize and validate the procedure , an electronic model of the ear , developed by gigure and woodland and applied in a previous work , is used to simulate teoae signals both in normal ears and in ears with different amounts of cochlear damage . taken together , the aims of this paper are : 1 ) to compare the measured teoae among normal and ih ears ; 2 ) to simulate normal and ih ears by using the electronic model of the ear with a selective damage corresponding to a 4 khz notch ; 3 ) to obtain simulated teoae and to compare them with the measured ones ; 4 ) to estimate the rad2d parameter in the electronic model and to compare the obtained rad2d values with those evaluated in the normal and ih recorded teoae . of these , 14 ears were from 11 impaired - hearing ( ih ) subjects , and the remaining 15 ears were from 10 subjects with normal hearing . conventional pure tone audiometric testing was carried out in a soundproof room and the pure - tone thresholds ( ptt ) of each ear were measured at 0.25 , 0.50 , 1 , 2 , 4 , and 8 khz with a clinical audiometer ( ac-40 , interacoustics co , assens , denmark ) . in the measured data , the mean snr given by the ilo92 was 13.04 db for the normal ears and 3.96 db for the ih ears . finally , to eliminate the initial ringing , the first 2.8 ms of the simulated teoae signals were excluded from the successive analysis . the wwr value of teoaes in ih subjects with a 4 khz notch can be > 70% as in normal hearing subjects . in the present study , teoae signals were analyzed by rqa and pca techniques to reduce inter - subject variability and the dynamical structure of signals were investigated rather than signal - level differences . finally , the recurrence plot was built , drawing a black dot ( named recurrent point ) in the represented space if the distance between the corresponding rows ( the distance between the j - th and the ( jth+1 ) row is i=1512(aj , i - aj+1,i)2 ) of the embedding matrix was lower than a fixed value ( radius ) . in the obtained plot , the horizontal and vertical axes represented the relative position of the 512 points into the teoae waveform . rqa descriptors were then calculated on the basis of the number and the location of dots in the recurrence plot . based on experience gained in previous research , the delay in the embedding procedure ( lag ) was set to 1 ; the number of the embedding matrix columns ( embedding dimension , n ) was 10 ; and the cut - off distance ( radius ) was 15 . moreover , having pc1 and pc2 , by construction , zero mean and standard deviation equal to 1 , if a set of teoae signals from normal ears are studied , 96% of them will fall within a circle centered in the origin of the pc1/pc2 plane , and with a radius equal to 2 ( reference circle ) . in the present study , the pc1/pc2 plane was defined starting from a representative data set made by 118 signals measured from normal hearing subjects , in agreement with results of a previous study . rad2d is defined in the pc1/pc2 plane as the euclidean distance of 1 point representing a teoae signal from the plane origin : to determine the amount of damage , the ear model was used to simulate different levels of cochlear damage by zeroing a growing number of cochlear partitions , and the relation between the rad2d value and the number of silenced partitions was evaluated . a statistical analysis was performed on 15 parameters : a ) the age of the participants , b ) the whole waveform reproducibility , c ) 6 audiometric levels ( at 250 hz , 500 hz , 1 khz , 2 khz , 4 khz and 8 khz ) , d ) 3 rqa parameters ( rec , det , ent ) , e ) 3 pca coordinates ( pc1 , pc2 and pc3 ) , and f ) the rad2d parameter calculated from the first 2 principal components pc1 and pc2 ( see eq . of these , 14 ears were from 11 impaired - hearing ( ih ) subjects , and the remaining 15 ears were from 10 subjects with normal hearing . the value of the automatically computed correlation or reproducibility between the 2 obtained waveforms ( a and b ) of an oae signal is termed repro or whole waveform reproducibility ( wwr ) ( pearson correlation coefficient * 100 ) . finally , to eliminate the initial ringing , the first 2.8 ms of the simulated teoae signals were excluded from the successive analysis . in the present study , teoae signals were analyzed by rqa and pca techniques to reduce inter - subject variability and the dynamical structure of signals were investigated rather than signal - level differences . finally , the recurrence plot was built , drawing a black dot ( named recurrent point ) in the represented space if the distance between the corresponding rows ( the distance between the j - th and the ( jth+1 ) row is i=1512(aj , i - aj+1,i)2 ) of the embedding matrix was lower than a fixed value ( radius ) . in the obtained plot , the horizontal and vertical axes represented the relative position of the 512 points into the teoae waveform . rqa descriptors were then calculated on the basis of the number and the location of dots in the recurrence plot . based on experience gained in previous research , the delay in the embedding procedure ( lag ) was set to 1 ; the number of the embedding matrix columns ( embedding dimension , n ) was 10 ; and the cut - off distance ( radius ) was 15 . as shown in a previous study , if a set of at least 70 representative teoae signals is chosen as input to the rqa - pca analysis , the first 2 main components ( pc1 , pc2 ) explain more than 90% of the total variability in the data set . moreover , having pc1 and pc2 , by construction , zero mean and standard deviation equal to 1 , if a set of teoae signals from normal ears are studied , 96% of them will fall within a circle centered in the origin of the pc1/pc2 plane , and with a radius equal to 2 ( reference circle ) . rad2d is defined in the pc1/pc2 plane as the euclidean distance of 1 point representing a teoae signal from the plane origin : to determine the amount of damage , the ear model was used to simulate different levels of cochlear damage by zeroing a growing number of cochlear partitions , and the relation between the rad2d value and the number of silenced partitions was evaluated . a statistical analysis was performed on 15 parameters : a ) the age of the participants , b ) the whole waveform reproducibility , c ) 6 audiometric levels ( at 250 hz , 500 hz , 1 khz , 2 khz , 4 khz and 8 khz ) , d ) 3 rqa parameters ( rec , det , ent ) , e ) 3 pca coordinates ( pc1 , pc2 and pc3 ) , and f ) the rad2d parameter calculated from the first 2 principal components pc1 and pc2 ( see eq . at 0.5 , 1 , 2 khz no differences were found on the contrary , the audiometric thresholds belonging to ih ears were characterized by a deep and narrow notch close to 4 khz , that in all patients was higher than 30 db hl with a mean value of 59.617.4 db hl , as compared to the flat shape of the normal hearing audiograms . bm volume velocity was obtained by using a pure tone stimulus at 4 khz and by plotting the current values evaluated in each cochlear section at a fixed instant of time ( 8.9 ms in figure 3 ) . in particular , the first 4 rows of table 1 refer to measured teoae from normal ( row 1 ) and ih ears ( row 2 ) , showing the values of the considered parameters and the results of the statistical analyses between normal and ih data . in this case , the ih simulated signal ( last row ) was obtained by zeroing the generation gain of 6 partitions around the 4 khz frequency . moreover , comparing rows 1 and 5 ( normal ) and rows 2 and 6 ( ih ) , it is clear that the rqa parameters of the simulated signals were close to the mean values calculated for real ears , validating the use of the ear model to study teoae characteristics both in normal and in ih ears . the dashed line in figure 4 corresponds to the value of rad2d ( 0.21 ) obtained by considering the mean values of the rqa parameters obtained from the 15 normal hearing ears , whereas the black bold line corresponds to the value of rad2d ( 1.78 ) obtained from the mean values of the rqa parameters of the 14 ih ears . figure 4 shows that rad2d grew approximately linearly with the number of zeroed partitions ( amount of damage ) , so that it is possible to represent the scaling of rad2d with the number of silenced partitions with a line having a slope of 0.199 and intercept at 0.204 ( r= 0.883 ) ( thin line ) . it is important to note that the intercept value ( 0.20 ) was very close to the rad2d value ( 0.21 ) evaluated from the mean values of the rqa parameters calculated on teoaes of normal ears . in table 2 the ptt at 4 khz , wwr , and the newly proposed rad2d parameter the rad2d calculated for each signal of the 2 groups ( normal and ih ) was statistically different ( t - test p=0.004 , the statistical power is 89.1% for =5% and =50% , and the minimum difference between means that can be observed with these samples is 0.35 ) . table 2 demonstrates that 9 ih signals ( 64% ) had a rad2d value > 1.78 , which was the mean value of the ih group . , audiometric and rqa parameters carry independent information ) ; and c ) the new parameter rad2d correlated negatively with wwr ( r=0.698 ) , confirming that at large distances from the origin of the reference plane pc1/pc2 , the auditory functionality was poorer . at 0.5 , 1 , 2 khz no differences were found on the contrary , the audiometric thresholds belonging to ih ears were characterized by a deep and narrow notch close to 4 khz , that in all patients was higher than 30 db hl with a mean value of 59.617.4 db hl , as compared to the flat shape of the normal hearing audiograms . bm volume velocity was obtained by using a pure tone stimulus at 4 khz and by plotting the current values evaluated in each cochlear section at a fixed instant of time ( 8.9 ms in figure 3 ) . in particular , the first 4 rows of table 1 refer to measured teoae from normal ( row 1 ) and ih ears ( row 2 ) , showing the values of the considered parameters and the results of the statistical analyses between normal and ih data . in this case , the ih simulated signal ( last row ) was obtained by zeroing the generation gain of 6 partitions around the 4 khz frequency . moreover , comparing rows 1 and 5 ( normal ) and rows 2 and 6 ( ih ) , it is clear that the rqa parameters of the simulated signals were close to the mean values calculated for real ears , validating the use of the ear model to study teoae characteristics both in normal and in ih ears . the dashed line in figure 4 corresponds to the value of rad2d ( 0.21 ) obtained by considering the mean values of the rqa parameters obtained from the 15 normal hearing ears , whereas the black bold line corresponds to the value of rad2d ( 1.78 ) obtained from the mean values of the rqa parameters of the 14 ih ears . figure 4 shows that rad2d grew approximately linearly with the number of zeroed partitions ( amount of damage ) , so that it is possible to represent the scaling of rad2d with the number of silenced partitions with a line having a slope of 0.199 and intercept at 0.204 ( r= 0.883 ) ( thin line ) . it is important to note that the intercept value ( 0.20 ) was very close to the rad2d value ( 0.21 ) evaluated from the mean values of the rqa parameters calculated on teoaes of normal ears . in this way , since each partition corresponds to a specific portion of the uncoiled cochlea and to a specific number of ohc cells , a descriptor of ohc integrity ( rad2d value ) is obtained . in table 2 the ptt at 4 khz , wwr , and the newly proposed rad2d parameter the rad2d calculated for each signal of the 2 groups ( normal and ih ) was statistically different ( t - test p=0.004 , the statistical power is 89.1% for =5% and =50% , and the minimum difference between means that can be observed with these samples is 0.35 ) . table 2 demonstrates that 9 ih signals ( 64% ) had a rad2d value > 1.78 , which was the mean value of the ih group . , audiometric and rqa parameters carry independent information ) ; and c ) the new parameter rad2d correlated negatively with wwr ( r=0.698 ) , confirming that at large distances from the origin of the reference plane pc1/pc2 , the auditory functionality was poorer . screening protocols are fundamental for the early diagnosis of hearing impairment , and neither audiogram tests nor the different teoae analyses provide sufficiently reliable diagnostic tools . the primary aim of this preliminary study was to propose an additional global parameter , rad2d , useful in clinical applications to improve the early detection of cochlear damage in ih subjects with notches by way of a rqa / pca procedure . specifically , our results can be summarized as follows : a ) the combined use of the rqa and pca methods discriminated ih teoae from normal ones ; b ) the rad2d was validated by adopting an ear model where the cochlea was assumed to be an active electronic circuit that includes ohc active processes ; and c ) the new parameter , rad2d , by correlating the waveform dynamic structure of teoaes with the amount of damage , defined the topology and quantified the hearing impairment . rqa parameters obtained from simulated signals were close to the mean values calculated for real ears , validating the use of the ear model to study teoae characteristics both in normal and in ih ears . in this context , it is important to observe that the loss of structure of the signals depends on the number of active resonators which have lost their normal function , and that the same loss occurs both in simulated ih and measured ih signals . on the basis of the above considerations , the combination of the rqa / pca techniques , focusing on the dynamic regime of teoae , reduced individual variability which , masking the relevant features of the signals , is the most relevant problem occurring in standard teoae technique , thus confirming previous results . , a deep notch at 4 khz ) and ih teoaes , often exhibiting high wwr , can be confused with ones from normal ears , as was the case in some of the patients examined in the present study ( e.g. a first validation of the proposed parameter was obtained by studying simulated signals . the evidence that rad2d value grew approximately linearly with the number of silenced amplifiers in the ear model confirmed that rad2d quantitatively described the level of damage of ih . by taking the rad2d value of 1.78 as a threshold value ( the intercept of the bold horizontal line of figure 4 ) the rad2d was able to identify 2 more cases with respect to wwr data of ih subjects ; the mean normal rad2d value ( the intercept of the dashed horizontal line of figure 4 ) was very close to the value predicted by the model simulating a normal ear . if such a value was used to evaluate the ih ears of the present study , 9 ears out of 14 would have been recognized as ih ears by the wwr value . however , the rad2d procedure would still be able to identify 2 more ih ears , thus confirming that the use of the 2 global parameters , wwr and rad2d , can improve the early detection of hearing impairments through teoae analysis . with reference to the value chosen as threshold for the new rad2d parameter , the 1.78 value was obtained as the mean value from the teoae recorded in the ih ears . thus , the use of the parameter obtained in the present research in conjunction with the teoae standard procedure can improve the percentage of successfully detected early stages hearing impairments that is fundamental for preventive measures to be applied . although preliminary , the findings of this study illustrate a global parameter to be used for the clinical screening of hearing impairments . the analysis of the teoaes of 4 khz notch subjects , as chosen in this study , establishes the validity of the rad2d parameter , as evidenced by : a ) combining wwr value with rad2d unrevealed 2 hearing impaired cases ; b ) the ear model , used to simulate the 4 khz damage and to reproduce the corresponding teoaes , has validated the measured human signals ; and c ) the entity and location of the damage has been obtained .

dissecting the interactions between bacterial adhesins and host surface receptors at the host - pathogen interface is an essential step towards our understanding of the underlying mechanisms driving bacterial adhesion . ultimately , this will help us to identify strategies to interfere with these processes during infections . in conducting such studies , we often face a dilemma : biochemical and biophysical analysis of the molecular mechanisms of adhesin binding requires their separation from other cell wall components , which may interfere with adhesion events . on the other hand , the use of recombinant soluble proteins over - simplifies the adhesion event , by disregarding protein anchoring in the bacterial cell wall and multivalency of binding achieved through bacterial surface display . equally , from the host cell s perspective , encountering and binding to single adhesin molecules does not always have the same impact in terms of plasma membrane organization , membrane fluidity and receptor clustering and this , ultimately , makes it difficult to evaluate the impact of adhesion on host cellular signaling and the outcome of bacteria - host interactions . recently a method was devised , whereby recombinant purified adhesins or adhesin fragments are directionally and covalently coupled to polymer particles similar in size to bacteria , thus mimicking bacterial surface display . this approach has been used on a range of different adhesins , from gram - negative multivalent adhesion molecules ( mams ) , to gram - positive adhesins including staphylococcus aureus fibronectin binding protein ( fnbpa ) and streptococcus pyogenes f1 . this method has allowed the dissection of adhesin fragments important for host cell binding , identification of host surface receptors and determination of their behavior on the host cell surface , while taking both binding affinity and avidity into consideration . additionally , this approach has been used to investigate the efficacy of immobilized adhesins and derivatives as inhibitors of host - pathogen interactions . it has been demonstrated that surface coupled derivatives of the vibrio parahaemolyticus multivalent adhesion molecule ( mam ) 7 can be used to attenuate a range of bacterial infections in vitro , including those caused by multidrug - resistant pathogens such as acinetobacter baumannii and methicillin - resistant s. aureus ( mrsa ) . herein , we describe different chemistries which can be used to immobilize adhesins to commercially available functionalized polystyrene particles . due to the wide array of available surface functionalities , labels and particle sizes , these are a useful scaffold for the production of bacteriomimetic materials to investigate adhesin - host interactions . we further describe methods for the initial characterization of the coupling reaction , and for the calculation of important properties of the resulting materials . finally , the use of bead - coupled adhesins as competitive inhibitors of in vitro bacterial infections is discussed as an example of their application , as well as the future scope and limitations of this approach . thiol - amine directional coupling note : this protocol is suitable for coupling of cysteine containing proteins to amine - functionalized polymer beads , using sulfosuccinimidyl 4-(p - maleimidophenyl)butyrate ( sulfo - smpb ) as cross - linking agent ( figure 1 ) . cross - linking strategy used for directional thiol - amine coupling of proteins to polymer beads . preparation of reagents : prepare pbs ( 100 mm sodium phosphate , 150 mm nacl , ph 7.0 ) and autoclave.prepare a 100x stock ( 0.5 m or 287 mg / ml in pbs ) of tcep ( tris(2-carboxyethyl)phosphine ) immediately before use.prepare a 5x stock ( 10 mm or 4.58 mg / ml in dh2o ) of sulfo - smpb immediately prior to use.prepare a 10x stock ( 500 mm or 88 mg / ml in pbs , ph 7.0 ) of cysteine immediately before use . bead activation : mix the bead suspension by gently inverting and transfer the required amount of bead suspension ( e.g. , 12 ml ) into a sterile 1.5 ml tube containing 1 ml sterile pbs , ph 7.0.gently pipette up and down to wash the beads and pellet by centrifugation in a microcentrifuge ( 2 min at 16,000 x g).carefully remove the supernatant with a pipette and discard . resuspend the bead pellet in 1 ml of fresh sterile pbs and repeat the washing step . resuspend the bead pellet in 0.8 ml of pbs.add 200 ml of freshly prepared 10 mm sulfo - smpb , to give a final concentration of 2 mm.incubate the bead suspension for 1 hr at 25 c on a rotating wheel . protein reduction : during the incubation period of the activation step , prepare the protein for the following coupling step so it can be immediately added to the activated beads . note : although this reduction step is not always required for gst ( glutathione s - transferase ) fusion proteins , it is recommended to ensure a high coupling efficiency . check the protein concentration , and adjust it to the final concentration required for the coupling reaction . note : 6 mm protein in pbs , and a volume of 1 ml are usually used.add tcep stock to give a final concentration of 5 mm . note : the reaction mixture can be directly used for the following coupling reaction.retain a small amount ( a few ml ) of the protein solution for determining the protein concentration and calculation of coupling efficiency ( see section 2 ) . protein coupling step : pellet the activated beads by centrifugation ( 2 min , 16,000 x g in a microcentrifuge ) , and wash the pellet once in 1 ml of fresh sterile pbs.resuspend the pellet in the prepared protein solution ( e.g. , 1 ml ) , to give the desired protein concentration . note : the protein concentration during the coupling step will depend on the average coupling efficiency ( see section 2 for determination of the coupling efficiency ) and desired coupling density ( as calculated in 1.1.4.3 ) . the efficiency is approximately 85% and the desired final concentration 5 mm in the 10x bead suspension , so protein concentration during the coupling step should be approximately 6 mm.calculate the coupling density using the following formula : where c coupling density [ number of protein molecules / nm ] , protein conc protein concentration [ mg / ml ] , protein mw protein molecular weight [ da ] , bead conc bead concentration [ number of beads / l ] , d bead diameter [ nm ] avogadro s numberuse the coupling density to calculate the average ligand spacing : incubate the protein - bead suspension for 2 hr at 25 c on a rotating wheel . the reaction can be carried out at 4 c o / n.deactivate remaining activated groups on the beads by adding cysteine stock to a final concentration of 50 mm and incubate the suspension for 30 min at 25 c on a rotating wheel . pellet the beads by centrifugation ( 2 min , 16,000 x g in a microcentrifuge).keep the supernatant for determining the protein concentration and calculation of coupling efficiency ( see section 2).wash the bead pellet twice with 1 ml pbs and resuspend in 1 ml fresh pbs to give the final product . note : the above protocol will typically give 1 ml of coupled protein , at a final concentration of 5 mm protein , which can be used as a 10x stock for subsequent experiments ( see section 3).to proceed to section 3 of the protocol , work with 100 ml / ml of bead stock , or at a final concentration of 500 nm protein . note : a good starting point for this procedure will be 210 beads / ml , resulting in an average coupling density of 310 proteins / nm or an average spacing of 57 nm on a bead of 2 mm diameter . preparation of reagents : prepare pbs ( 100 mm sodium phosphate , 150 mm nacl , ph 7.0 ) and autoclave.prepare a 100x stock ( 0.5 m or 287 mg / ml in pbs ) of tcep ( tris(2-carboxyethyl)phosphine ) immediately before use.prepare a 5x stock ( 10 mm or 4.58 mg / ml in dh2o ) of sulfo - smpb immediately prior to use.prepare a 10x stock ( 500 mm or 88 mg / ml in pbs , ph 7.0 ) of cysteine immediately before use . prepare pbs ( 100 mm sodium phosphate , 150 mm nacl , ph 7.0 ) and autoclave . prepare a 100x stock ( 0.5 m or 287 mg / ml in pbs ) of tcep ( tris(2-carboxyethyl)phosphine ) immediately before use . prepare a 5x stock ( 10 mm or 4.58 mg / ml in dh2o ) of sulfo - smpb immediately prior to use . prepare a 10x stock ( 500 mm or 88 mg / ml in pbs , ph 7.0 ) of cysteine immediately before use . bead activation : mix the bead suspension by gently inverting and transfer the required amount of bead suspension ( e.g. , 12 ml ) into a sterile 1.5 ml tube containing 1 ml sterile pbs , ph 7.0.gently pipette up and down to wash the beads and pellet by centrifugation in a microcentrifuge ( 2 min at 16,000 x g).carefully remove the supernatant with a pipette and discard . resuspend the bead pellet in 1 ml of fresh sterile pbs and repeat the washing step . resuspend the bead pellet in 0.8 ml of pbs.add 200 ml of freshly prepared 10 mm sulfo - smpb , to give a final concentration of 2 mm.incubate the bead suspension for 1 hr at 25 c on a rotating wheel . mix the bead suspension by gently inverting and transfer the required amount of bead suspension ( e.g. , 12 ml ) into a sterile 1.5 ml tube containing 1 ml sterile pbs , ph 7.0 . gently pipette up and down to wash the beads and pellet by centrifugation in a microcentrifuge ( 2 min at 16,000 x g ) . carefully resuspend the bead pellet in 1 ml of fresh sterile pbs and repeat the washing step . add 200 ml of freshly prepared 10 mm sulfo - smpb , to give a final concentration of 2 mm . incubate the bead suspension for 1 hr at 25 c on a rotating wheel . protein reduction : during the incubation period of the activation step , prepare the protein for the following coupling step so it can be immediately added to the activated beads . note : although this reduction step is not always required for gst ( glutathione s - transferase ) fusion proteins , it is recommended to ensure a high coupling efficiency . check the protein concentration , and adjust it to the final concentration required for the coupling reaction . note : 6 mm protein in pbs , and a volume of 1 ml are usually used.add tcep stock to give a final concentration of 5 mm . note : the reaction mixture can be directly used for the following coupling reaction.retain a small amount ( a few ml ) of the protein solution for determining the protein concentration and calculation of coupling efficiency ( see section 2 ) . during the incubation period of the activation step , prepare the protein for the following coupling step so it can be immediately added to the activated beads . note : although this reduction step is not always required for gst ( glutathione s - transferase ) fusion proteins , it is recommended to ensure a high coupling efficiency . check the protein concentration , and adjust it to the final concentration required for the coupling reaction . note : 6 mm protein in pbs , and a volume of 1 ml are usually used.add tcep stock to give a final concentration of 5 mm . note : the reaction mixture can be directly used for the following coupling reaction.retain a small amount ( a few ml ) of the protein solution for determining the protein concentration and calculation of coupling efficiency ( see section 2 ) . check the protein concentration , and adjust it to the final concentration required for the coupling reaction . note : 6 mm protein in pbs , and a volume of 1 ml are usually used . retain a small amount ( a few ml ) of the protein solution for determining the protein concentration and calculation of coupling efficiency ( see section 2 ) . protein coupling step : pellet the activated beads by centrifugation ( 2 min , 16,000 x g in a microcentrifuge ) , and wash the pellet once in 1 ml of fresh sterile pbs.resuspend the pellet in the prepared protein solution ( e.g. , 1 ml ) , to give the desired protein concentration . note : the protein concentration during the coupling step will depend on the average coupling efficiency ( see section 2 for determination of the coupling efficiency ) and desired coupling density ( as calculated in 1.1.4.3 ) . the efficiency is approximately 85% and the desired final concentration 5 mm in the 10x bead suspension , so protein concentration during the coupling step should be approximately 6 mm.calculate the coupling density using the following formula : where c coupling density [ number of protein molecules / nm ] , protein conc protein concentration [ mg / ml ] , protein mw protein molecular weight [ da ] , bead conc bead concentration [ number of beads / l ] , d bead diameter [ nm ] avogadro s numberuse the coupling density to calculate the average ligand spacing : incubate the protein - bead suspension for 2 hr at 25 c on a rotating wheel . in these cases , the reaction can be carried out at 4 c o / n.deactivate remaining activated groups on the beads by adding cysteine stock to a final concentration of 50 mm and incubate the suspension for 30 min at 25 c on a rotating wheel . pellet the beads by centrifugation ( 2 min , 16,000 x g in a microcentrifuge).keep the supernatant for determining the protein concentration and calculation of coupling efficiency ( see section 2).wash the bead pellet twice with 1 ml pbs and resuspend in 1 ml fresh pbs to give the final product . note : the above protocol will typically give 1 ml of coupled protein , at a final concentration of 5 mm protein , which can be used as a 10x stock for subsequent experiments ( see section 3).to proceed to section 3 of the protocol , work with 100 ml / ml of bead stock , or at a final concentration of 500 nm protein . note : a good starting point for this procedure will be 210 beads / ml , resulting in an average coupling density of 310 proteins / nm or an average spacing of 57 nm on a bead of 2 mm diameter . pellet the activated beads by centrifugation ( 2 min , 16,000 x g in a microcentrifuge ) , and wash the pellet once in 1 ml of fresh sterile pbs . resuspend the pellet in the prepared protein solution ( e.g. , 1 ml ) , to give the desired protein concentration . note : the protein concentration during the coupling step will depend on the average coupling efficiency ( see section 2 for determination of the coupling efficiency ) and desired coupling density ( as calculated in 1.1.4.3 ) . the efficiency is approximately 85% and the desired final concentration 5 mm in the 10x bead suspension , so protein concentration during the coupling step should be approximately 6 mm . calculate the coupling density using the following formula : where c coupling density [ number of protein molecules / nm ] , protein conc protein concentration [ mg / ml ] , protein mw protein molecular weight [ da ] , bead conc bead concentration [ number of beads / l ] , d bead diameter [ nm ] avogadro s number use the coupling density to calculate the average ligand spacing : incubate the protein - bead suspension for 2 hr at 25 c on a rotating wheel . the reaction can be carried out at 4 c o / n . deactivate remaining activated groups on the beads by adding cysteine stock to a final concentration of 50 mm and incubate the suspension for 30 min at 25 c on a rotating wheel . pellet the beads by centrifugation ( 2 min , 16,000 x g in a microcentrifuge ) . keep the supernatant for determining the protein concentration and calculation of coupling efficiency ( see section 2 ) . wash the bead pellet twice with 1 ml pbs and resuspend in 1 ml fresh pbs to give the final product . note : the above protocol will typically give 1 ml of coupled protein , at a final concentration of 5 mm protein , which can be used as a 10x stock for subsequent experiments ( see section 3 ) . to proceed to section 3 of the protocol , work with 100 ml / ml of bead stock , or at a final concentration of 500 nm protein . note : a good starting point for this procedure will be 210 beads / ml , resulting in an average coupling density of 310 proteins / nm or an average spacing of 57 nm on a bead of 2 mm diameter . thiol - carboxy directional coupling note : this protocol is suitable for coupling cysteine containing proteins to carboxyl - functionalized polystyrene beads . the carboxyl moiety is first activated using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide ( edc)/ n - hydroxysuccinimide ( nhs ) , amine modified and then cross - linked using sulfo - smpb ( figure 2 ) . cross - linking strategy used for directional thiol - carboxyl coupling of proteins to polymer beads . carboxylated polystyrene beads are first activated with edc and modified with nhs to form a semi - stable nhs - ester . subsequent amine - coupling of ethylenediamine results in a free amine group , which reacts with sulfo - smpb . maleimide activated beads can then react with free cysteines to directionally couple proteins to beads . preparation of reagents : prepare pbs ( 100 mm sodium phosphate , 150 mm nacl , ph 7.0 ) and autoclave.prepare a 100x stock ( 0.5 m or 287 mg / ml in pbs , ) of tcep immediately before use.prepare a 10x stock ( 20 mm , or 4 mg / ml in pbs ) of edc ( 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide ) immediately before use.prepare a 10x stock ( 50 mm , or 6 mg / ml in pbs ) of nhs ( n - hydroxysuccinimide ) immediately prior to use.prepare a 5x stock ( 10 mm or 4.58 mg / ml in dh2o ) of sulfo - smpb immediately prior to use.prepare a 10x stock ( 500 mm or 88 mg / ml in pbs , ph 7.0 ) of cysteine immediately before use . bead activation : mix bead suspension by gently inverting and transfer the required amount of bead suspension ( e.g. , 12 ml ) into a sterile 1.5 ml tube containing 1 ml sterile pbs , ph 7.0.gently pipette up and down to wash the beads and pellet by centrifugation in a microcentrifuge ( 2 min at 16,000 x g).carefully remove the supernatant with a pipette and discard.resuspend the bead pellet in 1 ml of fresh sterile pbs and repeat the washing step.resuspend the bead pellet in 0.8 ml of pbs.add 100 ml of 10x edc stock ( 2 mm final concentration ) and immediately 100 ml of 10x nhs stock solution ( 5 mm final concentration ) to the bead suspension.incubate the bead suspension for 30 min at 25 c on a rotating wheel.wash beads once in 1 ml of pbs and resuspend in 0.8 ml fresh sterile pbs.add 200 ml ethylenediamine , and incubate the bead suspension for 1 hr at 25 c on a rotating wheel.wash beads once with 1 ml pbs and resuspend the beads in 0.8 ml of fresh pbs.proceed from section 1.1.2.4 ( bead activation with sulfo - smpb ) as described under section 1.1.2 and follow the rest of the protocol described in section 1.1 ( thiol - amine directional coupling ) . perform protein preparation and protein coupling steps in a manner identical to those described in section 1.1 . 75% ) compared to section 1.1 , therefore adjust the initial protein concentration accordingly to achieve the same coupling density . preparation of reagents : prepare pbs ( 100 mm sodium phosphate , 150 mm nacl , ph 7.0 ) and autoclave.prepare a 100x stock ( 0.5 m or 287 mg / ml in pbs , ) of tcep immediately before use.prepare a 10x stock ( 20 mm , or 4 mg / ml in pbs ) of edc ( 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide ) immediately before use.prepare a 10x stock ( 50 mm , or 6 mg / ml in pbs ) of nhs ( n - hydroxysuccinimide ) immediately prior to use.prepare a 5x stock ( 10 mm or 4.58 mg / ml in dh2o ) of sulfo - smpb immediately prior to use.prepare a 10x stock ( 500 mm or 88 mg / ml in pbs , ph 7.0 ) of cysteine immediately before use . prepare pbs ( 100 mm sodium phosphate , 150 mm nacl , ph 7.0 ) and autoclave . prepare a 100x stock ( 0.5 m or 287 mg / ml in pbs , ) of tcep immediately before use . prepare a 10x stock ( 20 mm , or 4 mg / ml in pbs ) of edc ( 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide ) immediately before use . prepare a 10x stock ( 50 mm , or 6 mg / ml in pbs ) of nhs ( n - hydroxysuccinimide ) immediately prior to use . prepare a 5x stock ( 10 mm or 4.58 mg / ml in dh2o ) of sulfo - smpb immediately prior to use . prepare a 10x stock ( 500 mm or 88 mg / ml in pbs , ph 7.0 ) of cysteine immediately before use . bead activation : mix bead suspension by gently inverting and transfer the required amount of bead suspension ( e.g. , 12 ml ) into a sterile 1.5 ml tube containing 1 ml sterile pbs , ph 7.0.gently pipette up and down to wash the beads and pellet by centrifugation in a microcentrifuge ( 2 min at 16,000 x g).carefully remove the supernatant with a pipette and discard.resuspend the bead pellet in 1 ml of fresh sterile pbs and repeat the washing step.resuspend the bead pellet in 0.8 ml of pbs.add 100 ml of 10x edc stock ( 2 mm final concentration ) and immediately 100 ml of 10x nhs stock solution ( 5 mm final concentration ) to the bead suspension.incubate the bead suspension for 30 min at 25 c on a rotating wheel.wash beads once in 1 ml of pbs and resuspend in 0.8 ml fresh sterile pbs.add 200 ml ethylenediamine , and incubate the bead suspension for 1 hr at 25 c on a rotating wheel.wash beads once with 1 ml pbs and resuspend the beads in 0.8 ml of fresh pbs.proceed from section 1.1.2.4 ( bead activation with sulfo - smpb ) as described under section 1.1.2 and follow the rest of the protocol described in section 1.1 ( thiol - amine directional coupling ) . perform protein preparation and protein coupling steps in a manner identical to those described in section 1.1 . 75% ) compared to section 1.1 , therefore adjust the initial protein concentration accordingly to achieve the same coupling density . mix bead suspension by gently inverting and transfer the required amount of bead suspension ( e.g. , 12 ml ) into a sterile 1.5 ml tube containing 1 ml sterile pbs , ph 7.0 . gently pipette up and down to wash the beads and pellet by centrifugation in a microcentrifuge ( 2 min at 16,000 x g ) . carefully remove the supernatant with a pipette and discard . resuspend the bead pellet in 1 ml of fresh sterile pbs and repeat the washing step . add 100 ml of 10x edc stock ( 2 mm final concentration ) and immediately 100 ml of 10x nhs stock solution ( 5 mm final concentration ) to the bead suspension . incubate the bead suspension for 30 min at 25 c on a rotating wheel . wash beads once in 1 ml of pbs and resuspend in 0.8 ml fresh sterile pbs . add 200 ml ethylenediamine , and incubate the bead suspension for 1 hr at 25 c on a rotating wheel . wash beads once with 1 ml pbs and resuspend the beads in 0.8 ml of fresh pbs . proceed from section 1.1.2.4 ( bead activation with sulfo - smpb ) as described under section 1.1.2 and follow the rest of the protocol described in section 1.1 ( thiol - amine directional coupling ) . perform protein preparation and protein coupling steps in a manner identical to those described in section 1.1 . 75% ) compared to section 1.1 , therefore adjust the initial protein concentration accordingly to achieve the same coupling density . note : to determine protein concentration , use bradford reagent and colorimetric assays as follows : gently invert bradford reagent to ensure homogeneity of the reagent . using a 10 mg / ml bsa stock solution , prepare protein standards covering concentrations from 0.1 to 1.5 mg / ml bsa in buffer . prepare enough wells for all samples , protein standards and negative controls ( buffer only ) . add 5 ml of protein sample ( protein standards or buffer , for the negative control ) to the reagent in the 96-well plate . generate a standard curve of bsa concentration versus a595 nm and use this to determine protein concentrations in initial and supernatant samples . calculate the concentration of coupled protein as follows : calculate the coupling efficiency as : preparation : seed 1 ml per well of hela cells at a concentration of 150,000 cells / ml into a 24-well plate the day before the competition assay , to allow cells to reach approximately 80% confluency prior to starting the experiment.set up each experimental condition in triplicate . include wells for negative controls ( no bacteria added during competition assays ) , positive controls ( control beads coupled to fusion - tag only added during competition assays ) and lysis controls ( for cytotoxicity experiments).inoculate a 5 ml marine lb ( mlb ) culture with a fresh colony of v. parahaemolyticus and grow o / n at 30 c , shaking.prepare sufficient bead - coupled mam , as described in section 1 . seed 1 ml per well of hela cells at a concentration of 150,000 cells / ml into a 24-well plate the day before the competition assay , to allow cells to reach approximately 80% confluency prior to starting the experiment . include wells for negative controls ( no bacteria added during competition assays ) , positive controls ( control beads coupled to fusion - tag only added during competition assays ) and lysis controls ( for cytotoxicity experiments ) . inoculate a 5 ml marine lb ( mlb ) culture with a fresh colony of v. parahaemolyticus and grow o / n at 30 c , shaking . competition assay : on the day of the competition experiment , measure the od600 of the bacterial culture.prepare infection media by diluting bacterial cultures into colorless dmem without additives , pre - warmed to 37 c , containing 10% v / v bead suspension ( either adhesin - coupled beads or control beads ) , to give an moi of 10 . note : for the above mentioned conditions ( 24-well plate , v. parahaemolyticus , moi 10 ) , the necessary volume of o / n culture to be added per well ( ml / ml ) is calculated as 3/od600 of the culture . for example , 1 ml of infection medium will typically contain 100 ml bead suspension , a few ml of bacterial culture and be made up to 1 ml with colorless , unsupplemented dmem.remove old medium from wells and wash cultured hela cells by adding 1 ml of sterile pbs pre - warmed to 37 c to each well.remove pbs and add 1 ml of infection medium per well . also set up controls , by adding solutions containing control beads and bacteria ( positive control ) or adhesin beads and no bacteria ( negative controls ) , or dmem containing 0.1% triton x-100 ( lysis control , only necessary for cytotoxicity measurements).incubate the plate in a tissue culture incubator at 37 c for the desired amount of time ( e.g. 4 hr for cytotoxicity measurements or 1 hr for adhesion measurements ) . note : both bacterial adhesion and cytotoxicity on host cells can be used as read - outs for the efficacy of inhibition . if the time points of cytotoxicity and adhesion measurements coincide , both assays may be performed using samples from the same well , since cytotoxicity is determined using the culture supernatant and attachment assays use samples derived from the remaining cell layer . on the day of the competition experiment , prepare infection media by diluting bacterial cultures into colorless dmem without additives , pre - warmed to 37 c , containing 10% v / v bead suspension ( either adhesin - coupled beads or control beads ) , to give an moi of 10 . note : for the above mentioned conditions ( 24-well plate , v. parahaemolyticus , moi 10 ) , the necessary volume of o / n culture to be added per well ( ml / ml ) is calculated as 3/od600 of the culture . for example , 1 ml of infection medium will typically contain 100 ml bead suspension , a few ml of bacterial culture and be made up to 1 ml with colorless , unsupplemented dmem . remove old medium from wells and wash cultured hela cells by adding 1 ml of sterile pbs pre - warmed to 37 c to each well . remove pbs and add 1 ml of infection medium per well . also set up controls , by adding solutions containing control beads and bacteria ( positive control ) or adhesin beads and no bacteria ( negative controls ) , or dmem containing 0.1% triton x-100 ( lysis control , only necessary for cytotoxicity measurements ) . incubate the plate in a tissue culture incubator at 37 c for the desired amount of time ( e.g. 4 note : both bacterial adhesion and cytotoxicity on host cells can be used as read - outs for the efficacy of inhibition . if the time points of cytotoxicity and adhesion measurements coincide , both assays may be performed using samples from the same well , since cytotoxicity is determined using the culture supernatant and attachment assays use samples derived from the remaining cell layer . cytotoxicity measurements : at indicated time points ( e.g. , 4 hr post infection ) , remove three times 200 ml from each 24-well and transfer to a 96-well plate.spin 96-well plates at 1,500 x g , 5 min and transfer 100 ml from each well into a fresh 96-well plate . add 100 ml of the media used during infection experiments to fresh wells of the 96-well plate in triplicate ( these will be used as blanks).carry out the lactate dehydrogenase ( ldh ) release assay using a ldh cytotoxicity detection kit and following the manufacturer s instructions . briefly , calculate the amount of reagent needed in increments of 25 ( e.g. , if 62 samples are to be measured , make up enough reagent mix for 75 etc.).for example , for 100 samples , mix 11.25 ml of reagent a with 250 l of reagent b. invert , do not vortex to avoid foaming.put the mixture in a reservoir to be able to pipet with a multi - channel pipette . add 100 l of reagent mix to each sample.incubate plate at rt and read the absorbance at 490 nm on a plate reader at 10 , 20 , 30 min.analyze the data set for which the absorbance of the lysis control sample is high but still within the linear range of the plate reader ( typically , 2 - 3 absorbance units).express results as % cytotoxicity , using the following formula for conversion : at indicated time points ( e.g. , 4 hr post infection ) , remove three times 200 ml from each 24-well and transfer to a 96-well plate . spin 96-well plates at 1,500 x g , 5 min and transfer 100 ml from each well into a fresh 96-well plate . add 100 ml of the media used during infection experiments to fresh wells of the 96-well plate in triplicate ( these will be used as blanks ) . carry out the lactate dehydrogenase ( ldh ) release assay using a ldh cytotoxicity detection kit and following the manufacturer s instructions . briefly , calculate the amount of reagent needed in increments of 25 ( e.g. , if 62 samples are to be measured , make up enough reagent mix for 75 etc.).for example , for 100 samples , mix 11.25 ml of reagent a with 250 l of reagent b. invert , do not vortex to avoid foaming.put the mixture in a reservoir to be able to pipet with a multi - channel pipette . add 100 l of reagent mix to each sample.incubate plate at rt and read the absorbance at 490 nm on a plate reader at 10 , 20 , 30 min.analyze the data set for which the absorbance of the lysis control sample is high but still within the linear range of the plate reader ( typically , 2 - 3 absorbance units).express results as % cytotoxicity , using the following formula for conversion : briefly , calculate the amount of reagent needed in increments of 25 ( e.g. , if 62 samples are to be measured , make up enough reagent mix for 75 etc . ) . for example , for 100 samples , mix 11.25 ml of reagent a with 250 l of reagent b. invert , do not vortex to avoid foaming . put the mixture in a reservoir to be able to pipet with a multi - channel pipette . incubate plate at rt and read the absorbance at 490 nm on a plate reader at 10 , 20 , 30 min . analyze the data set for which the absorbance of the lysis control sample is high but still within the linear range of the plate reader ( typically , 2 - 3 absorbance units ) . express results as % cytotoxicity , using the following formula for conversion : measurement of bacterial adhesion : at indicated time points ( e.g. , 1 hr post infection ) , remove media from the cell layer.thoroughly wash the cell layer with sterile , pre - warmed pbs ( at least 3 - 4 washes of 1 ml pbs each ) to remove any un - attached cells.lyse host cells by adding 1 ml of a sterile 1 % v / v triton x-100 solution in pbs per well . incubate the plate at 37 c for 5 min.pipette each sample up and down several times before transferring the contents of each well to separate 1.5 ml tubes . prepare 10-fold serial dilutions of samples into sterile pbs ( e.g. , use 100 ml of sample and 900 ml of pbs).plate 100 ml of each sample on mlb agar and spread using a cell spreader . note : for the described experimental setup , 10 or 10 fold dilutions give a suitable number of cfus.incubate plates at 37 c o / n and enumerate bacteria by colony counting . at indicated time points ( e.g. , 1 hr post infection ) , remove media from the cell layer . thoroughly wash the cell layer with sterile , pre - warmed pbs ( at least 3 - 4 washes of 1 ml pbs each ) to remove any un - attached cells . lyse host cells by adding 1 ml of a sterile 1 % v / v triton x-100 solution in pbs per well . pipette each sample up and down several times before transferring the contents of each well to separate 1.5 ml tubes . prepare 10-fold serial dilutions of samples into sterile pbs ( e.g. , use 100 ml of sample and 900 ml of pbs ) . plate 100 ml of each sample on mlb agar and spread using a cell spreader . optimize which dilutions to plate depending on the bacterial strains and time point . note : for the described experimental setup , 10 or 10 fold dilutions give a suitable number of cfus . thiol - amine directional coupling note : this protocol is suitable for coupling of cysteine containing proteins to amine - functionalized polymer beads , using sulfosuccinimidyl 4-(p - maleimidophenyl)butyrate ( sulfo - smpb ) as cross - linking agent ( figure 1 ) . cross - linking strategy used for directional thiol - amine coupling of proteins to polymer beads . preparation of reagents : prepare pbs ( 100 mm sodium phosphate , 150 mm nacl , ph 7.0 ) and autoclave.prepare a 100x stock ( 0.5 m or 287 mg / ml in pbs ) of tcep ( tris(2-carboxyethyl)phosphine ) immediately before use.prepare a 5x stock ( 10 mm or 4.58 mg / ml in dh2o ) of sulfo - smpb immediately prior to use.prepare a 10x stock ( 500 mm or 88 mg / ml in pbs , ph 7.0 ) of cysteine immediately before use . bead activation : mix the bead suspension by gently inverting and transfer the required amount of bead suspension ( e.g. , 12 ml ) into a sterile 1.5 ml tube containing 1 ml sterile pbs , ph 7.0.gently pipette up and down to wash the beads and pellet by centrifugation in a microcentrifuge ( 2 min at 16,000 x g).carefully remove the supernatant with a pipette and discard . resuspend the bead pellet in 1 ml of fresh sterile pbs and repeat the washing step . resuspend the bead pellet in 0.8 ml of pbs.add 200 ml of freshly prepared 10 mm sulfo - smpb , to give a final concentration of 2 mm.incubate the bead suspension for 1 hr at 25 c on a rotating wheel . protein reduction : during the incubation period of the activation step , prepare the protein for the following coupling step so it can be immediately added to the activated beads . note : although this reduction step is not always required for gst ( glutathione s - transferase ) fusion proteins , it is recommended to ensure a high coupling efficiency . check the protein concentration , and adjust it to the final concentration required for the coupling reaction . note : 6 mm protein in pbs , and a volume of 1 ml are usually used.add tcep stock to give a final concentration of 5 mm . note : the reaction mixture can be directly used for the following coupling reaction.retain a small amount ( a few ml ) of the protein solution for determining the protein concentration and calculation of coupling efficiency ( see section 2 ) . protein coupling step : pellet the activated beads by centrifugation ( 2 min , 16,000 x g in a microcentrifuge ) , and wash the pellet once in 1 ml of fresh sterile pbs.resuspend the pellet in the prepared protein solution ( e.g. , 1 ml ) , to give the desired protein concentration . note : the protein concentration during the coupling step will depend on the average coupling efficiency ( see section 2 for determination of the coupling efficiency ) and desired coupling density ( as calculated in 1.1.4.3 ) . the efficiency is approximately 85% and the desired final concentration 5 mm in the 10x bead suspension , so protein concentration during the coupling step should be approximately 6 mm.calculate the coupling density using the following formula : where c coupling density [ number of protein molecules / nm ] , protein conc protein concentration [ mg / ml ] , protein mw protein molecular weight [ da ] , bead conc bead concentration [ number of beads / l ] , d bead diameter [ nm ] avogadro s numberuse the coupling density to calculate the average ligand spacing : incubate the protein - bead suspension for 2 hr at 25 c on a rotating wheel . the reaction can be carried out at 4 c o / n.deactivate remaining activated groups on the beads by adding cysteine stock to a final concentration of 50 mm and incubate the suspension for 30 min at 25 c on a rotating wheel . pellet the beads by centrifugation ( 2 min , 16,000 x g in a microcentrifuge).keep the supernatant for determining the protein concentration and calculation of coupling efficiency ( see section 2).wash the bead pellet twice with 1 ml pbs and resuspend in 1 ml fresh pbs to give the final product . note : the above protocol will typically give 1 ml of coupled protein , at a final concentration of 5 mm protein , which can be used as a 10x stock for subsequent experiments ( see section 3).to proceed to section 3 of the protocol , work with 100 ml / ml of bead stock , or at a final concentration of 500 nm protein . note : a good starting point for this procedure will be 210 beads / ml , resulting in an average coupling density of 310 proteins / nm or an average spacing of 57 nm on a bead of 2 mm diameter . preparation of reagents : prepare pbs ( 100 mm sodium phosphate , 150 mm nacl , ph 7.0 ) and autoclave.prepare a 100x stock ( 0.5 m or 287 mg / ml in pbs ) of tcep ( tris(2-carboxyethyl)phosphine ) immediately before use.prepare a 5x stock ( 10 mm or 4.58 mg / ml in dh2o ) of sulfo - smpb immediately prior to use.prepare a 10x stock ( 500 mm or 88 mg / ml in pbs , ph 7.0 ) of cysteine immediately before use . prepare pbs ( 100 mm sodium phosphate , 150 mm nacl , ph 7.0 ) and autoclave . prepare a 100x stock ( 0.5 m or 287 mg / ml in pbs ) of tcep ( tris(2-carboxyethyl)phosphine ) immediately before use . prepare a 5x stock ( 10 mm or 4.58 mg / ml in dh2o ) of sulfo - smpb immediately prior to use . prepare a 10x stock ( 500 mm or 88 mg / ml in pbs , ph 7.0 ) of cysteine immediately before use . bead activation : mix the bead suspension by gently inverting and transfer the required amount of bead suspension ( e.g. , 12 ml ) into a sterile 1.5 ml tube containing 1 ml sterile pbs , ph 7.0.gently pipette up and down to wash the beads and pellet by centrifugation in a microcentrifuge ( 2 min at 16,000 x g).carefully remove the supernatant with a pipette and discard . resuspend the bead pellet in 1 ml of fresh sterile pbs and repeat the washing step . resuspend the bead pellet in 0.8 ml of pbs.add 200 ml of freshly prepared 10 mm sulfo - smpb , to give a final concentration of 2 mm.incubate the bead suspension for 1 hr at 25 c on a rotating wheel . mix the bead suspension by gently inverting and transfer the required amount of bead suspension ( e.g. , 12 ml ) into a sterile 1.5 ml tube containing 1 ml sterile pbs , ph 7.0 . gently pipette up and down to wash the beads and pellet by centrifugation in a microcentrifuge ( 2 min at 16,000 x g ) . carefully resuspend the bead pellet in 1 ml of fresh sterile pbs and repeat the washing step . add 200 ml of freshly prepared 10 mm sulfo - smpb , to give a final concentration of 2 mm . incubate the bead suspension for 1 hr at 25 c on a rotating wheel . protein reduction : during the incubation period of the activation step , prepare the protein for the following coupling step so it can be immediately added to the activated beads . note : although this reduction step is not always required for gst ( glutathione s - transferase ) fusion proteins , it is recommended to ensure a high coupling efficiency . check the protein concentration , and adjust it to the final concentration required for the coupling reaction . note : 6 mm protein in pbs , and a volume of 1 ml are usually used.add tcep stock to give a final concentration of 5 mm . note : the reaction mixture can be directly used for the following coupling reaction.retain a small amount ( a few ml ) of the protein solution for determining the protein concentration and calculation of coupling efficiency ( see section 2 ) . during the incubation period of the activation step , prepare the protein for the following coupling step so it can be immediately added to the activated beads . note : although this reduction step is not always required for gst ( glutathione s - transferase ) fusion proteins , it is recommended to ensure a high coupling efficiency . check the protein concentration , and adjust it to the final concentration required for the coupling reaction . note : 6 mm protein in pbs , and a volume of 1 ml are usually used.add tcep stock to give a final concentration of 5 mm . note : the reaction mixture can be directly used for the following coupling reaction.retain a small amount ( a few ml ) of the protein solution for determining the protein concentration and calculation of coupling efficiency ( see section 2 ) . check the protein concentration , and adjust it to the final concentration required for the coupling reaction . note : 6 mm protein in pbs , and a volume of 1 ml are usually used . retain a small amount ( a few ml ) of the protein solution for determining the protein concentration and calculation of coupling efficiency ( see section 2 ) . protein coupling step : pellet the activated beads by centrifugation ( 2 min , 16,000 x g in a microcentrifuge ) , and wash the pellet once in 1 ml of fresh sterile pbs.resuspend the pellet in the prepared protein solution ( e.g. , 1 ml ) , to give the desired protein concentration . note : the protein concentration during the coupling step will depend on the average coupling efficiency ( see section 2 for determination of the coupling efficiency ) and desired coupling density ( as calculated in 1.1.4.3 ) . the efficiency is approximately 85% and the desired final concentration 5 mm in the 10x bead suspension , so protein concentration during the coupling step should be approximately 6 mm.calculate the coupling density using the following formula : where c coupling density [ number of protein molecules / nm ] , protein conc protein concentration [ mg / ml ] , protein mw protein molecular weight [ da ] , bead conc bead concentration [ number of beads / l ] , d bead diameter [ nm ] avogadro s numberuse the coupling density to calculate the average ligand spacing : incubate the protein - bead suspension for 2 hr at 25 c on a rotating wheel . in these cases , the reaction can be carried out at 4 c o / n.deactivate remaining activated groups on the beads by adding cysteine stock to a final concentration of 50 mm and incubate the suspension for 30 min at 25 c on a rotating wheel . pellet the beads by centrifugation ( 2 min , 16,000 x g in a microcentrifuge).keep the supernatant for determining the protein concentration and calculation of coupling efficiency ( see section 2).wash the bead pellet twice with 1 ml pbs and resuspend in 1 ml fresh pbs to give the final product . note : the above protocol will typically give 1 ml of coupled protein , at a final concentration of 5 mm protein , which can be used as a 10x stock for subsequent experiments ( see section 3).to proceed to section 3 of the protocol , work with 100 ml / ml of bead stock , or at a final concentration of 500 nm protein . note : a good starting point for this procedure will be 210 beads / ml , resulting in an average coupling density of 310 proteins / nm or an average spacing of 57 nm on a bead of 2 mm diameter . pellet the activated beads by centrifugation ( 2 min , 16,000 x g in a microcentrifuge ) , and wash the pellet once in 1 ml of fresh sterile pbs . resuspend the pellet in the prepared protein solution ( e.g. , 1 ml ) , to give the desired protein concentration . note : the protein concentration during the coupling step will depend on the average coupling efficiency ( see section 2 for determination of the coupling efficiency ) and desired coupling density ( as calculated in 1.1.4.3 ) . the efficiency is approximately 85% and the desired final concentration 5 mm in the 10x bead suspension , so protein concentration during the coupling step should be approximately 6 mm . calculate the coupling density using the following formula : where c coupling density [ number of protein molecules / nm ] , protein conc protein concentration [ mg / ml ] , protein mw protein molecular weight [ da ] , bead conc bead concentration [ number of beads / l ] , d bead diameter [ nm ] avogadro s number use the coupling density to calculate the average ligand spacing : incubate the protein - bead suspension for 2 hr at 25 c on a rotating wheel . the reaction can be carried out at 4 c o / n . deactivate remaining activated groups on the beads by adding cysteine stock to a final concentration of 50 mm and incubate the suspension for 30 min at 25 c on a rotating wheel . pellet the beads by centrifugation ( 2 min , 16,000 x g in a microcentrifuge ) . keep the supernatant for determining the protein concentration and calculation of coupling efficiency ( see section 2 ) . wash the bead pellet twice with 1 ml pbs and resuspend in 1 ml fresh pbs to give the final product . note : the above protocol will typically give 1 ml of coupled protein , at a final concentration of 5 mm protein , which can be used as a 10x stock for subsequent experiments ( see section 3 ) . to proceed to section 3 of the protocol , work with 100 ml / ml of bead stock , or at a final concentration of 500 nm protein . note : a good starting point for this procedure will be 210 beads / ml , resulting in an average coupling density of 310 proteins / nm or an average spacing of 57 nm on a bead of 2 mm diameter . thiol - carboxy directional coupling note : this protocol is suitable for coupling cysteine containing proteins to carboxyl - functionalized polystyrene beads . the carboxyl moiety is first activated using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide ( edc)/ n - hydroxysuccinimide ( nhs ) , amine modified and then cross - linked using sulfo - smpb ( figure 2 ) . cross - linking strategy used for directional thiol - carboxyl coupling of proteins to polymer beads . carboxylated polystyrene beads are first activated with edc and modified with nhs to form a semi - stable nhs - ester . subsequent amine - coupling of ethylenediamine results in a free amine group , which reacts with sulfo - smpb . maleimide activated beads can then react with free cysteines to directionally couple proteins to beads . preparation of reagents : prepare pbs ( 100 mm sodium phosphate , 150 mm nacl , ph 7.0 ) and autoclave.prepare a 100x stock ( 0.5 m or 287 mg / ml in pbs , ) of tcep immediately before use.prepare a 10x stock ( 20 mm , or 4 mg / ml in pbs ) of edc ( 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide ) immediately before use.prepare a 10x stock ( 50 mm , or 6 mg / ml in pbs ) of nhs ( n - hydroxysuccinimide ) immediately prior to use.prepare a 5x stock ( 10 mm or 4.58 mg / ml in dh2o ) of sulfo - smpb immediately prior to use.prepare a 10x stock ( 500 mm or 88 mg / ml in pbs , ph 7.0 ) of cysteine immediately before use . bead activation : mix bead suspension by gently inverting and transfer the required amount of bead suspension ( e.g. , 12 ml ) into a sterile 1.5 ml tube containing 1 ml sterile pbs , ph 7.0.gently pipette up and down to wash the beads and pellet by centrifugation in a microcentrifuge ( 2 min at 16,000 x g).carefully remove the supernatant with a pipette and discard.resuspend the bead pellet in 1 ml of fresh sterile pbs and repeat the washing step.resuspend the bead pellet in 0.8 ml of pbs.add 100 ml of 10x edc stock ( 2 mm final concentration ) and immediately 100 ml of 10x nhs stock solution ( 5 mm final concentration ) to the bead suspension.incubate the bead suspension for 30 min at 25 c on a rotating wheel.wash beads once in 1 ml of pbs and resuspend in 0.8 ml fresh sterile pbs.add 200 ml ethylenediamine , and incubate the bead suspension for 1 hr at 25 c on a rotating wheel.wash beads once with 1 ml pbs and resuspend the beads in 0.8 ml of fresh pbs.proceed from section 1.1.2.4 ( bead activation with sulfo - smpb ) as described under section 1.1.2 and follow the rest of the protocol described in section 1.1 ( thiol - amine directional coupling ) . perform protein preparation and protein coupling steps in a manner identical to those described in section 1.1 . 75% ) compared to section 1.1 , therefore adjust the initial protein concentration accordingly to achieve the same coupling density . preparation of reagents : prepare pbs ( 100 mm sodium phosphate , 150 mm nacl , ph 7.0 ) and autoclave.prepare a 100x stock ( 0.5 m or 287 mg / ml in pbs , ) of tcep immediately before use.prepare a 10x stock ( 20 mm , or 4 mg / ml in pbs ) of edc ( 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide ) immediately before use.prepare a 10x stock ( 50 mm , or 6 mg / ml in pbs ) of nhs ( n - hydroxysuccinimide ) immediately prior to use.prepare a 5x stock ( 10 mm or 4.58 mg / ml in dh2o ) of sulfo - smpb immediately prior to use.prepare a 10x stock ( 500 mm or 88 mg / ml in pbs , ph 7.0 ) of cysteine immediately before use . prepare pbs ( 100 mm sodium phosphate , 150 mm nacl , ph 7.0 ) and autoclave . prepare a 100x stock ( 0.5 m or 287 mg / ml in pbs , ) of tcep immediately before use . prepare a 10x stock ( 20 mm , or 4 mg / ml in pbs ) of edc ( 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide ) immediately before use . prepare a 10x stock ( 50 mm , or 6 mg / ml in pbs ) of nhs ( n - hydroxysuccinimide ) immediately prior to use . prepare a 5x stock ( 10 mm or 4.58 mg / ml in dh2o ) of sulfo - smpb immediately prior to use . prepare a 10x stock ( 500 mm or 88 mg / ml in pbs , ph 7.0 ) of cysteine immediately before use . bead activation : mix bead suspension by gently inverting and transfer the required amount of bead suspension ( e.g. , 12 ml ) into a sterile 1.5 ml tube containing 1 ml sterile pbs , ph 7.0.gently pipette up and down to wash the beads and pellet by centrifugation in a microcentrifuge ( 2 min at 16,000 x g).carefully remove the supernatant with a pipette and discard.resuspend the bead pellet in 1 ml of fresh sterile pbs and repeat the washing step.resuspend the bead pellet in 0.8 ml of pbs.add 100 ml of 10x edc stock ( 2 mm final concentration ) and immediately 100 ml of 10x nhs stock solution ( 5 mm final concentration ) to the bead suspension.incubate the bead suspension for 30 min at 25 c on a rotating wheel.wash beads once in 1 ml of pbs and resuspend in 0.8 ml fresh sterile pbs.add 200 ml ethylenediamine , and incubate the bead suspension for 1 hr at 25 c on a rotating wheel.wash beads once with 1 ml pbs and resuspend the beads in 0.8 ml of fresh pbs.proceed from section 1.1.2.4 ( bead activation with sulfo - smpb ) as described under section 1.1.2 and follow the rest of the protocol described in section 1.1 ( thiol - amine directional coupling ) . perform protein preparation and protein coupling steps in a manner identical to those described in section 1.1 . 75% ) compared to section 1.1 , therefore adjust the initial protein concentration accordingly to achieve the same coupling density . mix bead suspension by gently inverting and transfer the required amount of bead suspension ( e.g. , 12 ml ) into a sterile 1.5 ml tube containing 1 ml sterile pbs , ph 7.0 . gently pipette up and down to wash the beads and pellet by centrifugation in a microcentrifuge ( 2 min at 16,000 x g ) . carefully remove the supernatant with a pipette and discard . resuspend the bead pellet in 1 ml of fresh sterile pbs and repeat the washing step . add 100 ml of 10x edc stock ( 2 mm final concentration ) and immediately 100 ml of 10x nhs stock solution ( 5 mm final concentration ) to the bead suspension . incubate the bead suspension for 30 min at 25 c on a rotating wheel . wash beads once in 1 ml of pbs and resuspend in 0.8 ml fresh sterile pbs . add 200 ml ethylenediamine , and incubate the bead suspension for 1 hr at 25 c on a rotating wheel . wash beads once with 1 ml pbs and resuspend the beads in 0.8 ml of fresh pbs . proceed from section 1.1.2.4 ( bead activation with sulfo - smpb ) as described under section 1.1.2 and follow the rest of the protocol described in section 1.1 ( thiol - amine directional coupling ) . perform protein preparation and protein coupling steps in a manner identical to those described in section 1.1 . 75% ) compared to section 1.1 , therefore adjust the initial protein concentration accordingly to achieve the same coupling density . note : to determine protein concentration , use bradford reagent and colorimetric assays as follows : gently invert bradford reagent to ensure homogeneity of the reagent . using a 10 mg / ml bsa stock solution , prepare protein standards covering concentrations from 0.1 to 1.5 mg / ml bsa in buffer . prepare enough wells for all samples , protein standards and negative controls ( buffer only ) . add 5 ml of protein sample ( protein standards or buffer , for the negative control ) to the reagent in the 96-well plate . generate a standard curve of bsa concentration versus a595 nm and use this to determine protein concentrations in initial and supernatant samples . calculate the concentration of coupled protein as follows : calculate the coupling efficiency as : preparation : seed 1 ml per well of hela cells at a concentration of 150,000 cells / ml into a 24-well plate the day before the competition assay , to allow cells to reach approximately 80% confluency prior to starting the experiment.set up each experimental condition in triplicate . include wells for negative controls ( no bacteria added during competition assays ) , positive controls ( control beads coupled to fusion - tag only added during competition assays ) and lysis controls ( for cytotoxicity experiments).inoculate a 5 ml marine lb ( mlb ) culture with a fresh colony of v. parahaemolyticus and grow o / n at 30 c , shaking.prepare sufficient bead - coupled mam , as described in section 1 . seed 1 ml per well of hela cells at a concentration of 150,000 cells / ml into a 24-well plate the day before the competition assay , to allow cells to reach approximately 80% confluency prior to starting the experiment . include wells for negative controls ( no bacteria added during competition assays ) , positive controls ( control beads coupled to fusion - tag only added during competition assays ) and lysis controls ( for cytotoxicity experiments ) . inoculate a 5 ml marine lb ( mlb ) culture with a fresh colony of v. parahaemolyticus and grow o / n at 30 c , shaking . competition assay : on the day of the competition experiment , measure the od600 of the bacterial culture.prepare infection media by diluting bacterial cultures into colorless dmem without additives , pre - warmed to 37 c , containing 10% v / v bead suspension ( either adhesin - coupled beads or control beads ) , to give an moi of 10 . note : for the above mentioned conditions ( 24-well plate , v. parahaemolyticus , moi 10 ) , the necessary volume of o / n culture to be added per well ( ml / ml ) is calculated as 3/od600 of the culture . for example , 1 ml of infection medium will typically contain 100 ml bead suspension , a few ml of bacterial culture and be made up to 1 ml with colorless , unsupplemented dmem.remove old medium from wells and wash cultured hela cells by adding 1 ml of sterile pbs pre - warmed to 37 c to each well.remove pbs and add 1 ml of infection medium per well . also set up controls , by adding solutions containing control beads and bacteria ( positive control ) or adhesin beads and no bacteria ( negative controls ) , or dmem containing 0.1% triton x-100 ( lysis control , only necessary for cytotoxicity measurements).incubate the plate in a tissue culture incubator at 37 c for the desired amount of time ( e.g. 4 hr for cytotoxicity measurements or 1 hr for adhesion measurements ) . note : both bacterial adhesion and cytotoxicity on host cells can be used as read - outs for the efficacy of inhibition . if the time points of cytotoxicity and adhesion measurements coincide , both assays may be performed using samples from the same well , since cytotoxicity is determined using the culture supernatant and attachment assays use samples derived from the remaining cell layer . on the day of the competition experiment , prepare infection media by diluting bacterial cultures into colorless dmem without additives , pre - warmed to 37 c , containing 10% v / v bead suspension ( either adhesin - coupled beads or control beads ) , to give an moi of 10 . note : for the above mentioned conditions ( 24-well plate , v. parahaemolyticus , moi 10 ) , the necessary volume of o / n culture to be added per well ( ml / ml ) is calculated as 3/od600 of the culture . for example , 1 ml of infection medium will typically contain 100 ml bead suspension , a few ml of bacterial culture and be made up to 1 ml with colorless , unsupplemented dmem . remove old medium from wells and wash cultured hela cells by adding 1 ml of sterile pbs pre - warmed to 37 c to each well . remove pbs and add 1 ml of infection medium per well . also set up controls , by adding solutions containing control beads and bacteria ( positive control ) or adhesin beads and no bacteria ( negative controls ) , or dmem containing 0.1% triton x-100 ( lysis control , only necessary for cytotoxicity measurements ) . incubate the plate in a tissue culture incubator at 37 c for the desired amount of time ( e.g. 4 hr for cytotoxicity measurements or 1 hr for adhesion measurements ) . note : both bacterial adhesion and cytotoxicity on host cells can be used as read - outs for the efficacy of inhibition . if the time points of cytotoxicity and adhesion measurements coincide , both assays may be performed using samples from the same well , since cytotoxicity is determined using the culture supernatant and attachment assays use samples derived from the remaining cell layer . cytotoxicity measurements : at indicated time points ( e.g. , 4 hr post infection ) , remove three times 200 ml from each 24-well and transfer to a 96-well plate.spin 96-well plates at 1,500 x g , 5 min and transfer 100 ml from each well into a fresh 96-well plate . add 100 ml of the media used during infection experiments to fresh wells of the 96-well plate in triplicate ( these will be used as blanks).carry out the lactate dehydrogenase ( ldh ) release assay using a ldh cytotoxicity detection kit and following the manufacturer s instructions . briefly , calculate the amount of reagent needed in increments of 25 ( e.g. , if 62 samples are to be measured , make up enough reagent mix for 75 etc.).for example , for 100 samples , mix 11.25 ml of reagent a with 250 l of reagent b. invert , do not vortex to avoid foaming.put the mixture in a reservoir to be able to pipet with a multi - channel pipette . add 100 l of reagent mix to each sample.incubate plate at rt and read the absorbance at 490 nm on a plate reader at 10 , 20 , 30 min.analyze the data set for which the absorbance of the lysis control sample is high but still within the linear range of the plate reader ( typically , 2 - 3 absorbance units).express results as % cytotoxicity , using the following formula for conversion : at indicated time points ( e.g. , 4 hr post infection ) , remove three times 200 ml from each 24-well and transfer to a 96-well plate . spin 96-well plates at 1,500 x g , 5 min and transfer 100 ml from each well into a fresh 96-well plate . add 100 ml of the media used during infection experiments to fresh wells of the 96-well plate in triplicate ( these will be used as blanks ) . carry out the lactate dehydrogenase ( ldh ) release assay using a ldh cytotoxicity detection kit and following the manufacturer s instructions . briefly , calculate the amount of reagent needed in increments of 25 ( e.g. , if 62 samples are to be measured , make up enough reagent mix for 75 etc.).for example , for 100 samples , mix 11.25 ml of reagent a with 250 l of reagent b. invert , do not vortex to avoid foaming.put the mixture in a reservoir to be able to pipet with a multi - channel pipette . add 100 l of reagent mix to each sample.incubate plate at rt and read the absorbance at 490 nm on a plate reader at 10 , 20 , 30 min.analyze the data set for which the absorbance of the lysis control sample is high but still within the linear range of the plate reader ( typically , 2 - 3 absorbance units).express results as % cytotoxicity , using the following formula for conversion : briefly , calculate the amount of reagent needed in increments of 25 ( e.g. , if 62 samples are to be measured , make up enough reagent mix for 75 etc . ) . for example , for 100 samples , mix 11.25 ml of reagent a with 250 l of reagent b. invert , do not vortex to avoid foaming . put the mixture in a reservoir to be able to pipet with a multi - channel pipette . incubate plate at rt and read the absorbance at 490 nm on a plate reader at 10 , 20 , 30 min . analyze the data set for which the absorbance of the lysis control sample is high but still within the linear range of the plate reader ( typically , 2 - 3 absorbance units ) . express results as % cytotoxicity , using the following formula for conversion : measurement of bacterial adhesion : at indicated time points ( e.g. , 1 hr post infection ) , remove media from the cell layer.thoroughly wash the cell layer with sterile , pre - warmed pbs ( at least 3 - 4 washes of 1 ml pbs each ) to remove any un - attached cells.lyse host cells by adding 1 ml of a sterile 1 % v / v triton x-100 solution in pbs per well . incubate the plate at 37 c for 5 min.pipette each sample up and down several times before transferring the contents of each well to separate 1.5 ml tubes . prepare 10-fold serial dilutions of samples into sterile pbs ( e.g. , use 100 ml of sample and 900 ml of pbs).plate 100 ml of each sample on mlb agar and spread using a cell spreader . optimize which dilutions to plate depending on the bacterial strains and time point . note : for the described experimental setup , 10 or 10 fold dilutions give a suitable number of cfus.incubate plates at 37 c o / n and enumerate bacteria by colony counting . at indicated time points ( e.g. , 1 hr post infection ) , remove media from the cell layer . thoroughly wash the cell layer with sterile , pre - warmed pbs ( at least 3 - 4 washes of 1 ml pbs each ) to remove any un - attached cells . lyse host cells by adding 1 ml of a sterile 1 % v / v triton x-100 solution in pbs per well . pipette each sample up and down several times before transferring the contents of each well to separate 1.5 ml tubes . prepare 10-fold serial dilutions of samples into sterile pbs ( e.g. , use 100 ml of sample and 900 ml of pbs ) . plate 100 ml of each sample on mlb agar and spread using a cell spreader . optimize which dilutions to plate depending on the bacterial strains and time point . note : for the described experimental setup , 10 or 10 fold dilutions give a suitable number of cfus . the v. parahaemolyticus adhesin mam7 contains seven tandem mammalian cell entry ( mce ) domains involved in recognition of host surface receptors . we used polystyrene beads coupled to recombinant , purified fragments encompassing either all seven tandem mce domains ( mam7 ) or only the first mce domain ( mam1 ) to test the ability of these materials to compete with v. parahaemolyticus for host cell binding and the resulting efficacy of these materials as adhesion inhibitors . hela cells were infected with v. parahaemolyticus strain por1 , and cytotoxicity resulting from in vitro infection was evaluated after 4 hr ( figure 3 ) . treatment of hela cells with 0.1% triton x-100 ( positive lysis control ) resulted in complete cell lysis , uninfected cells displayed very low levels of cytotoxicity . in vitro infection of untreated cells with por1 resulted in very high levels of cell lysis , and this was inhibited by mam7-coupled beads but not mam1-coupled or gst - coupled control beads ( figure 3 ) . cells were treated with v. parahaemolyticus ( v.p ) , indicated with ( + ) , or no bacteria ( - ) in the presence of different competing entities ( comp . ) as indicated . ( - ) , mam7 beads ( mam7 ) , mam1 beads ( mam1 ) or gst control beads ( gst ) . as controls , cells were treated either with triton x-100 ( triton , lysis control ) , or left uninfected ( uninf ) . ldh release after 4 hr was measured as described in section 3 , and results normalized to triton controls ( 100 % ) and blanks ( 0 % ) as described above . enumeration of v. parahaemolyticus attached to either untreated hela cells , or cells incubated with mam7- , mam1- , or gst control beads , revealed that mam7-beads but not mam1- or gst- control beads outcompete v. parahaemolyticus for attachment to host cell surface receptors ( figure 4 ) . + ) , in the absence ( - ) or presence of competing entities ( comp . ) as follows : mam7 beads ( mam7 ) , mam1 beads ( mam1 ) or gst control beads ( gst ) . means ( f.l.t.r . in cfu / ml ) are 2.3010 , 1.5310 , 2.0510 , 2.1210 adhesins coupled to fluorophore labeled beads result in the generation of materials that mimic bacterial adhesion to host cells and are powerful tools for cellular imaging ( figure 5 ) . using mam7-coupled fluorescent blue beads , we characterized the process of mam7-mediated attachment to epithelial cells . attachment of mam7 to host cells resulted in actin rearrangements and formation of stress fibers , which were co - visualized using rhodamine - phalloidin to stain for f - actin ( figure 5b ) . figure 5 . preparation and use of fluorophore labeled biomimetic beads for imaging purposes.(a ) suspension of fluorescent blue biomimetic beads ( right tube , 10x stock ) and buffer control ( left tube ) . ( b ) attachment of mam7-coupled beads to hela cells results in actin rearrangements and stress fiber formation . attachment of fluorescent blue beads and resulting actin stress fibers ( red , stained with rhodamine - phalloidin ) were imaged by microscopy . bar , 10 m . images in panel b were adapted from lim et al . and reproduced under the creative commons attribution license herein , we describe two protocols , which can be used to couple thiol - containing proteins to amine- or carboxylate - modified polystyrene beads , respectively . due to ease of the procedure , thiol - amine coupling is preferable , but depending on the desired bead specification ( diameter , fluorescence properties ) , use of amine - functionalized beads may not be possible and we have therefore included a protocol which will convert the carboxylate- into an amine moiety to give the researcher the greatest possible flexibility in choice of scaffold . although both thiol - amine and thiol - carboxyl coupling work for any cysteine containing protein , directional coupling ( i.e. , immobilization of the protein per its n - terminus , mimicking surface display ) requires a protein without cysteine residues , that is produced as a gst fusion protein , or that contains a single terminal cysteine residue introduced by site directed mutagenesis . if multiple reactive cysteines are contained within the protein , this will lead to random immobilization which may impede protein function . these may be removed by site directed mutagenesis , although this would require extensive assays to ensure native structure and function are retained in the mutant . for gst fusion proteins , purified gst - tag coupled to beads can be used as a suitable negative control . using uncoupled beads as a control should be avoided , as these often have a higher tendency to clump together or adhere to cells non - specifically . a simplified version of protocol 1.2 . , using only edc , can be used to couple proteins to carboxyl - functionalized polymer beads , however in this case coupling takes place via primary amines in the protein and therefore does not guarantee directional coupling . tcep as a reducing agent must not be replaced with other commercially available and commonly used reducing agents , such as dithiothreitol ( dtt ) or 2-mercaptoethanol ( bme ) , as the thiols contained within them will compete with protein coupling in the thiol - maleimide coupling step . pbs may be replaced with other buffers , but with the following considerations : buffers may not contain primary amines ( so tris - containing buffers are not suitable ) . use of buffers containing very low ( < 10 mm ) salt concentrations leads to bead clumping and should also be avoided . protein purity is also an important factor to consider during this procedure , and to achieve high quality data , pure proteins should be used . we routinely purify proteins in multiple steps , including at least an affinity purification and gel filtration step , but in some cases ion exchange chromatography is done as a third step . as a result the purity of proteins used for coupling is usually 90% or higher , as judged by sds - page . it is recommended to determine protein concentrations both in the initial reaction mixture as well as of the supernatant after reaction completion . this will help to determine the apparent concentration of bead - coupled protein , and thus coupling density . this can be taken into account when preparing the initial protein solution in subsequent reactions , to achieve the desired final concentration and coupling density . bradford reagent is particularly suited for determination of protein concentration before and after the coupling reaction , as none of the substances in the reaction interferes with the dye complex formation at the concentrations used . if lower protein concentrations are to be used , this method may have to be replaced by a more sensitive detection method , however attention has to be paid to the fact it has to be compatible with the substances contained within the coupling reaction . it is also recommended to use freshly prepared reagents and handle powdered reagents with care ( e.g. , store in a sealed container and use silica beads , to avoid the reagents drawing moisture ) since the quality of reagents will influence the coupling efficiency . if the coupling efficiency is lower than expected , possible remedies include increasing the initial bead and protein concentrations . if higher concentrations are being used , the concentration of coupling reagents has to be increased proportionally to ensure sufficient molar excess . modifying bead / protein concentrations is usually a better step towards optimization rather than increasing reaction times . since the protocols for bead coupling are lengthy , we commonly prepare a large batch of material . aliquots of the suspension can be snap - frozen in liquid nitrogen and stored at -20 c for several months . thawed aliquots should not be refrozen and should be kept at 4 c and used within 1 - 2 days . however , this will vary with the nature and stability of the protein used as should be tested on a small batch initially . this protocol described an assay that is commonly used to measure inhibition of bacterial binding and pathogen - mediated cytotoxicity on host cells . the assay is commonly performed to measure the capacity of bead - coupled mams to competitively inhibit infection of hela epithelial cells with the sea - food borne pathogen vibrio parahaemolyticus , using either a decrease in bacterial attachment to host cells or reduced cytotoxicity as a read - out . in both cases , preparation and competition assays follow the same protocol . depending on the readout , different strains of v. parahaemolyticus are being used : the cytotoxic strain por1 is used for cytotoxicity measurements , while the non - cytotoxic strain por2 is used for measuring bacterial adhesion , since cell death and cell detachment compromises the procedure for quantifying attached bacteria . initially , competition experiments were set up as a step - wise protocol , where host cells were first pre - incubated with beads prior to the addition of bacteria . for v. parahaemolyticus and the bead specifications used ( 2 m beads coupled to mam7 ) , both beads and bacteria can be added at the same time without changes in the resulting cytotoxicity . i.e. , in this experimental setup , beads outcompete bacteria for host cell binding . depending on the bacterial species and bead geometry used , there may be good reasons for maintaining the bead adhesion and bacterial infection as two separate steps . for example , to infect cells with non - motile bacteria , plates are commonly centrifuged after addition of the infection media . however , centrifugation of plates containing bead suspensions should be avoided , since this leads to a highly uneven distribution of beads on the cell layer . if smaller particle sizes are being used , beads will take longer to settle on the cell surface , in which case sufficient time should be allowed for bead attachment prior to the infection . when bacterial adhesion is used as a read out , samples should be taken at time points where host cells are not significantly damaged by the infecting strain , as cell detachment and lysis can compromise the quantification of attached bacteria . instead of enumerating bacterial adhesion by dilution plating , samples may alternatively be processed for imaging ( figure 5 ) . in this case , tissue culture cells should be seeded onto glass cover slips , rather than directly into wells . additionally , fluorescent beads and bacteria expressing a fluorescent protein may be used , along with infection - specific host cell markers . for example , competition experiments are commonly imaged using fluorescent red rhodamine - phalloidin to stain the host cells actin cytoskeleton and assess morphological changes resulting from infection , together with fluorescent blue beads and fluorescent green ( gfp expressing or syto18 stained ) bacteria . a range of bead - coupled adhesins , including staphylococcus aureus fnbpa , streptococcus pyogenes f1 fud and vibrio parahaemolyticus mam , have been used as biomimetic materials to study adhesion , adhesion inhibition and the contribution of adhesion to pathogen - mediated cytotoxicity . one of the advantages of using this approach is the ease of visualization of attachment events , since the polymer beads used as scaffolds are available in a wide range of colors ( e.g. , blue , fluorescent red , blue , green , orange ) . thus , direct protein labeling , which may interfere with function , can be avoided . additionally , surface coupling mimics the multivalent display of adhesins on the bacterial surface , thus reflecting a more physiologically relevant conformation compared to soluble proteins . compared to studies using intact bacteria or bacterial mutants , the bead approach circumvents problems associated with bacterial growth . for example , longer - term ( e.g. , o / n ) studies of bacterial adhesion to host cells using intact bacteria are often compromised by phenomena accompanying bacterial growth acidification of the growth medium and nutrient depletion negatively affect host cells , and bacterial replication eventually compromises imaging quality . more recently , the use of bead - coupled adhesins has been extended to include their use as tools for affinity purifications of host cellular factors involved in signaling processes downstream of bacterial attachment . v. parahaemolyticus mam7 , via binding to phosphatidic acids in the host cell membrane , triggers rhoa activation and actin rearrangements . mam - coupled beads are being used to purify and identify proteins involved in the signaling platforms assembled as a consequence of mam - host cell binding . since beads can easily be separated from the supernatant by a short centrifugation step , and the protein of interest is covalently coupled , this is a good method to achieve separation from contaminant proteins and enrich relevant protein complexes , which can be used for downstream applications such as proteomics or western blotting .

bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection . the biochemical and functional characterization of adhesins mediating these initial bacteria - host interactions is often compromised by the presence of other bacterial factors , such as cell wall components or secreted molecules , which interfere with the analysis . this protocol describes the production and use of biomimetic materials , consisting of pure recombinant adhesins chemically coupled to commercially available , functionalized polystyrene beads , which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors . protocols for different coupling chemistries , allowing directional immobilization of recombinant adhesins on polymer scaffolds , and for assessment of the coupling efficiency of the resulting bacteriomimetic materials are also discussed . we further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope , limitations and further applications of this approach in studying bacterial - host interactions .

Introduction Protocol 1. Chemical Coupling of Proteins to Polymer Beads 2. Determination of Coupling Efficiency 3. Use of Bead-coupled Adhesins in Competition Assays Representative Results Discussion Disclosures

dissecting the interactions between bacterial adhesins and host surface receptors at the host - pathogen interface is an essential step towards our understanding of the underlying mechanisms driving bacterial adhesion . in conducting such studies , we often face a dilemma : biochemical and biophysical analysis of the molecular mechanisms of adhesin binding requires their separation from other cell wall components , which may interfere with adhesion events . on the other hand , the use of recombinant soluble proteins over - simplifies the adhesion event , by disregarding protein anchoring in the bacterial cell wall and multivalency of binding achieved through bacterial surface display . equally , from the host cell s perspective , encountering and binding to single adhesin molecules does not always have the same impact in terms of plasma membrane organization , membrane fluidity and receptor clustering and this , ultimately , makes it difficult to evaluate the impact of adhesion on host cellular signaling and the outcome of bacteria - host interactions . this approach has been used on a range of different adhesins , from gram - negative multivalent adhesion molecules ( mams ) , to gram - positive adhesins including staphylococcus aureus fibronectin binding protein ( fnbpa ) and streptococcus pyogenes f1 . this method has allowed the dissection of adhesin fragments important for host cell binding , identification of host surface receptors and determination of their behavior on the host cell surface , while taking both binding affinity and avidity into consideration . additionally , this approach has been used to investigate the efficacy of immobilized adhesins and derivatives as inhibitors of host - pathogen interactions . it has been demonstrated that surface coupled derivatives of the vibrio parahaemolyticus multivalent adhesion molecule ( mam ) 7 can be used to attenuate a range of bacterial infections in vitro , including those caused by multidrug - resistant pathogens such as acinetobacter baumannii and methicillin - resistant s. aureus ( mrsa ) . herein , we describe different chemistries which can be used to immobilize adhesins to commercially available functionalized polystyrene particles . due to the wide array of available surface functionalities , labels and particle sizes , these are a useful scaffold for the production of bacteriomimetic materials to investigate adhesin - host interactions . we further describe methods for the initial characterization of the coupling reaction , and for the calculation of important properties of the resulting materials . finally , the use of bead - coupled adhesins as competitive inhibitors of in vitro bacterial infections is discussed as an example of their application , as well as the future scope and limitations of this approach . protein reduction : during the incubation period of the activation step , prepare the protein for the following coupling step so it can be immediately added to the activated beads . note : the reaction mixture can be directly used for the following coupling reaction.retain a small amount ( a few ml ) of the protein solution for determining the protein concentration and calculation of coupling efficiency ( see section 2 ) . note : the protein concentration during the coupling step will depend on the average coupling efficiency ( see section 2 for determination of the coupling efficiency ) and desired coupling density ( as calculated in 1.1.4.3 ) . note : the above protocol will typically give 1 ml of coupled protein , at a final concentration of 5 mm protein , which can be used as a 10x stock for subsequent experiments ( see section 3).to proceed to section 3 of the protocol , work with 100 ml / ml of bead stock , or at a final concentration of 500 nm protein . note : the reaction mixture can be directly used for the following coupling reaction.retain a small amount ( a few ml ) of the protein solution for determining the protein concentration and calculation of coupling efficiency ( see section 2 ) . check the protein concentration , and adjust it to the final concentration required for the coupling reaction . note : the reaction mixture can be directly used for the following coupling reaction.retain a small amount ( a few ml ) of the protein solution for determining the protein concentration and calculation of coupling efficiency ( see section 2 ) . retain a small amount ( a few ml ) of the protein solution for determining the protein concentration and calculation of coupling efficiency ( see section 2 ) . note : the protein concentration during the coupling step will depend on the average coupling efficiency ( see section 2 for determination of the coupling efficiency ) and desired coupling density ( as calculated in 1.1.4.3 ) . note : the above protocol will typically give 1 ml of coupled protein , at a final concentration of 5 mm protein , which can be used as a 10x stock for subsequent experiments ( see section 3).to proceed to section 3 of the protocol , work with 100 ml / ml of bead stock , or at a final concentration of 500 nm protein . note : the protein concentration during the coupling step will depend on the average coupling efficiency ( see section 2 for determination of the coupling efficiency ) and desired coupling density ( as calculated in 1.1.4.3 ) . note : the above protocol will typically give 1 ml of coupled protein , at a final concentration of 5 mm protein , which can be used as a 10x stock for subsequent experiments ( see section 3 ) . thiol - carboxy directional coupling note : this protocol is suitable for coupling cysteine containing proteins to carboxyl - functionalized polystyrene beads . , 12 ml ) into a sterile 1.5 ml tube containing 1 ml sterile pbs , ph 7.0.gently pipette up and down to wash the beads and pellet by centrifugation in a microcentrifuge ( 2 min at 16,000 x g).carefully remove the supernatant with a pipette and discard.resuspend the bead pellet in 1 ml of fresh sterile pbs and repeat the washing step.resuspend the bead pellet in 0.8 ml of pbs.add 100 ml of 10x edc stock ( 2 mm final concentration ) and immediately 100 ml of 10x nhs stock solution ( 5 mm final concentration ) to the bead suspension.incubate the bead suspension for 30 min at 25 c on a rotating wheel.wash beads once in 1 ml of pbs and resuspend in 0.8 ml fresh sterile pbs.add 200 ml ethylenediamine , and incubate the bead suspension for 1 hr at 25 c on a rotating wheel.wash beads once with 1 ml pbs and resuspend the beads in 0.8 ml of fresh pbs.proceed from section 1.1.2.4 ( bead activation with sulfo - smpb ) as described under section 1.1.2 and follow the rest of the protocol described in section 1.1 ( thiol - amine directional coupling ) . , 12 ml ) into a sterile 1.5 ml tube containing 1 ml sterile pbs , ph 7.0.gently pipette up and down to wash the beads and pellet by centrifugation in a microcentrifuge ( 2 min at 16,000 x g).carefully remove the supernatant with a pipette and discard.resuspend the bead pellet in 1 ml of fresh sterile pbs and repeat the washing step.resuspend the bead pellet in 0.8 ml of pbs.add 100 ml of 10x edc stock ( 2 mm final concentration ) and immediately 100 ml of 10x nhs stock solution ( 5 mm final concentration ) to the bead suspension.incubate the bead suspension for 30 min at 25 c on a rotating wheel.wash beads once in 1 ml of pbs and resuspend in 0.8 ml fresh sterile pbs.add 200 ml ethylenediamine , and incubate the bead suspension for 1 hr at 25 c on a rotating wheel.wash beads once with 1 ml pbs and resuspend the beads in 0.8 ml of fresh pbs.proceed from section 1.1.2.4 ( bead activation with sulfo - smpb ) as described under section 1.1.2 and follow the rest of the protocol described in section 1.1 ( thiol - amine directional coupling ) . calculate the concentration of coupled protein as follows : calculate the coupling efficiency as : preparation : seed 1 ml per well of hela cells at a concentration of 150,000 cells / ml into a 24-well plate the day before the competition assay , to allow cells to reach approximately 80% confluency prior to starting the experiment.set up each experimental condition in triplicate . note : both bacterial adhesion and cytotoxicity on host cells can be used as read - outs for the efficacy of inhibition . 4 note : both bacterial adhesion and cytotoxicity on host cells can be used as read - outs for the efficacy of inhibition . add 100 ml of the media used during infection experiments to fresh wells of the 96-well plate in triplicate ( these will be used as blanks).carry out the lactate dehydrogenase ( ldh ) release assay using a ldh cytotoxicity detection kit and following the manufacturer s instructions . add 100 ml of the media used during infection experiments to fresh wells of the 96-well plate in triplicate ( these will be used as blanks ) . note : the reaction mixture can be directly used for the following coupling reaction.retain a small amount ( a few ml ) of the protein solution for determining the protein concentration and calculation of coupling efficiency ( see section 2 ) . note : the protein concentration during the coupling step will depend on the average coupling efficiency ( see section 2 for determination of the coupling efficiency ) and desired coupling density ( as calculated in 1.1.4.3 ) . note : the above protocol will typically give 1 ml of coupled protein , at a final concentration of 5 mm protein , which can be used as a 10x stock for subsequent experiments ( see section 3).to proceed to section 3 of the protocol , work with 100 ml / ml of bead stock , or at a final concentration of 500 nm protein . protein reduction : during the incubation period of the activation step , prepare the protein for the following coupling step so it can be immediately added to the activated beads . note : the reaction mixture can be directly used for the following coupling reaction.retain a small amount ( a few ml ) of the protein solution for determining the protein concentration and calculation of coupling efficiency ( see section 2 ) . during the incubation period of the activation step , prepare the protein for the following coupling step so it can be immediately added to the activated beads . check the protein concentration , and adjust it to the final concentration required for the coupling reaction . note : the reaction mixture can be directly used for the following coupling reaction.retain a small amount ( a few ml ) of the protein solution for determining the protein concentration and calculation of coupling efficiency ( see section 2 ) . note : the protein concentration during the coupling step will depend on the average coupling efficiency ( see section 2 for determination of the coupling efficiency ) and desired coupling density ( as calculated in 1.1.4.3 ) . note : the above protocol will typically give 1 ml of coupled protein , at a final concentration of 5 mm protein , which can be used as a 10x stock for subsequent experiments ( see section 3).to proceed to section 3 of the protocol , work with 100 ml / ml of bead stock , or at a final concentration of 500 nm protein . note : the protein concentration during the coupling step will depend on the average coupling efficiency ( see section 2 for determination of the coupling efficiency ) and desired coupling density ( as calculated in 1.1.4.3 ) . note : the above protocol will typically give 1 ml of coupled protein , at a final concentration of 5 mm protein , which can be used as a 10x stock for subsequent experiments ( see section 3 ) . thiol - carboxy directional coupling note : this protocol is suitable for coupling cysteine containing proteins to carboxyl - functionalized polystyrene beads . , 12 ml ) into a sterile 1.5 ml tube containing 1 ml sterile pbs , ph 7.0.gently pipette up and down to wash the beads and pellet by centrifugation in a microcentrifuge ( 2 min at 16,000 x g).carefully remove the supernatant with a pipette and discard.resuspend the bead pellet in 1 ml of fresh sterile pbs and repeat the washing step.resuspend the bead pellet in 0.8 ml of pbs.add 100 ml of 10x edc stock ( 2 mm final concentration ) and immediately 100 ml of 10x nhs stock solution ( 5 mm final concentration ) to the bead suspension.incubate the bead suspension for 30 min at 25 c on a rotating wheel.wash beads once in 1 ml of pbs and resuspend in 0.8 ml fresh sterile pbs.add 200 ml ethylenediamine , and incubate the bead suspension for 1 hr at 25 c on a rotating wheel.wash beads once with 1 ml pbs and resuspend the beads in 0.8 ml of fresh pbs.proceed from section 1.1.2.4 ( bead activation with sulfo - smpb ) as described under section 1.1.2 and follow the rest of the protocol described in section 1.1 ( thiol - amine directional coupling ) . note : both bacterial adhesion and cytotoxicity on host cells can be used as read - outs for the efficacy of inhibition . note : both bacterial adhesion and cytotoxicity on host cells can be used as read - outs for the efficacy of inhibition . add 100 ml of the media used during infection experiments to fresh wells of the 96-well plate in triplicate ( these will be used as blanks).carry out the lactate dehydrogenase ( ldh ) release assay using a ldh cytotoxicity detection kit and following the manufacturer s instructions . add 100 ml of the media used during infection experiments to fresh wells of the 96-well plate in triplicate ( these will be used as blanks ) . we used polystyrene beads coupled to recombinant , purified fragments encompassing either all seven tandem mce domains ( mam7 ) or only the first mce domain ( mam1 ) to test the ability of these materials to compete with v. parahaemolyticus for host cell binding and the resulting efficacy of these materials as adhesion inhibitors . cells were treated with v. parahaemolyticus ( v.p ) , indicated with ( + ) , or no bacteria ( - ) in the presence of different competing entities ( comp . ) enumeration of v. parahaemolyticus attached to either untreated hela cells , or cells incubated with mam7- , mam1- , or gst control beads , revealed that mam7-beads but not mam1- or gst- control beads outcompete v. parahaemolyticus for attachment to host cell surface receptors ( figure 4 ) . in cfu / ml ) are 2.3010 , 1.5310 , 2.0510 , 2.1210 adhesins coupled to fluorophore labeled beads result in the generation of materials that mimic bacterial adhesion to host cells and are powerful tools for cellular imaging ( figure 5 ) . attachment of mam7 to host cells resulted in actin rearrangements and formation of stress fibers , which were co - visualized using rhodamine - phalloidin to stain for f - actin ( figure 5b ) . preparation and use of fluorophore labeled biomimetic beads for imaging purposes. and reproduced under the creative commons attribution license herein , we describe two protocols , which can be used to couple thiol - containing proteins to amine- or carboxylate - modified polystyrene beads , respectively . due to ease of the procedure , thiol - amine coupling is preferable , but depending on the desired bead specification ( diameter , fluorescence properties ) , use of amine - functionalized beads may not be possible and we have therefore included a protocol which will convert the carboxylate- into an amine moiety to give the researcher the greatest possible flexibility in choice of scaffold . , immobilization of the protein per its n - terminus , mimicking surface display ) requires a protein without cysteine residues , that is produced as a gst fusion protein , or that contains a single terminal cysteine residue introduced by site directed mutagenesis . for gst fusion proteins , purified gst - tag coupled to beads can be used as a suitable negative control . , using only edc , can be used to couple proteins to carboxyl - functionalized polymer beads , however in this case coupling takes place via primary amines in the protein and therefore does not guarantee directional coupling . tcep as a reducing agent must not be replaced with other commercially available and commonly used reducing agents , such as dithiothreitol ( dtt ) or 2-mercaptoethanol ( bme ) , as the thiols contained within them will compete with protein coupling in the thiol - maleimide coupling step . bradford reagent is particularly suited for determination of protein concentration before and after the coupling reaction , as none of the substances in the reaction interferes with the dye complex formation at the concentrations used . if lower protein concentrations are to be used , this method may have to be replaced by a more sensitive detection method , however attention has to be paid to the fact it has to be compatible with the substances contained within the coupling reaction . , store in a sealed container and use silica beads , to avoid the reagents drawing moisture ) since the quality of reagents will influence the coupling efficiency . however , this will vary with the nature and stability of the protein used as should be tested on a small batch initially . this protocol described an assay that is commonly used to measure inhibition of bacterial binding and pathogen - mediated cytotoxicity on host cells . the assay is commonly performed to measure the capacity of bead - coupled mams to competitively inhibit infection of hela epithelial cells with the sea - food borne pathogen vibrio parahaemolyticus , using either a decrease in bacterial attachment to host cells or reduced cytotoxicity as a read - out . for v. parahaemolyticus and the bead specifications used ( 2 m beads coupled to mam7 ) , both beads and bacteria can be added at the same time without changes in the resulting cytotoxicity . when bacterial adhesion is used as a read out , samples should be taken at time points where host cells are not significantly damaged by the infecting strain , as cell detachment and lysis can compromise the quantification of attached bacteria . additionally , fluorescent beads and bacteria expressing a fluorescent protein may be used , along with infection - specific host cell markers . a range of bead - coupled adhesins , including staphylococcus aureus fnbpa , streptococcus pyogenes f1 fud and vibrio parahaemolyticus mam , have been used as biomimetic materials to study adhesion , adhesion inhibition and the contribution of adhesion to pathogen - mediated cytotoxicity . one of the advantages of using this approach is the ease of visualization of attachment events , since the polymer beads used as scaffolds are available in a wide range of colors ( e.g. thus , direct protein labeling , which may interfere with function , can be avoided . , o / n ) studies of bacterial adhesion to host cells using intact bacteria are often compromised by phenomena accompanying bacterial growth acidification of the growth medium and nutrient depletion negatively affect host cells , and bacterial replication eventually compromises imaging quality . more recently , the use of bead - coupled adhesins has been extended to include their use as tools for affinity purifications of host cellular factors involved in signaling processes downstream of bacterial attachment . mam - coupled beads are being used to purify and identify proteins involved in the signaling platforms assembled as a consequence of mam - host cell binding . since beads can easily be separated from the supernatant by a short centrifugation step , and the protein of interest is covalently coupled , this is a good method to achieve separation from contaminant proteins and enrich relevant protein complexes , which can be used for downstream applications such as proteomics or western blotting .

an expert consultation organized by the world health organization in 2005 made an inventory of available research on births spacing . the experts recommended an interpregnancy interval ( ipi ) of at least 6 months after a miscarriage before attempting a next pregnancy , in order to reduce morbidity and mortality risks for mother , fetus , and newborn . an ipi of at least 24 months was recommended after a live birth , corresponding to a birth interval of at least 33 months . the consultation team also concluded that future research is needed on the mechanisms underlying the relation between interval length and pregnancy outcomes . more studies using datasets from both rich and poor countries could contribute to more in - depth knowledge . contradicting results of research on the effect of short intervals on the risk of adverse pregnancy outcomes [ 16 ] conducted after 2005 confirmed the relevance of these statements . the few studies on the effect of intervals after a previous pregnancy disruption show a large variation by country . davanzo and colleagues emphasized that studies on the effects of interpregnancy intervals should take into account the outcome of the previous pregnancy . our study contributes to the debate on pregnancy loss and interpregnancy intervals ( ipis ) in line with these recommendations . we focus on the effect of the duration of the ipi on pregnancy losses by combining the effect of the interval duration and the type of previous pregnancy outcome ( pregnancy loss , live births that survived infancy or died in the first year ) , and controlling for important confounders . in this study , the term pregnancy loss includes all pregnancy outcomes ( spontaneous and induced abortion , fetal death , and stillbirth ) opposite to a live birth . fetal loss has got limited attention compared to other issues , neither in the field of reproductive health nor in development debates among policy makers , nor in debates among scholars in population studies . this lack of attention is regrettable , because for many women the loss of a pregnancy is an emotional experience which affects their subsequent reproductive health and behavior . fetal loss and stillbirths constitute the majority of the world 's perinatal deaths and , yet , the absence of easy accessible and reliable secondary data on pregnancy loss is mentioned as a reason for neglecting the topic of fetal deaths by scientists [ 7 , 8 ] . reducing adverse pregnancy outcomes contributes to the health of the mother . contrary to the reduction of maternal morbidity and of infant mortality , reducing pregnancy loss is not a policy objective but should become so in the future . the outcome of our analysis will be discussed in the framework of results of a few [ 1 , 4 , 7 , 911 ] available studies that followed the same approach by including the ipi duration and the previous pregnancy outcome to estimate the risk of a pregnancy loss . the majority of these studies focus on the effect of ipi duration on adverse pregnancy outcomes after a previous spontaneous or induced abortion while only a few have a broader perspective and include also other prior pregnancy outcomes ( see table 4 ) . the studies differ on essential points , such as various types of mothers in the sample , nulliparous or multiparous women ; various types of adverse pregnancy outcomes , pregnancy loss , preterm births , and low birth weight ; reference group ; categories of ipi ; data collection method ; and geographical region , which could contribute to explain the variety in the findings . only two studies , one from the usa and the other from latin america , did not find significant associations between ipi duration and a fetal or neonatal death after a spontaneous or induced abortion [ 7 , 9 ] . the other studies do find an association between ipi duration and adverse maternal and pregnancy outcomes after a previous fetal loss , but the associations between ipi duration and recurrent pregnancy losses are weak or nonexistent for late fetal deaths ( stillbirths ) [ 911 ] . some results from usa and scotland are even the opposite of what is expected on the who recommendations after early fetal deaths ( miscarriages ) [ 5 , 6 ] . they find that , after a previous early fetal loss , short ipi intervals are associated with a higher likelihood on a live birth compared to longer ipis . for bangladesh , no differences between these likelihoods were found after a fetal loss according to ipi length except after very long ipi ( > 74 months ) . however , the studies [ 10 , 11 ] that focus on the association between ipi duration and the risk of a fetal death ( and other adverse pregnancy outcomes ) show higher risks of an adverse outcome after a fetal loss and a short ipi relative to a previous live birth combined with a healthy ipi . the meta - analyses included many countries from all over the world and consequently examples with good and poor health care systems . a study on sweden , a country with an advanced medical health care system , however , stated that risks are only found for long intervals and that the impact of short intervals may have been overestimated in other studies . from all those studies , we learn that it is important to include the outcome of the previous pregnancy in the analysis and to focus on several previous pregnancy outcomes when analyzing the effect of short ipis on fetal losses . therefore , we will follow this approach in our analysis about pregnancy loss in rwanda . since 2000 , rwanda is experiencing a steady economic growth and after 2005 a rapid demographic and health transition [ 13 , 14 ] thanks to the extension of access to reproductive health facilities . the health service infrastructure , which was badly damaged during the civil war of the ninety - nineties , has been rebuilt to a large extent . at local level , community health centers are established and more than 45,000 community health care workers , male and female , have been trained to provide basic medical care and drugs and to give information on health matters . the number of qualified medical staffs increased yet is still insufficient according to international standards : one medical doctor and one professional midwife per 16,500 and 23,400 inhabitants , respectively . the government improved access to community health care also by introducing a community based insurance system . today , more than 90% of the population participates in these mutuelles de sant health mutualities which give access to community level health services and , with additional payment , to a package of extra health care at district hospitals . this percentage of more than 90% explains an impressive increase in access to health care , given the fact that this percentage was only 7% in 2003 . the prenatal checks and the costs of a normal delivery assisted by a nurse or midwife are covered by the basic health insurance schemes but exclude the 200 rwf per visit to a health center . yet , the health - seeking behavior among pregnant women still needs improvement although , according to the 2010 demographic and health survey ( dhs ) , less than two percent of the women did not have any antenatal medical test before the delivery . however , only 35 percent of them went for four antenatal checks , as recommended by the who . most pregnant women got their first medical examination in the second trimester of their pregnancy and some even later [ 1619 ] . the demographic and health survey ( dhs ) is an internationally recognized data collection method that provides current and reliable data based on a national representative sample . data from three successive rwanda demographic and health surveys ( rdhs 2000 , rdhs 2005 , and rdhs 2010 ) were merged in this study to analyze the last pregnancy outcome of women within the dhs calendar periods of five years preceding the moment of interview . pregnancies and pregnancy outcomes that occurred in the eight months before the month of the interview were not included in our analysis to be sure that all pregnancies in the analysis had the same probability of ending in a pregnancy termination or a live birth after nine months . to identify the moment of the start of the last pregnancy , we used the detailed recording in the calendar of the dhs , which gives the pregnancy status for each month over a period of 59 months before the month of the interview . the nature and timing of the previous event the exact months of all births , deaths , and pregnancy terminations are recorded in the dhs . the duration of the ipi is measured by subtracting the date of the previous pregnancy outcome from the date of the start of the last pregnancy . the month of the previous outcome is registered at any time before the start of the last pregnancy . in total , 21532 women had at least one pregnancy outcome in the three reduced calendar periods before 2000 , 2005 , and 2010 ; for 3631 women , it was their first pregnancy ( primigravida ) ; for the other 17901 women , we calculated the date and type of the previous pregnancy outcome . in case of a long interpregnancy interval , the dhs datasets enable the calculation of the exact date ( in terms of month and year ) of the events in the reproductive history of women in the sample if one combines answers to various questions in the questionnaire . we constructed century month codes ( cmc ) , the number of months elapsed since january 1900 , of the pregnancy outcomes ( pregnancy loss , infant death , and live birth ) reported by the mothers to calculate the ipis . in case of a live birth as last pregnancy outcome , the pregnancy was supposed to start 9 months before the cmc of the birth . in case of a fetal loss as last pregnancy outcome , the mother did report the duration of the pregnancy in months . data from retrospective studies , like the demographic and health surveys , are biased by errors due to memory lapses as the respondents have to report the number and date of the events in the past . this is in particular the case when one asks for matters as pregnancy losses and induced abortions . early pregnancy losses may not be noted or easily forgotten and induced abortions may not be reported as these are illegal in many societies . by focusing on the two last pregnancies of which the last one ( and in many cases the previous as well ) occurred during the calendar period , we reduced the risk of memory errors . for our analysis , we calibrated a binary logistic regression model using the statistical package stata 12 . the dependent variable is the outcome of the last pregnancy ( fetal loss coded as 1 , live birth coded as 0 ) . we checked whether a distinction between early and late losses gave different results , but this turned out not to be the case . to construct a more powerful model we defined the two main independent variables : length of the interpregnancy interval and previous pregnancy outcome as follows . interpregnancy intervals ( ipi ) were calculated as the time between the outcome of the previous pregnancy that ended either in a pregnancy loss or live birth and the last conception . short intervals are defined as shorter than 4 months or 4 up to 12 months after a previous pregnancy loss and shorter than 1 year or between 1 and 2 years after a live birth . a healthy interval after a live we categorized the live births of the previous pregnancy in two groups : infants that survived the first year of their life or infants that did not survive . the reason behind this categorization relates to the maternal depletion hypothesis and the quick return of the ovulation combined with the replacement strategy . in regard to the maternal depletion , the idea is to test whether a surviving breastfed infant will increase the depletion of maternal resources and therefore affect the survival of the next pregnancy that is conceived after a short ipi . secondly , the death of the previous infant might be related to an unhealthy physiological status of the mother and the wish to quickly replace the infant , thus shortening the ipi . subsequently , we constructed variables to represent the interaction between those two main independent variables in which we used different classifications for the ipi duration after the previous pregnancy outcomes . we tested for several confounding factors but , in the final model , included only four control variables that turned out to be of significance . the first is the inevitable biodemographic control variable age of the mother at conception which is an indicator for her physiological condition at the start of and during her pregnancy . we specified mother 's age as a categorical variable to allow for nonlinear effects as we will focus on broad age categories and compare young ( below the age of 21 ) and especially older mothers ( over the age of 35 ) with women of a more optimal reproductive age . age refers to the reproductive condition that contributes to a healthy pregnancy and the birth of a healthy infant . in particular , we want to test if older mothers have higher pregnancy loss risks compared to younger ones . question v228 of the dhs women 's questionnaire asks the responding woman whether , at the time she became pregnant for the last pregnancy , she wished to become pregnant , she wished to wait until later , or she did not want to have any more children at all . response categories included the following : wanting pregnancy then , wanting pregnancy later , wanting no more children , or unknown / vague answer . the latter category is used as a proxy for intended pregnancy losses together with the third included variable : place of residence that distinguishes between urban and rural residence . the available dataset does not make a distinction between induced and spontaneous pregnancy losses , which is an omission seen in the different relations between the two types of abortions at the beginning of the ipi , length of the ipi , and pregnancy outcome found in other researches . however , we expect that rwandan women will not easily indicate that they had an induced abortion as it is illegal , except when the physical health of the mother is in great danger . the study of basinga and colleagues estimated the rate of induced abortion in rwanda in 2009 between 18 and 31 per 1,000 women in the 1544-year - old group . their study also revealed that the induced abortion rate was remarkably higher in the capital city kigali compared to the situation in the provinces . in most african rural settings , induced abortion remains a taboo and social control in communities that watch over virginity as a core value , the reason why in particular rural women support legalization of abortion less compared to urban women . . stated that in the early nineties induced abortion did not occur traditionally in this country . for that reason , we expected that in case this situation changed during the last two decades abortions will be chiefly an urban phenomenon . we tried to control for it by including place of residence ( urban versus rural ) in the analysis . the percentages of unknown / vague answers in our dataset were , respectively , 27.9 and 23.6 for urban and rural women . finally , we included the year of the interview to check for changes over time in reproductive health : notably , the extent of the possible reduction of fetal mortality . the results presented in table 1 illustrate that many rwandan women still have to deal with pregnancy losses , the death of an infant , and unwanted pregnancies : 36 out of 1000 last pregnancies in our sample population ended in a pregnancy loss . among the women with at least two pregnancies , 15 percent mourned a fatal outcome of the previous pregnancy : five percent had a pregnancy loss and nearly ten percent got a child that died in infancy . the results indicate also that the percentages of pregnancies ending in a pregnancy loss are the highest after an ipi shorter than 24 months that started after a pregnancy loss . higher percentages of pregnancy loss than the mean of 3.6 per cent were found after a live - born infant that died in its infancy and an ipi of more than two years and after a surviving live birth and a very long ipi ( > 60 months ) . the descriptive statistics in table 1 show a modest decline of the rate of fetal losses during the period under study . for the three consecutive research periods , the rate of pregnancy loss diminished from 41 out of 1000 pregnancies ( 19962000 ) to 36 ( 20012005 ) and finally to 33 in the most recent period 20062010 . it is difficult to assess if the total number of reported pregnancy losses in the three dhss used in this study , 36 per 1000 pregnancies , is in line with expectations or not . the frequency fits within an indication given in medical literature that states that the number of fetal losses in the month after conception is high but that after a gestation of 8 weeks the loss is about 3 percent . this could mean that women in rwanda did not mention losses that occurred in the first one or two months of a pregnancy , when they were not fully aware of being pregnant . the early pregnancy losses reported in the dhs are probably underestimated as in poor countries the number of stillbirths ( after a gestation of 28 weeks ) is higher compared to that of rich countries and the stillbirth rate in countries in the central part of sub - saharan africa varies between 25 and 40 or more per 1000 births . with women who were pregnant for the first time reported the highest percentage of wanted pregnancies ( nearly 60% , see table 2 ) . of all last pregnancies by the other women , only 40 percent were wanted at that time , while more than a third were unwanted or the mother gave an unclear answer ( or answer not known ) . the cross tabulation presented in table 2 shows that after a pregnancy loss or the loss of an infant a large portion of the women want to replace this loss . very low numbers of wanted pregnancies are found among women who became pregnant within two years after the birth of the previous child that survived the first year of its life . those two groups of women had liked to become pregnant later in time ( indicated by 40 and 31% , resp . ) . a large portion of the women became pregnant again before the recommended time ( by who ) for recovery was over . from the women whose previous pregnancy ended with a fetal loss , 43 percent were expecting a child again within half a year . for women who had a live - born child that died afterwards in infancy , 71 percent were pregnant again within two years after the last delivery , which could point at a replacement effect or at a lack of protection against pregnancy . for women whose child survived the first year of its life , this percentage was much lower ( 47% ) . this group of women is probably temporarily subfecund due to a longer amenorrhea period caused by lactation . the constant ( table 3 ) reflects the risk of a pregnancy loss for the reference category : rural women in 2000 , in age category of 2135 years at the time of the last conception , whose last pregnancy was wanted and started after a healthy ipi ( 2559 months ) , and whose previous pregnancy resulted in a child that survived its infancy . the estimate of the risk of experiencing a pregnancy termination for these women is very low ( 2% ) . the other variables exp( ) give the odds ratios for women in the categories that deviate from the reference category . linking the risk of a pregnancy termination with both the outcome of the previous pregnancy and the length of the ipi ( interaction variables ) shows significant deviations from the risk estimated for the reference group : all except one point at a higher risk . the highest odds are found for women who became pregnant shortly after a previous pregnancy loss . women who conceived again within 3 months after the previous pregnancy loss are 3.68 times more likely to lose the next pregnancy than the reference group with a healthy interval . the odds ratio is 2.648 for those that waited 412 months and even women who waited 1214 months were almost twice as likely to lose the next pregnancy . women with an ipi of more than two years after a previous fetal loss had a lower risk compared to the reference group , but the association is not significant . the higher odds of pregnancy loss for all groups with a previous pregnancy loss suggest that some women are prone to repeated losses , regardless of ipi duration . repeated or recurrent pregnancy loss is phenomenon that puzzles medical experts like gynecologist already for decades and that is probably associated with more than genetic factors of the woman alone [ 25 , 26 ] . after a live birth , regardless of whether the newborn survived its infancy or not , the likelihood of a pregnancy loss after an ipi considered as unhealthy ( < 2 years ) is not higher compared to the reference group with a recommended ipi duration . for the mother that became pregnant within , respectively , one to two years after the previous birth , the signs of the coefficients are negative but only significant for women whose infant stayed alive and conceived within one year . any pregnancy after a live birth seems to prepare for a successful next pregnancy , regardless of the interpregnancy interval . this mechanism vanishes after some years , as an ipi of more than 5 years results in a substantial higher likelihood of a pregnancy loss ( 1.6 times more likely ) . the risk of a pregnancy loss for mothers who are pregnant for the first time is of the same magnitude ( 1.5 times more likely ) . the physiological regression hypothesis states that after a very long ipi the body of a women has lost the beneficial physiological adaptations in her reproductive system that occur after a pregnancy [ 10 , 12 ] . this is reflected in the higher likelihood of a pregnancy loss ( 2.3 times more likely ) for women who were older than 35 years when they became pregnant . the positive coefficients found for urban women and for women who gave a vague answer or did not answer the question on whether they wanted the last pregnancy could point at the occurrence of induced pregnancy terminations . as induced abortions are prohibited and a taboo , women who had an illegal abortion will probably answer evasively when asked for their pregnancy timing . the higher risk of pregnancy losses among urban women in our sample fits in with research findings by basinga and colleagues who calculated that induced abortions occur more frequently in the capital city of kigali compared to other regions of rwanda . the finding that women who explicitly declared that the last pregnancy was not wanted have a significant lower risk of losing the next pregnancy gives food for thought . maybe these are highly fecund women who become pregnant easily and therefore more often unwanted , and who do not encounter pregnancy problems . we remark that the likelihood of a pregnancy termination decreased significantly between 2000 and 2010 . for 2005 , the sign of the coefficient ( ) is negative , but the decrease is not significant . in 2010 , however , the decrease is significant . further analyses , not shown here , showed that this decrease pertained to late pregnancy loss only . this may be seen as an indication that improved health - seeking behaviour among pregnant women in particular during the second half of their pregnancy contributed to less pregnancy losses . the first important result of our analyses is that one needs to take the previous pregnancy outcome into account when estimating the effects of ipis on the risk of a pregnancy loss . the second main finding is that negative outcomes ( in terms of a higher risk of recurrent pregnancy loss ) were found for ipis up to 24 months after a prior pregnancy loss , a period four times as long as the recommended healthy ipi of only 6 months . in contrast , an ipi shorter than 2 years after a live birth does not seem to increase significantly the risk of a pregnancy loss . we are aware that a pregnancy loss is not the only possible adverse pregnancy outcome . shorter ipis than two years after a live birth do not give higher risks of a pregnancy loss , but they will affect other pregnancy outcomes such as preterm birth , low birth weight , low apgar scores , and a higher neonatal death . we found clear indications for negative effects of the replacement mechanism after the loss of a pregnancy . the replacement wish after a fetal death leads to shorter ipis and therefore to a higher risk of another pregnancy loss . finally , the results of our study confirm the physiological regression hypothesis : a higher risk of a fetal death when ipis are longer than 5 years . also older women have a higher likelihood of a pregnancy loss compared to younger ones . our results are partially in line with the ones from davanzo and colleagues in bangladesh [ 1 , 11 ] based also on a general sample of women with all types of prior pregnancy outcomes . to avoid a higher risk of a next miscarriage or stillbirth also in bangladesh , women should wait longer than the recommended 6 months ( up to 15 months ) to become pregnant again after a former pregnancy loss . the researchers found a significant increased risk of a pregnancy loss after a live birth and an ipi < 6 months . for longer ipi durations up to 74 months after a live birth , no significant higher risks of a pregnancy loss were found . based on the results of this study on bangladesh and ours on rwanda , one could conclude that , in societies without an advanced health care system , the who recommendations concerning spacing after a fetal loss still count . workers in the health care system should advise women , even if they are eager to become pregnant again , to take actions to prevent a quick new pregnancy and wait even longer than a year to become pregnant again . the improvements in the rwandan health care system between 2000 and 2010 and in particular the increased access to this system contributed to a lower pregnancy loss frequency . probably , the increased antenatal checks during the last pregnancy period had an impact , as the significant decrease in pregnancy losses between 2000 and 2010 resulted in particular in fewer late fetal losses ( after a pregnancy duration of 20 weeks ) . with a policy that recommends to women an ipi of at least a year to two years after a fetal death and more early pregnancy visits to the community health facility , a decrease in an early fetal death

in 2005 , a who consultation meeting on pregnancy intervals recommended a minimum interval of 6 months after a pregnancy disruption and an interval of two years after a live birth before attempting another pregnancy . since then , studies have found contradictory evidence on the effect of shorter intervals after a pregnancy disruption . a binary regression analysis on 21532 last pregnancy outcomes from the 2000 , 2005 , and 2010 rwanda demographic and health surveys was done to assess the combined effects of the preceding pregnancy outcome and the interpregnancy intervals ( ipis ) on fetal mortality in rwanda . risks of pregnancy loss are higher for primigravida and for mothers who lost the previous pregnancy and conceived again within 24 months . after a live birth , interpregnancy intervals less than two years do not increase the risk of a pregnancy loss . this study also confirms higher risks of fetal death when ipis are beyond 5 years . an ipi of longer than 12 months after a fetal death is recommended in rwanda . particular attention needs to be directed to postpregnancy abortion care and family planning programs geared to spacing pregnancies should also include spacing after a fetal death .

1. Introduction 2. Material and Method 3. Results and Discussion 4. Conclusion and Policy Recommendations

the experts recommended an interpregnancy interval ( ipi ) of at least 6 months after a miscarriage before attempting a next pregnancy , in order to reduce morbidity and mortality risks for mother , fetus , and newborn . an ipi of at least 24 months was recommended after a live birth , corresponding to a birth interval of at least 33 months . the consultation team also concluded that future research is needed on the mechanisms underlying the relation between interval length and pregnancy outcomes . contradicting results of research on the effect of short intervals on the risk of adverse pregnancy outcomes [ 16 ] conducted after 2005 confirmed the relevance of these statements . the few studies on the effect of intervals after a previous pregnancy disruption show a large variation by country . davanzo and colleagues emphasized that studies on the effects of interpregnancy intervals should take into account the outcome of the previous pregnancy . our study contributes to the debate on pregnancy loss and interpregnancy intervals ( ipis ) in line with these recommendations . we focus on the effect of the duration of the ipi on pregnancy losses by combining the effect of the interval duration and the type of previous pregnancy outcome ( pregnancy loss , live births that survived infancy or died in the first year ) , and controlling for important confounders . in this study , the term pregnancy loss includes all pregnancy outcomes ( spontaneous and induced abortion , fetal death , and stillbirth ) opposite to a live birth . this lack of attention is regrettable , because for many women the loss of a pregnancy is an emotional experience which affects their subsequent reproductive health and behavior . fetal loss and stillbirths constitute the majority of the world 's perinatal deaths and , yet , the absence of easy accessible and reliable secondary data on pregnancy loss is mentioned as a reason for neglecting the topic of fetal deaths by scientists [ 7 , 8 ] . reducing adverse pregnancy outcomes contributes to the health of the mother . the outcome of our analysis will be discussed in the framework of results of a few [ 1 , 4 , 7 , 911 ] available studies that followed the same approach by including the ipi duration and the previous pregnancy outcome to estimate the risk of a pregnancy loss . the majority of these studies focus on the effect of ipi duration on adverse pregnancy outcomes after a previous spontaneous or induced abortion while only a few have a broader perspective and include also other prior pregnancy outcomes ( see table 4 ) . the studies differ on essential points , such as various types of mothers in the sample , nulliparous or multiparous women ; various types of adverse pregnancy outcomes , pregnancy loss , preterm births , and low birth weight ; reference group ; categories of ipi ; data collection method ; and geographical region , which could contribute to explain the variety in the findings . only two studies , one from the usa and the other from latin america , did not find significant associations between ipi duration and a fetal or neonatal death after a spontaneous or induced abortion [ 7 , 9 ] . the other studies do find an association between ipi duration and adverse maternal and pregnancy outcomes after a previous fetal loss , but the associations between ipi duration and recurrent pregnancy losses are weak or nonexistent for late fetal deaths ( stillbirths ) [ 911 ] . some results from usa and scotland are even the opposite of what is expected on the who recommendations after early fetal deaths ( miscarriages ) [ 5 , 6 ] . they find that , after a previous early fetal loss , short ipi intervals are associated with a higher likelihood on a live birth compared to longer ipis . for bangladesh , no differences between these likelihoods were found after a fetal loss according to ipi length except after very long ipi ( > 74 months ) . however , the studies [ 10 , 11 ] that focus on the association between ipi duration and the risk of a fetal death ( and other adverse pregnancy outcomes ) show higher risks of an adverse outcome after a fetal loss and a short ipi relative to a previous live birth combined with a healthy ipi . from all those studies , we learn that it is important to include the outcome of the previous pregnancy in the analysis and to focus on several previous pregnancy outcomes when analyzing the effect of short ipis on fetal losses . therefore , we will follow this approach in our analysis about pregnancy loss in rwanda . since 2000 , rwanda is experiencing a steady economic growth and after 2005 a rapid demographic and health transition [ 13 , 14 ] thanks to the extension of access to reproductive health facilities . at local level , community health centers are established and more than 45,000 community health care workers , male and female , have been trained to provide basic medical care and drugs and to give information on health matters . today , more than 90% of the population participates in these mutuelles de sant health mutualities which give access to community level health services and , with additional payment , to a package of extra health care at district hospitals . the prenatal checks and the costs of a normal delivery assisted by a nurse or midwife are covered by the basic health insurance schemes but exclude the 200 rwf per visit to a health center . yet , the health - seeking behavior among pregnant women still needs improvement although , according to the 2010 demographic and health survey ( dhs ) , less than two percent of the women did not have any antenatal medical test before the delivery . most pregnant women got their first medical examination in the second trimester of their pregnancy and some even later [ 1619 ] . the demographic and health survey ( dhs ) is an internationally recognized data collection method that provides current and reliable data based on a national representative sample . data from three successive rwanda demographic and health surveys ( rdhs 2000 , rdhs 2005 , and rdhs 2010 ) were merged in this study to analyze the last pregnancy outcome of women within the dhs calendar periods of five years preceding the moment of interview . pregnancies and pregnancy outcomes that occurred in the eight months before the month of the interview were not included in our analysis to be sure that all pregnancies in the analysis had the same probability of ending in a pregnancy termination or a live birth after nine months . to identify the moment of the start of the last pregnancy , we used the detailed recording in the calendar of the dhs , which gives the pregnancy status for each month over a period of 59 months before the month of the interview . the nature and timing of the previous event the exact months of all births , deaths , and pregnancy terminations are recorded in the dhs . the duration of the ipi is measured by subtracting the date of the previous pregnancy outcome from the date of the start of the last pregnancy . the month of the previous outcome is registered at any time before the start of the last pregnancy . in total , 21532 women had at least one pregnancy outcome in the three reduced calendar periods before 2000 , 2005 , and 2010 ; for 3631 women , it was their first pregnancy ( primigravida ) ; for the other 17901 women , we calculated the date and type of the previous pregnancy outcome . in case of a long interpregnancy interval , the dhs datasets enable the calculation of the exact date ( in terms of month and year ) of the events in the reproductive history of women in the sample if one combines answers to various questions in the questionnaire . we constructed century month codes ( cmc ) , the number of months elapsed since january 1900 , of the pregnancy outcomes ( pregnancy loss , infant death , and live birth ) reported by the mothers to calculate the ipis . in case of a live birth as last pregnancy outcome , the pregnancy was supposed to start 9 months before the cmc of the birth . in case of a fetal loss as last pregnancy outcome , the mother did report the duration of the pregnancy in months . data from retrospective studies , like the demographic and health surveys , are biased by errors due to memory lapses as the respondents have to report the number and date of the events in the past . by focusing on the two last pregnancies of which the last one ( and in many cases the previous as well ) occurred during the calendar period , we reduced the risk of memory errors . for our analysis , we calibrated a binary logistic regression model using the statistical package stata 12 . the dependent variable is the outcome of the last pregnancy ( fetal loss coded as 1 , live birth coded as 0 ) . we checked whether a distinction between early and late losses gave different results , but this turned out not to be the case . to construct a more powerful model we defined the two main independent variables : length of the interpregnancy interval and previous pregnancy outcome as follows . interpregnancy intervals ( ipi ) were calculated as the time between the outcome of the previous pregnancy that ended either in a pregnancy loss or live birth and the last conception . short intervals are defined as shorter than 4 months or 4 up to 12 months after a previous pregnancy loss and shorter than 1 year or between 1 and 2 years after a live birth . a healthy interval after a live we categorized the live births of the previous pregnancy in two groups : infants that survived the first year of their life or infants that did not survive . the reason behind this categorization relates to the maternal depletion hypothesis and the quick return of the ovulation combined with the replacement strategy . in regard to the maternal depletion , the idea is to test whether a surviving breastfed infant will increase the depletion of maternal resources and therefore affect the survival of the next pregnancy that is conceived after a short ipi . secondly , the death of the previous infant might be related to an unhealthy physiological status of the mother and the wish to quickly replace the infant , thus shortening the ipi . subsequently , we constructed variables to represent the interaction between those two main independent variables in which we used different classifications for the ipi duration after the previous pregnancy outcomes . we tested for several confounding factors but , in the final model , included only four control variables that turned out to be of significance . age refers to the reproductive condition that contributes to a healthy pregnancy and the birth of a healthy infant . question v228 of the dhs women 's questionnaire asks the responding woman whether , at the time she became pregnant for the last pregnancy , she wished to become pregnant , she wished to wait until later , or she did not want to have any more children at all . the available dataset does not make a distinction between induced and spontaneous pregnancy losses , which is an omission seen in the different relations between the two types of abortions at the beginning of the ipi , length of the ipi , and pregnancy outcome found in other researches . the study of basinga and colleagues estimated the rate of induced abortion in rwanda in 2009 between 18 and 31 per 1,000 women in the 1544-year - old group . their study also revealed that the induced abortion rate was remarkably higher in the capital city kigali compared to the situation in the provinces . finally , we included the year of the interview to check for changes over time in reproductive health : notably , the extent of the possible reduction of fetal mortality . the results presented in table 1 illustrate that many rwandan women still have to deal with pregnancy losses , the death of an infant , and unwanted pregnancies : 36 out of 1000 last pregnancies in our sample population ended in a pregnancy loss . among the women with at least two pregnancies , 15 percent mourned a fatal outcome of the previous pregnancy : five percent had a pregnancy loss and nearly ten percent got a child that died in infancy . the results indicate also that the percentages of pregnancies ending in a pregnancy loss are the highest after an ipi shorter than 24 months that started after a pregnancy loss . higher percentages of pregnancy loss than the mean of 3.6 per cent were found after a live - born infant that died in its infancy and an ipi of more than two years and after a surviving live birth and a very long ipi ( > 60 months ) . the descriptive statistics in table 1 show a modest decline of the rate of fetal losses during the period under study . for the three consecutive research periods , the rate of pregnancy loss diminished from 41 out of 1000 pregnancies ( 19962000 ) to 36 ( 20012005 ) and finally to 33 in the most recent period 20062010 . it is difficult to assess if the total number of reported pregnancy losses in the three dhss used in this study , 36 per 1000 pregnancies , is in line with expectations or not . the frequency fits within an indication given in medical literature that states that the number of fetal losses in the month after conception is high but that after a gestation of 8 weeks the loss is about 3 percent . this could mean that women in rwanda did not mention losses that occurred in the first one or two months of a pregnancy , when they were not fully aware of being pregnant . the early pregnancy losses reported in the dhs are probably underestimated as in poor countries the number of stillbirths ( after a gestation of 28 weeks ) is higher compared to that of rich countries and the stillbirth rate in countries in the central part of sub - saharan africa varies between 25 and 40 or more per 1000 births . the cross tabulation presented in table 2 shows that after a pregnancy loss or the loss of an infant a large portion of the women want to replace this loss . very low numbers of wanted pregnancies are found among women who became pregnant within two years after the birth of the previous child that survived the first year of its life . from the women whose previous pregnancy ended with a fetal loss , 43 percent were expecting a child again within half a year . for women who had a live - born child that died afterwards in infancy , 71 percent were pregnant again within two years after the last delivery , which could point at a replacement effect or at a lack of protection against pregnancy . the constant ( table 3 ) reflects the risk of a pregnancy loss for the reference category : rural women in 2000 , in age category of 2135 years at the time of the last conception , whose last pregnancy was wanted and started after a healthy ipi ( 2559 months ) , and whose previous pregnancy resulted in a child that survived its infancy . the estimate of the risk of experiencing a pregnancy termination for these women is very low ( 2% ) . linking the risk of a pregnancy termination with both the outcome of the previous pregnancy and the length of the ipi ( interaction variables ) shows significant deviations from the risk estimated for the reference group : all except one point at a higher risk . the highest odds are found for women who became pregnant shortly after a previous pregnancy loss . women who conceived again within 3 months after the previous pregnancy loss are 3.68 times more likely to lose the next pregnancy than the reference group with a healthy interval . women with an ipi of more than two years after a previous fetal loss had a lower risk compared to the reference group , but the association is not significant . the higher odds of pregnancy loss for all groups with a previous pregnancy loss suggest that some women are prone to repeated losses , regardless of ipi duration . repeated or recurrent pregnancy loss is phenomenon that puzzles medical experts like gynecologist already for decades and that is probably associated with more than genetic factors of the woman alone [ 25 , 26 ] . after a live birth , regardless of whether the newborn survived its infancy or not , the likelihood of a pregnancy loss after an ipi considered as unhealthy ( < 2 years ) is not higher compared to the reference group with a recommended ipi duration . for the mother that became pregnant within , respectively , one to two years after the previous birth , the signs of the coefficients are negative but only significant for women whose infant stayed alive and conceived within one year . any pregnancy after a live birth seems to prepare for a successful next pregnancy , regardless of the interpregnancy interval . this mechanism vanishes after some years , as an ipi of more than 5 years results in a substantial higher likelihood of a pregnancy loss ( 1.6 times more likely ) . the risk of a pregnancy loss for mothers who are pregnant for the first time is of the same magnitude ( 1.5 times more likely ) . the physiological regression hypothesis states that after a very long ipi the body of a women has lost the beneficial physiological adaptations in her reproductive system that occur after a pregnancy [ 10 , 12 ] . this is reflected in the higher likelihood of a pregnancy loss ( 2.3 times more likely ) for women who were older than 35 years when they became pregnant . the positive coefficients found for urban women and for women who gave a vague answer or did not answer the question on whether they wanted the last pregnancy could point at the occurrence of induced pregnancy terminations . the higher risk of pregnancy losses among urban women in our sample fits in with research findings by basinga and colleagues who calculated that induced abortions occur more frequently in the capital city of kigali compared to other regions of rwanda . the finding that women who explicitly declared that the last pregnancy was not wanted have a significant lower risk of losing the next pregnancy gives food for thought . maybe these are highly fecund women who become pregnant easily and therefore more often unwanted , and who do not encounter pregnancy problems . we remark that the likelihood of a pregnancy termination decreased significantly between 2000 and 2010 . for 2005 , the sign of the coefficient ( ) is negative , but the decrease is not significant . further analyses , not shown here , showed that this decrease pertained to late pregnancy loss only . the first important result of our analyses is that one needs to take the previous pregnancy outcome into account when estimating the effects of ipis on the risk of a pregnancy loss . the second main finding is that negative outcomes ( in terms of a higher risk of recurrent pregnancy loss ) were found for ipis up to 24 months after a prior pregnancy loss , a period four times as long as the recommended healthy ipi of only 6 months . in contrast , an ipi shorter than 2 years after a live birth does not seem to increase significantly the risk of a pregnancy loss . we are aware that a pregnancy loss is not the only possible adverse pregnancy outcome . shorter ipis than two years after a live birth do not give higher risks of a pregnancy loss , but they will affect other pregnancy outcomes such as preterm birth , low birth weight , low apgar scores , and a higher neonatal death . we found clear indications for negative effects of the replacement mechanism after the loss of a pregnancy . the replacement wish after a fetal death leads to shorter ipis and therefore to a higher risk of another pregnancy loss . finally , the results of our study confirm the physiological regression hypothesis : a higher risk of a fetal death when ipis are longer than 5 years . also older women have a higher likelihood of a pregnancy loss compared to younger ones . to avoid a higher risk of a next miscarriage or stillbirth also in bangladesh , women should wait longer than the recommended 6 months ( up to 15 months ) to become pregnant again after a former pregnancy loss . the researchers found a significant increased risk of a pregnancy loss after a live birth and an ipi < 6 months . for longer ipi durations up to 74 months after a live birth , no significant higher risks of a pregnancy loss were found . based on the results of this study on bangladesh and ours on rwanda , one could conclude that , in societies without an advanced health care system , the who recommendations concerning spacing after a fetal loss still count . workers in the health care system should advise women , even if they are eager to become pregnant again , to take actions to prevent a quick new pregnancy and wait even longer than a year to become pregnant again . the improvements in the rwandan health care system between 2000 and 2010 and in particular the increased access to this system contributed to a lower pregnancy loss frequency . probably , the increased antenatal checks during the last pregnancy period had an impact , as the significant decrease in pregnancy losses between 2000 and 2010 resulted in particular in fewer late fetal losses ( after a pregnancy duration of 20 weeks ) . with a policy that recommends to women an ipi of at least a year to two years after a fetal death and more early pregnancy visits to the community health facility , a decrease in an early fetal death

depending on its pathomechanism , diabetes can be classified into two main types , namely , type-1 and type-2 diabetes mellitus ( dm ) . it is a well - known phenomenon that type-2 diabetes , most common form of diabetes , is dominated by hyperinsulinaemia , hyperglycaemia , and dyslipidaemia , while type-1 diabetes , due to the autoimmune - mediated destruction of the cells of the langerhans islets in the pancreas , is characterized by insulinopenia , hyperglycaemia , and a largely unaffected lipid profile . furthermore , the onset of type-1 diabetes is acute , unlike that of type-2 diabetes , which is characterized by a period of insulin resistance , hyperinsulinaemia , and euglycaemia preceding the onset of hyperglycaemia . although increased atherosclerosis of the coronary arteries is thought to be responsible for a major portion of the mortality in diabetic patients , there is evidence supporting the existence of diabetes - induced direct structural and functional alterations at the level of the myocardium . the development of diabetic cardiomyopathy has been described in both types of dm , also in the absence of coronary artery disease and hypertension , and is suggested to be a consequence of altered cellular metabolism of the myocardium , which increases the risk of heart failure . for decades , experimental studies on diabetic cardiomyopathy were performed on streptozotocin- ( stz- ) induced type-1-diabetic rodents , the most widely used , standard animal model of dm . nevertheless , the majority of diabetic patients have type-2 diabetes with a different pathophysiological background . therefore a number of animal models of type-2 dm have been developed and become object of intensive cardiovascular investigations recent years [ 35 ] . our research group previously provided a detailed hemodynamic characterisation of rat models of type-1 ( stz - induced ) and type-2 ( zucker diabetic fatty rats ) dm by the sophisticated method of left ventricular ( lv ) pressure - volume ( p - v ) analysis . different characteristics of type-1 and type-2 diabetic cardiac dysfunction could be demonstrated : decreased systolic performance and delayed relaxation , mainly in type-1 dm ; accompanied by increased diastolic stiffness of the heart , more remarkably in type-2 dm . the underlying pathophysiological features and subcellular mechanisms responsible for the development of diabetic cardiomyopathy are not completely understood . metabolic abnormalities and overproduction of reactive oxygen and nitrogen species may trigger various intracellular pathways and alter myocardial expression of different genes . increased nitrooxidative stress , cardiomyocyte hypertrophy , profibrotic signalling , and myocardial remodelling along with apoptotic processes have been reported to play a critical role in the development of cardiomyopathy in both types of dm [ 79 ] . to what extent these characteristic molecular and cellular changes develop in type-1 versus type-2 dm and how they correspond with the described differences in several aspects of cardiac dysfunction also remain to be elucidated . therefore , in the present study we aimed at describing myocardial histological and molecular changes which may be involved in different characteristics of lv dysfunction in the well - established rat models of type-1 and type-2 dm [ 6 , 10 ] . male 12-week - old sprague - dawley ( sd ; charles river , sulzfeld , germany ) , zucker diabetic fatty ( zdf ) , and zdf lean rats ( charles river , kingston , ny ) were housed in a room at 22 2c under 12-h light / dark cycles . sprague - dawley rats were fed a standard laboratory rat diet and zdf rats a diet of purina 5008 as recommended by the supplier and water ad libitum . all animals received humane care in compliance with the principles of laboratory animal care formulated by the national society for medical research and the guide for the care and use of laboratory animals prepared by the institute of laboratory animal resources and published by the national institutes of health ( nih publication no . type-1 dm was induced in rats ( n = 8) with a single injection of streptozotocin ( stz , 60 mg / kg , i.p . ) freshly dissolved in 0.1 m citrate buffer , ph = 4.5 . vehicle - treated animals served as nondiabetic controls ( stz control group , n = 10 ) . seventy - two hours after stz injection , a drop of blood was collected from the tail vein , and the blood glucose concentration was determined using a digital blood glucose meter and test strips ( accu - chek sensor , roche , mannheim , germany ) . animals with a random blood glucose level of > 15 mm were considered as diabetic and were included into the study ( stz - induced diabetic group ) . according to the pathophysiology and disease characteristics of type-1 dm , based on literature data and results of our previous studies , experiments on the rats the zdf rat is an inbred rat model that through genetic mutation and a special diet develops type-2 diabetes and related complications . homozygous recessive males ( fa / fa ) develop obesity , fasting hyperglycemia , and type-2 diabetes ( zdf diabetic group , n = 8) . homozygous dominant ( + /+ ) and heterozygous ( fa/+ ) lean genotypes remain normoglycemic ( zdf lean group , n = 9 ) . according to the pathophysiology and disease characteristics of type-2 dm , based on literature data and results of our previous studies , experiments on the rats urine samples were collected by punctuation of the urine bladder after hemodynamic measurements had been completed . blood and urine glucose levels were determined by a digital blood glucose meter and test strips ( accu - chek sensor , roche ) . rats were anesthetized with a mixture of ketamine ( 100 mg / kg ) and xylazine ( 3 mg / kg ) i.p . the animals were placed on controlled heating pads and core temperature measured via a rectal probe was maintained at 37c . a polyethylene catheter was inserted into the left external jugular vein for fluid administration . a 2f microtip p - v catheter ( spr-838 , millar instruments , houston , tx , usa ) was inserted into the right carotid artery and advanced into the ascending aorta . after stabilization for 5 min , the signals were continuously recorded at a sampling rate of 1000/s using a p - v conductance system ( mpvs-400 , millar instruments , houston , tx , usa ) , stored , and displayed on a personal computer by the powerlab chart5 software system ( adinstruments inc . in addition , p - v - loops at different preloads were registered by transiently compressing the inferior caval vein ( reducing preload ) under the diaphragm with a cotton - tipped applicator . with the help of a special p - v analysis program ( pvan , millar instruments , houston , tx , usa ) ejection fraction ( ef ) and the slope of the lv end - systolic p - v relationship ( espvr ) ( according to the parabolic curvilinear model ) lv end - diastolic pressure ( lvedp ) and the slope of the lv end - diastolic p - v relationship ( edpvr ) were calculated as reliable indexes of lv stiffness . the in vivo and in vitro volume calibrations of the conductance system were performed as described previously . hearts were fixed in buffered paraformaldehyde solution ( 4% ) and embedded in paraffin . then , 5-m thick sections were placed on adhesive slides and stained with hematoxylin - eosin and masson 's trichrome . light microscopic examination was performed with a zeiss microscope ( axio observer.z1 , carl zeiss , jena , germany ) and digital images were captured using an imaging software ( qcapture pro 6.0 , qimaging , canada ) . the amount of myocardial collagen was determined by semiquantitative morphometry scoring of masson 's trichrome - stained sections by one blinded observer as follows : 0 : absent , 1 : slight , 2 : moderate , and 3 : intense . the mean value of twenty randomly selected visual fields ( magnification 400x ) of free lv wall represents each sample . the detection was carried out using a commercial kit following the protocol provided by the manufacturer ( chemicon international , temecula , ca , usa ) . rehydrated sections were treated with 20 g / ml dnase - free proteinase - k ( sigma - aldrich , germany ) to retrieve antigenic epitopes , followed by 3% hydrogen peroxide to quench endogenous peroxidase activity . free 3-oh termini were labeled with digoxigenin - dutp for 1 h at 37c utilizing a terminal deoxynucleotidyl transferase reaction mixture ( chemicon international , temecula , ca , usa ) . incorporated digoxigenin - conjugated nucleotides were detected using a horseradish peroxidase conjugated anti - digoxigenin antibody and 3,3-diaminobenzidine . dehydrated sections were cleared in xylene , mounted with permount ( fischer scientific , germany ) , and coverslips were applied . based on the intensity and distribution of labelling , semiquantitative histomorphological assessment was performed using conventional microscopy . for assessment of tunel - labeled cells , the number of tunel positive and total cardiomyocyte nuclei was counted ( four microscopic examination fields characterizing each specimen ) , and an average percent value was calculated . data were expressed as fold change to the mean value of the corresponding nondiabetic control group . according to previously described methods we performed immunohistochemical staining for nitrotyrosine , a marker of nitrooxidative stress . semiquantitative histomorphological assessment was performed based on the intensity and distribution of labelling using conventional microscopy . after initially evaluating all corresponding tissue sections ( magnification 200x ) , the tissue section with the most intense labelling signals was used as a reference for maximum labelling intensity . nitrotyrosine levels were scored as follows : 0 : complete absence of immunoreactivity , 1 : weak area of staining , 2 : intermediate staining , and 3 : extensive staining . using the cella software ( olympus soft imaging solutions gmbh , germany ) , we measured the area of the objects in each class in each field , assigned an area score ( 1 10% positive cells , 2 = 1150% positive cells , 3 = 5180% positive cells , and 4 80% positive cells ) , and calculated an average score for the whole picture ( intensity score multiplied by area score , 012 ) . total rna was extracted from lv myocardial samples using the rneasy fibrous tissue mini kit ( qiagen , hilden , germany ) according to the manufacturer 's instructions . rna concentration and purity were determined photometrically ( 260 , 280 , and 230 nm ) . reverse transcription was performed with the quantitect reverse transcription kit ( qiagen , hilden , germany ) using 400 ng rna in a volume of 20 l . quantitative real - time pcrs were performed on the steponeplus real - time pcr system with taqman universal pcr mastermix and taqman gene expression assays ( applied biosystems , foster city , usa ) in case of endogenous antioxidant genes , or on the lightcycler480 system with the lightcycler480 probes master and universal probe library ( upl ) probes ( roche , mannheim , germany ) in case of all other genes . sample quantifications were normalized to glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) expression in case of endogenous antioxidants and to -actin expression in case of all other genes by using a pool of all cdnas from the control groups ( positive calibrator ) . identification numbers of taqman gene expression assays ( endogenous antioxidant gene targets ) as well as upl probes and sequences of primers used ( tib molbiol , berlin , germany ) ( all other gene targets ) are represented in table 1 . evaluation was performed with the stepone software 2.2.2 ( applied biosystems , foster city , usa ) and lightcycler 480 sw 1.5 software ( roche , mannheim , germany ) , respectively . myocardial proteins were extracted into a solution containing 8 m urea , 5 mm edta , 0.002% trasylol , 0.05 mm pmsf , 0.003% triton x-100 containing protease inhibitors ( roche , mannheim , germany ) . protein concentration was determined by a commercial kit according to the manufacturer 's protocol ( bca protein assay kit ; thermo scientific , rockford , usa ) . total protein homogenates ( 30 g ) were denatured , separated on sds - page gradient gels ( invitrogen , darmstadt , germany ) , and transferred to pvdf membrane ( invitrogen , darmstadt , germany ) . the blots were probed with an antibody specific to tgf-1 ( 1 : 1000 , santa cruz biotechnology , heidelberg , germany ) . the immunoreactive protein bands were developed using enhanced chemiluminescence system ( ecl plus , perkinelmer , rodgau - juegesheim , germany ) and measured by an imaging densitometer with image analysis software ( image j. nih , bethesda , md , usa ) . densitometric data of each sample was normalized to the mean value of the corresponding nondiabetic control group . all data are expressed as means standard error of the mean ( sem ) . an unpaired two - sided student 's t - test was used to compare parameters of diabetic and control rats in both models . compared with the corresponding control group , dm was associated with decreased body weight and increased blood and urine glucose levels in both models ( table 2 ) . heart weight to body weight ratio showed a marked tendency towards increased values in the diabetic animals of the type-2 dm model , while it reached a statistical significance in the type-1 dm model ( table 2 ) . mean arterial pressure did not significantly differ between diabetic rats and corresponding controls ( data not shown ) . p - v loop derived load - dependent and -independent contractility parameters ( ef and slope of espvr ) were significantly lower in stz - induced diabetic animals compared to nondiabetic controls , suggesting impaired systolic performance in type-1 dm . in contrast , compared with the corresponding control , type-2 diabetic zdf rats showed unaltered lv contractility ( figure 1(a ) ) . lvedp and the slope of edpvr were significantly increased in diabetic animals of the type-2 dm model , indicating a marked increase in end - diastolic stiffness . these changes could be observed only to a lower extent in type-1 diabetic rats ( figure 1(b ) ) . the characteristics of diabetic cardiomyopathy were found in lv sections of the diabetic groups with hematoxylin - eosin staining ( figure 2(a ) ) . disarray and collapse of myofibers and myocardial mean cardiomyocyte width , as a marker of cardiomyocyte hypertrophy was significantly increased in both diabetic groups when compared to the corresponding controls ( figure 3(a ) ) . compared with the corresponding controls , diabetic hearts were associated with increased density of tunel - positive nuclei , indicating dna fragmentation ( figures 2(b ) and 3(b ) ) and increased immunoreactivity against the nitrooxidative stress marker nitrotyrosine , as evidenced by increased brown staining ( figure 2(c ) ) . moreover , both tunel positivity and nitrooxidative stress were found to be more pronounced in type-1 when compared with the type-2 dm group ( figure 3(c ) ) . quantitative real - time pcr from lv myocardial rna extracts revealed that mrna - expression for endogenous antioxidants cytosolic superoxide dismutase 1 ( sod1 ) , catalase , glutathione reductase ( gsr ) , and thioredoxin was significantly upregulated in type-1 dm , while these changes were less pronounced or absent in the type-2 dm model . mrna expression for the mitochondrial superoxide dismutase 2 ( sod2 ) was not altered in any groups studied . myocardial fibrotic remodelling , as reflected by masson 's trichrome staining of myocardial sections , was significantly more pronounced in the type-1 dm model , when compared to type-2 dm ( figure 2(d ) ) . semiquantitative scoring of the staining showed a marked difference in lv fibrosis between the two diabetic models ( figure 3(d ) ) . quantitative real - time pcr from lv myocardial rna extracts revealed that mrna expression for -myosin heavy chain ( -mhc ) was significantly decreased whereas -mhc and endothelin-1 mrna - levels were increased in both diabetic groups compared to corresponding controls ( figure 4 ) . whereas c - fos , c - jun , and caspase-12 mrna - expression was significantly upregulated , collagen-1 , collagen-3 , and endothelial nos mrna - levels were downregulated only in type-1 dm when compared with the control group ( figure 4 ) . atrial natriuretic factor ( anf ) mrna expression was upregulated in both types of dm , but the change reached the level of statistical significance only in the type-2 model ( figure 4 ) . the gene expression changes for -mhc , -mhc , c - fos , c - jun , caspase-12 , collagen-1 , collagen-3 , and endothelial nos were significantly more pronounced in type-1 when compared to the type-2 dm group ( figure 4 ) . in western blots , we detected tgf-1 protein in rat myocardium as a single band at 25 kda . densitometric analysis of the bands revealed a significant increase in relative tgf-1 protein content in myocardium from type-1 diabetic rats in comparison with the corresponding controls . in contrast , significantly decreased tgf-1 protein expression could be detected in the type-2 dm model ( figure 5 ) . in this study , a comparative investigation of functional , histological , and molecular changes in the heart of type-1 and type-2 diabetic rats was performed . the reported differences in cardiac performance between the two diabetes models were accompanied by an altered pattern of myocardial gene and protein expression and markers of nitrooxidative stress , apoptosis , cardiomyocyte hypertrophy , and fibrotic remodelling of the myocardium . this is the first study reporting characteristic differences on the cellular and subcellular level between rat models of type-1 and type-2 diabetic cardiomyopathy . in spite of the clinical importance of diabetic cardiomyopathy as a distinct disease entity , the cellular and molecular pathomechanisms triggering the adverse changes in diabetic myocardial structure and function have not been completely understood . oxidative imbalance and increased nitrooxidative stress , cardiomyocyte apoptosis , and hypertrophy , as well as myocardial fibrosis , have been suggested to be the key pathophysiological features of development and progression of diabetic cardiac complications . immunohistochemical staining against nitrotyrosine ( nt ) , a marker for nitrooxidative stress , clearly showed increased nt - immunoreactivity in the diabetic myocardium in both diabetes models , which is in line with previous data [ 13 , 14 ] . according to the semiquantitative analysis of the staining , more pronounced nitrooxidative stress could be documented in the type-1 dm model , when compared with type-2 dm . the compensatory overexpression of endogenous antioxidant systems ( such as the sod - catalase and glutathione systems or thioredoxin ) further confirmed a more significant increase of nitrooxidative stress in the myocardium of our type-1 diabetic animals when compared with type-2 dm . increased nitrooxidative stress dysregulation of apoptosis , a programmed form of cell death , is implicated in several cardiovascular pathologies , including diabetic cardiomyopathy . increasing body of evidence from both clinical and experimental observations suggests that progressive loss of cardiomyocytes via apoptosis plays an important causal role in the development of diabetic cardiomyopathy [ 8 , 9 , 15 ] . increased cardiomyocyte apoptosis is involved in the process of transition from compensated to decompensated ventricular dysfunction in the diabetic heart as dead cardiomyocytes are replaced by extracellular matrix components , which leads to the development of collagen deposition and myocardial fibrosis [ 17 , 18 ] . tunel assay is a widely used method for assessing dna breaks and dna fragmentation in various cells and thus it has been proposed for detection of cells undergoing apoptosis . our current results regarding tunel - positive cardiomyocyte nuclei ( figures 2 and 3(b ) ) as well as myocardial expression of the proapoptotic mediator caspase-12 ( figure 4 ) show increased dna fragmentation and cardiomyocyte apoptosis in diabetic animals , more remarkably in the type-1 dm model . correspondingly , previous studies reported diabetes - associated increase in tunel positive cardiomyocyte cell nuclei both in type-1 and type-2 diabetic rats , but a direct comparison of the two dm models was not available yet . cardiomyocyte width showed a significant increase in diabetic rats which was comparable in both models ( figure 3(a ) ) . an increasing tendency in heart weight / body weight ratio was observed in type-2 diabetic rats , which reached the level of statistical significance only in the type-1 dm model ; however , this index is severely influenced by marked body weight changes ( that were more pronounced in the type-1 dm model ) . molecular mechanisms of the hypertrophic transcriptional program include the transient activation of immediate - early genes that encode transcription factors such as c - jun and c - fos [ 21 , 22 ] . c - jun and c - fos have been described to activate the fetal gene program ( transcription of genes such as -mhc or anf ) , which play a key role in adaptive hypertrophy and also in maladaptive changes of cardiomyocytes , and have been widely used as markers of pathological hypertrophy . decreased expression of -mhc and increase in the fetal type -mhc , as molecular markers of pathological hypertrophy , were present in both dm models , being more prominent in type-1 dm ( figure 4 ) . the rate of myocardial overexpression of the hypertrophy marker anf was found to be comparable in the two dm models ( figure 4 ) . these results clearly reflect the existence of myocardial hypertrophy associated with diabetes in type-1 and also in type-2 dm models and are in correspondence with previous data [ 9 , 24 , 25 ] . fibrotic remodelling of the myocardium has been reported to be another key pathophysiological feature in diabetic cardiomyopathy . profibrotic signalling and interstitial collagen deposition are triggered by overproduction of reactive oxygen and nitrogen species ( ros / rns ) and/or could be a consequence of apoptotic cardiomyocyte death ( replacement fibrosis ) . a more pronounced increase in nitrooxidative stress and apoptosis , along with markedly elevated myocardial content of the profibrotic protein mediator tgf-1 ( figure 5 ) , and significantly higher histopathological fibrosis score values ( figure 3(d ) ) have been demonstrated in type-1 diabetic rats ( fibrosis + ~260% ) when compared to zdf rats with type-2 dm ( fibrosis + ~45% ) . although the existence of myocardial fibrosis in stz - induced type-1 dm has been well recognized and widely described [ 9 , 25 , 26 ] , there are controversial data regarding the zdf type-2 dm model . reported no myocardial collagen deposition in zdf diabetic animals at 14 weeks of age ; other studies demonstrated only perivascular but not interstitial myocardial fibrosis at the age of 19 weeks , while a robust fibrotic remodelling of the left ventricle has been described in 45-week - old zdf rats . differences regarding the age of the animals and thus different diabetes duration could be the reason for these discrepancies . our present results showing ~45% increase in myocardial interstitial fibrosis score in 3032-week - old zdf diabetic rats ( figure 3(d ) ) are in accordance with these literature data . interestingly , a much shorter diabetes duration ( 8 weeks ) leads to a more severe myocardial fibrosis in the type-1 compared to the type-2 dm model ( 3032 weeks of age corresponds to 2325 weeks of diabetes duration ) . although the severity of dm ( degree of hyperglycemia ) was found to be the same at the end of our investigations ( table 2 ) , pathophysiological / metabolic differences ( insulinopenia versus hyperinsulinaemia and insulin resistance in the first phase ) and the different time course of dm between the two types of the disease might explain these characteristics . in accordance with previous observations increased fibrosis score values of the masson 's trichrome staining in the diabetic myocardium were associated with depressed mrna expression of collagens 1 and 3 ( figure 4 ) , suggesting that altered rate of collagen degradation rather than enhanced synthesis is responsible for the observed interstitial fibrosis . diabetic cardiomyopathy is associated with changes in the expression and bioavailability of vasoactive factors released from the endothelium of coronary microvasculature , including upregulation of endothelin-1 ( et-1 ) and downregulation of nitric oxide ( no ) . in accordance with these observations we detected a marked increase in the expression of et-1 in diabetic heart of both dm models ( more pronounced in type-1 dm ) along with decreased enos expression only in animals with type-1 diabetes ( figure 4 ) . these results suggest more severe cardiac microvascular abnormalities in type-1 versus type-2 dm . increased et-1 levels in dm have been associated with the development of myocardial fibrotic remodeling through the accumulation of fibroblasts , mediated by the et-1-induced endothelial - to - mesenchymal transition process . this mechanism might be reflected by the above discussed corresponding differences in the severity of myocardial fibrosis between the two dm models . the functional alterations described in the diabetic heart are closely associated with molecular and histopathological evidence of cardiomyocyte hypertrophy , apoptosis , and fibrosis as discussed above . the contributing components of these three major features to diastolic ( heart failure with preserved ejection fraction , hfpef ) versus systolic dysfunction may be somewhat different . both cardiomyocyte hypertrophy and cardiac fibrosis are evident in hfpef , but the latter along with increased cardiomyocyte apoptosis is reported to be more prominent in systolic heart failure [ 9 , 17 , 18 ] . in contrast to diabetes - associated hfpef , myocardial systolic dysfunction in dm may have a greater reliance on cardiomyocyte - generated ros , cardiomyocyte apoptosis and subsequent extracellular matrix deposition , and fibrotic remodelling of the myocardium [ 17 , 31 ] . although not investigated in the present study , additional molecular mechanisms ( such as advanced glycation end products ( age ) deposition , collagen cross - linking , and oxidative modifications of sarcomeric proteins ) might also contribute to the development of diastolic dysfunction in diabetic animals . according to the above discussed pathophysiological changes , our previously published and current results on in vivo cardiac function ( lv p - v analysis , figure 1 ) showed corresponding differences in the characteristics of type-1 and type-2 diabetic cardiac dysfunction . a marked impairment of lv contractility and systolic dysfunction ( decreased ef and slope of espvr ) could be demonstrated only in type-1 dm , which was accompanied by severe nitrooxidative stress , cardiomyocyte apoptosis , and myocardial fibrosis . in contrast to the type-1 dm model , these pathological features were significantly less pronounced or absent , while cardiomyocyte hypertrophy was comparable in type-2 dm . zdf type-2 diabetic animals showed a marked increase in diastolic stiffness ( increased lvedp and slope of edpvr ) without impairment of systolic performance of the heart ( characteristic for hfpef ) . we hypothesize that pathophysiological differences in the disease development , severity , and progression between the two different types of dm could be the key factors in the observed functional and molecular characteristics of diabetic cardiomyopathy in type-1 versus type-2 dm . in the present study we have provided for the first time a direct comparison of type-1 and type-2 diabetic cardiomyopathy in widely used rodent models . we have shown characteristic functional differences between the two models by lv p - v analysis and revealed the underlying mechanisms : an altered pattern of key pathophysiological features ( nitrooxidative stress , cardiomyocyte apoptosis and hypertrophy , and myocardial fibrosis ) in the diabetic heart has been observed . our current results help to understand the pathomechanisms of diabetic cardiomyopathy in type-1 and type-2 dm and can serve as a basis for further experimental research .

increasing evidence suggests that both types of diabetes mellitus ( dm ) lead to cardiac structural and functional changes . in this study we investigated and compared functional characteristics and underlying subcellular pathological features in rat models of type-1 and type-2 diabetic cardiomyopathy . type-1 dm was induced by streptozotocin . for type-2 dm , zucker diabetic fatty ( zdf ) rats were used . left ventricular pressure - volume analysis was performed to assess cardiac function . myocardial nitrotyrosine immunohistochemistry , tunel assay , hematoxylin - eosin , and masson 's trichrome staining were performed . mrna and protein expression were quantified by qrt - pcr and western blot . marked systolic dysfunction in type-1 dm was associated with severe nitrooxidative stress , apoptosis , and fibrosis . these pathological features were less pronounced or absent , while cardiomyocyte hypertrophy was comparable in type-2 dm , which was associated with unaltered systolic function and increased diastolic stiffness . mrna - expression of hypertrophy markers c - fos , c - jun , and -mhc , as well as pro - apoptotic caspase-12 , was elevated in type-1 , while it remained unaltered or only slightly increased in type-2 dm . expression of the profibrotic tgf-1 was upregulated in type-1 and showed a decrease in type-2 dm . we compared type-1 and type-2 diabetic cardiomyopathy in standard rat models and described an altered pattern of key pathophysiological features in the diabetic heart and corresponding functional consequences .

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

depending on its pathomechanism , diabetes can be classified into two main types , namely , type-1 and type-2 diabetes mellitus ( dm ) . it is a well - known phenomenon that type-2 diabetes , most common form of diabetes , is dominated by hyperinsulinaemia , hyperglycaemia , and dyslipidaemia , while type-1 diabetes , due to the autoimmune - mediated destruction of the cells of the langerhans islets in the pancreas , is characterized by insulinopenia , hyperglycaemia , and a largely unaffected lipid profile . furthermore , the onset of type-1 diabetes is acute , unlike that of type-2 diabetes , which is characterized by a period of insulin resistance , hyperinsulinaemia , and euglycaemia preceding the onset of hyperglycaemia . although increased atherosclerosis of the coronary arteries is thought to be responsible for a major portion of the mortality in diabetic patients , there is evidence supporting the existence of diabetes - induced direct structural and functional alterations at the level of the myocardium . the development of diabetic cardiomyopathy has been described in both types of dm , also in the absence of coronary artery disease and hypertension , and is suggested to be a consequence of altered cellular metabolism of the myocardium , which increases the risk of heart failure . for decades , experimental studies on diabetic cardiomyopathy were performed on streptozotocin- ( stz- ) induced type-1-diabetic rodents , the most widely used , standard animal model of dm . therefore a number of animal models of type-2 dm have been developed and become object of intensive cardiovascular investigations recent years [ 35 ] . our research group previously provided a detailed hemodynamic characterisation of rat models of type-1 ( stz - induced ) and type-2 ( zucker diabetic fatty rats ) dm by the sophisticated method of left ventricular ( lv ) pressure - volume ( p - v ) analysis . different characteristics of type-1 and type-2 diabetic cardiac dysfunction could be demonstrated : decreased systolic performance and delayed relaxation , mainly in type-1 dm ; accompanied by increased diastolic stiffness of the heart , more remarkably in type-2 dm . the underlying pathophysiological features and subcellular mechanisms responsible for the development of diabetic cardiomyopathy are not completely understood . increased nitrooxidative stress , cardiomyocyte hypertrophy , profibrotic signalling , and myocardial remodelling along with apoptotic processes have been reported to play a critical role in the development of cardiomyopathy in both types of dm [ 79 ] . to what extent these characteristic molecular and cellular changes develop in type-1 versus type-2 dm and how they correspond with the described differences in several aspects of cardiac dysfunction also remain to be elucidated . therefore , in the present study we aimed at describing myocardial histological and molecular changes which may be involved in different characteristics of lv dysfunction in the well - established rat models of type-1 and type-2 dm [ 6 , 10 ] . male 12-week - old sprague - dawley ( sd ; charles river , sulzfeld , germany ) , zucker diabetic fatty ( zdf ) , and zdf lean rats ( charles river , kingston , ny ) were housed in a room at 22 2c under 12-h light / dark cycles . type-1 dm was induced in rats ( n = 8) with a single injection of streptozotocin ( stz , 60 mg / kg , i.p . ) according to the pathophysiology and disease characteristics of type-1 dm , based on literature data and results of our previous studies , experiments on the rats the zdf rat is an inbred rat model that through genetic mutation and a special diet develops type-2 diabetes and related complications . homozygous recessive males ( fa / fa ) develop obesity , fasting hyperglycemia , and type-2 diabetes ( zdf diabetic group , n = 8) . according to the pathophysiology and disease characteristics of type-2 dm , based on literature data and results of our previous studies , experiments on the rats urine samples were collected by punctuation of the urine bladder after hemodynamic measurements had been completed . the in vivo and in vitro volume calibrations of the conductance system were performed as described previously . then , 5-m thick sections were placed on adhesive slides and stained with hematoxylin - eosin and masson 's trichrome . the amount of myocardial collagen was determined by semiquantitative morphometry scoring of masson 's trichrome - stained sections by one blinded observer as follows : 0 : absent , 1 : slight , 2 : moderate , and 3 : intense . using the cella software ( olympus soft imaging solutions gmbh , germany ) , we measured the area of the objects in each class in each field , assigned an area score ( 1 10% positive cells , 2 = 1150% positive cells , 3 = 5180% positive cells , and 4 80% positive cells ) , and calculated an average score for the whole picture ( intensity score multiplied by area score , 012 ) . identification numbers of taqman gene expression assays ( endogenous antioxidant gene targets ) as well as upl probes and sequences of primers used ( tib molbiol , berlin , germany ) ( all other gene targets ) are represented in table 1 . compared with the corresponding control group , dm was associated with decreased body weight and increased blood and urine glucose levels in both models ( table 2 ) . heart weight to body weight ratio showed a marked tendency towards increased values in the diabetic animals of the type-2 dm model , while it reached a statistical significance in the type-1 dm model ( table 2 ) . p - v loop derived load - dependent and -independent contractility parameters ( ef and slope of espvr ) were significantly lower in stz - induced diabetic animals compared to nondiabetic controls , suggesting impaired systolic performance in type-1 dm . lvedp and the slope of edpvr were significantly increased in diabetic animals of the type-2 dm model , indicating a marked increase in end - diastolic stiffness . the characteristics of diabetic cardiomyopathy were found in lv sections of the diabetic groups with hematoxylin - eosin staining ( figure 2(a ) ) . disarray and collapse of myofibers and myocardial mean cardiomyocyte width , as a marker of cardiomyocyte hypertrophy was significantly increased in both diabetic groups when compared to the corresponding controls ( figure 3(a ) ) . compared with the corresponding controls , diabetic hearts were associated with increased density of tunel - positive nuclei , indicating dna fragmentation ( figures 2(b ) and 3(b ) ) and increased immunoreactivity against the nitrooxidative stress marker nitrotyrosine , as evidenced by increased brown staining ( figure 2(c ) ) . moreover , both tunel positivity and nitrooxidative stress were found to be more pronounced in type-1 when compared with the type-2 dm group ( figure 3(c ) ) . quantitative real - time pcr from lv myocardial rna extracts revealed that mrna - expression for endogenous antioxidants cytosolic superoxide dismutase 1 ( sod1 ) , catalase , glutathione reductase ( gsr ) , and thioredoxin was significantly upregulated in type-1 dm , while these changes were less pronounced or absent in the type-2 dm model . myocardial fibrotic remodelling , as reflected by masson 's trichrome staining of myocardial sections , was significantly more pronounced in the type-1 dm model , when compared to type-2 dm ( figure 2(d ) ) . semiquantitative scoring of the staining showed a marked difference in lv fibrosis between the two diabetic models ( figure 3(d ) ) . quantitative real - time pcr from lv myocardial rna extracts revealed that mrna expression for -myosin heavy chain ( -mhc ) was significantly decreased whereas -mhc and endothelin-1 mrna - levels were increased in both diabetic groups compared to corresponding controls ( figure 4 ) . whereas c - fos , c - jun , and caspase-12 mrna - expression was significantly upregulated , collagen-1 , collagen-3 , and endothelial nos mrna - levels were downregulated only in type-1 dm when compared with the control group ( figure 4 ) . atrial natriuretic factor ( anf ) mrna expression was upregulated in both types of dm , but the change reached the level of statistical significance only in the type-2 model ( figure 4 ) . the gene expression changes for -mhc , -mhc , c - fos , c - jun , caspase-12 , collagen-1 , collagen-3 , and endothelial nos were significantly more pronounced in type-1 when compared to the type-2 dm group ( figure 4 ) . in contrast , significantly decreased tgf-1 protein expression could be detected in the type-2 dm model ( figure 5 ) . in this study , a comparative investigation of functional , histological , and molecular changes in the heart of type-1 and type-2 diabetic rats was performed . the reported differences in cardiac performance between the two diabetes models were accompanied by an altered pattern of myocardial gene and protein expression and markers of nitrooxidative stress , apoptosis , cardiomyocyte hypertrophy , and fibrotic remodelling of the myocardium . this is the first study reporting characteristic differences on the cellular and subcellular level between rat models of type-1 and type-2 diabetic cardiomyopathy . in spite of the clinical importance of diabetic cardiomyopathy as a distinct disease entity , the cellular and molecular pathomechanisms triggering the adverse changes in diabetic myocardial structure and function have not been completely understood . oxidative imbalance and increased nitrooxidative stress , cardiomyocyte apoptosis , and hypertrophy , as well as myocardial fibrosis , have been suggested to be the key pathophysiological features of development and progression of diabetic cardiac complications . immunohistochemical staining against nitrotyrosine ( nt ) , a marker for nitrooxidative stress , clearly showed increased nt - immunoreactivity in the diabetic myocardium in both diabetes models , which is in line with previous data [ 13 , 14 ] . according to the semiquantitative analysis of the staining , more pronounced nitrooxidative stress could be documented in the type-1 dm model , when compared with type-2 dm . the compensatory overexpression of endogenous antioxidant systems ( such as the sod - catalase and glutathione systems or thioredoxin ) further confirmed a more significant increase of nitrooxidative stress in the myocardium of our type-1 diabetic animals when compared with type-2 dm . increased nitrooxidative stress dysregulation of apoptosis , a programmed form of cell death , is implicated in several cardiovascular pathologies , including diabetic cardiomyopathy . increasing body of evidence from both clinical and experimental observations suggests that progressive loss of cardiomyocytes via apoptosis plays an important causal role in the development of diabetic cardiomyopathy [ 8 , 9 , 15 ] . increased cardiomyocyte apoptosis is involved in the process of transition from compensated to decompensated ventricular dysfunction in the diabetic heart as dead cardiomyocytes are replaced by extracellular matrix components , which leads to the development of collagen deposition and myocardial fibrosis [ 17 , 18 ] . our current results regarding tunel - positive cardiomyocyte nuclei ( figures 2 and 3(b ) ) as well as myocardial expression of the proapoptotic mediator caspase-12 ( figure 4 ) show increased dna fragmentation and cardiomyocyte apoptosis in diabetic animals , more remarkably in the type-1 dm model . correspondingly , previous studies reported diabetes - associated increase in tunel positive cardiomyocyte cell nuclei both in type-1 and type-2 diabetic rats , but a direct comparison of the two dm models was not available yet . cardiomyocyte width showed a significant increase in diabetic rats which was comparable in both models ( figure 3(a ) ) . an increasing tendency in heart weight / body weight ratio was observed in type-2 diabetic rats , which reached the level of statistical significance only in the type-1 dm model ; however , this index is severely influenced by marked body weight changes ( that were more pronounced in the type-1 dm model ) . molecular mechanisms of the hypertrophic transcriptional program include the transient activation of immediate - early genes that encode transcription factors such as c - jun and c - fos [ 21 , 22 ] . c - jun and c - fos have been described to activate the fetal gene program ( transcription of genes such as -mhc or anf ) , which play a key role in adaptive hypertrophy and also in maladaptive changes of cardiomyocytes , and have been widely used as markers of pathological hypertrophy . decreased expression of -mhc and increase in the fetal type -mhc , as molecular markers of pathological hypertrophy , were present in both dm models , being more prominent in type-1 dm ( figure 4 ) . the rate of myocardial overexpression of the hypertrophy marker anf was found to be comparable in the two dm models ( figure 4 ) . these results clearly reflect the existence of myocardial hypertrophy associated with diabetes in type-1 and also in type-2 dm models and are in correspondence with previous data [ 9 , 24 , 25 ] . fibrotic remodelling of the myocardium has been reported to be another key pathophysiological feature in diabetic cardiomyopathy . a more pronounced increase in nitrooxidative stress and apoptosis , along with markedly elevated myocardial content of the profibrotic protein mediator tgf-1 ( figure 5 ) , and significantly higher histopathological fibrosis score values ( figure 3(d ) ) have been demonstrated in type-1 diabetic rats ( fibrosis + ~260% ) when compared to zdf rats with type-2 dm ( fibrosis + ~45% ) . although the existence of myocardial fibrosis in stz - induced type-1 dm has been well recognized and widely described [ 9 , 25 , 26 ] , there are controversial data regarding the zdf type-2 dm model . reported no myocardial collagen deposition in zdf diabetic animals at 14 weeks of age ; other studies demonstrated only perivascular but not interstitial myocardial fibrosis at the age of 19 weeks , while a robust fibrotic remodelling of the left ventricle has been described in 45-week - old zdf rats . interestingly , a much shorter diabetes duration ( 8 weeks ) leads to a more severe myocardial fibrosis in the type-1 compared to the type-2 dm model ( 3032 weeks of age corresponds to 2325 weeks of diabetes duration ) . although the severity of dm ( degree of hyperglycemia ) was found to be the same at the end of our investigations ( table 2 ) , pathophysiological / metabolic differences ( insulinopenia versus hyperinsulinaemia and insulin resistance in the first phase ) and the different time course of dm between the two types of the disease might explain these characteristics . in accordance with previous observations increased fibrosis score values of the masson 's trichrome staining in the diabetic myocardium were associated with depressed mrna expression of collagens 1 and 3 ( figure 4 ) , suggesting that altered rate of collagen degradation rather than enhanced synthesis is responsible for the observed interstitial fibrosis . diabetic cardiomyopathy is associated with changes in the expression and bioavailability of vasoactive factors released from the endothelium of coronary microvasculature , including upregulation of endothelin-1 ( et-1 ) and downregulation of nitric oxide ( no ) . in accordance with these observations we detected a marked increase in the expression of et-1 in diabetic heart of both dm models ( more pronounced in type-1 dm ) along with decreased enos expression only in animals with type-1 diabetes ( figure 4 ) . these results suggest more severe cardiac microvascular abnormalities in type-1 versus type-2 dm . the functional alterations described in the diabetic heart are closely associated with molecular and histopathological evidence of cardiomyocyte hypertrophy , apoptosis , and fibrosis as discussed above . in contrast to diabetes - associated hfpef , myocardial systolic dysfunction in dm may have a greater reliance on cardiomyocyte - generated ros , cardiomyocyte apoptosis and subsequent extracellular matrix deposition , and fibrotic remodelling of the myocardium [ 17 , 31 ] . although not investigated in the present study , additional molecular mechanisms ( such as advanced glycation end products ( age ) deposition , collagen cross - linking , and oxidative modifications of sarcomeric proteins ) might also contribute to the development of diastolic dysfunction in diabetic animals . according to the above discussed pathophysiological changes , our previously published and current results on in vivo cardiac function ( lv p - v analysis , figure 1 ) showed corresponding differences in the characteristics of type-1 and type-2 diabetic cardiac dysfunction . a marked impairment of lv contractility and systolic dysfunction ( decreased ef and slope of espvr ) could be demonstrated only in type-1 dm , which was accompanied by severe nitrooxidative stress , cardiomyocyte apoptosis , and myocardial fibrosis . in contrast to the type-1 dm model , these pathological features were significantly less pronounced or absent , while cardiomyocyte hypertrophy was comparable in type-2 dm . zdf type-2 diabetic animals showed a marked increase in diastolic stiffness ( increased lvedp and slope of edpvr ) without impairment of systolic performance of the heart ( characteristic for hfpef ) . we hypothesize that pathophysiological differences in the disease development , severity , and progression between the two different types of dm could be the key factors in the observed functional and molecular characteristics of diabetic cardiomyopathy in type-1 versus type-2 dm . in the present study we have provided for the first time a direct comparison of type-1 and type-2 diabetic cardiomyopathy in widely used rodent models . we have shown characteristic functional differences between the two models by lv p - v analysis and revealed the underlying mechanisms : an altered pattern of key pathophysiological features ( nitrooxidative stress , cardiomyocyte apoptosis and hypertrophy , and myocardial fibrosis ) in the diabetic heart has been observed . our current results help to understand the pathomechanisms of diabetic cardiomyopathy in type-1 and type-2 dm and can serve as a basis for further experimental research .

obesity has recently been recognized as a chronic disease condition necessitating appropriate medical treatment and not merely a transient condition that can be ameliorated simply with diet and exercise alone . the increase in obesity , coupled with increased insulin resistance ( ir ) and t2 dm , represents a healthcare crisis in developed and developing countries . obesity is also associated with a host of other comorbidities including sleep apnoea , hypogonadism ( testosterone deficiency , td ) , dyslipidaemia , and hypertension . the prevalence of obesity and t2 dm in the usa is higher than reported in other parts of the world and obesity is now considered a disease condition to be treated as any other disease conditions . the prevalence of diabetes increased by 7.3% and obesity increased by 7.8% during the past decade . this increased risk was attributed to release of nonesterified fatty acids , glycerol , hormones , proinflammatory cytokines , and other factors from adipose tissue in obese individuals . the risk of developing t2 dm may be reduced by 5060% with a modest weight loss ( wl ) of 57% of body weight . huang et al . projected the distribution of newly diagnosed , undiagnosed , and established cases of diabetes in the usa from 2009 to 2034 and estimated that the number of people with diagnosed and undiagnosed , diabetes will increase from 23.7 million to 44.1 million during this period . the obesity distribution in the population without diabetes will remain stable over time with ~65% of individuals of the population being overweight or obese . reported that visceral fat accumulation is associated with increased insulin and c - peptide and with glucose intolerance . in the early 1970s , sims et al . coined the term diabesity to describe the strong common link between diabetes and obesity , when they exist in the same individual . the risk of t2 dm increases with body weight gain and obesity [ 812 ] and , more importantly , visceral fat accumulation reduces insulin sensitivity and increases ir , thus increasing risk of t2 dm . it is estimated that the risk of diabetes worldwide will exceed 171 million and may reach 366 million by the year 2030 . because increases in obesity are paralleled with increases in t2 dm , the diagnosis of obesity and t2 dm in the same individual presents clinical and therapeutic challenges to healthcare providers . it should be recognized that t2 dm complications contribute to cardiovascular disease ( cvd ) , stroke , neuropathy , nephropathy , and retinopathy . on the other hand , obesity confers increased hypertension , dyslipidaemia , stroke , cancer , depression , and obstructive sleep apnoea in addition to t2 dm . thus , the combination of t2 dm and obesity in the same individual ( diabesity ) will have even more complications than either condition alone . the presence of obesity and t2 dm in the same individual represents a complex relationship between these conditions . clearly , it is established that the increased incidence of obesity and t2 dm contributes to higher incidence of cvd , hypertension , stroke , cancer , and increased mortality [ 17 , 18 ] . the frequency of cvd and t2 dm increased with increased body mass index ( bmi ) and wc . this relationship between wc , cvd , and t2 dm was noted even in patients with bmi 25 kg / m . recent studies have provided a critical assessment of the therapeutic options in diabetes and obesity and highlighted the various approaches used to date , including ( a ) antidiabetics , ( b ) incretin and glucagon like peptide-1 ( glp-1 ) receptor agonists and dipeptidyl peptidase inhibitors , ( c ) lifestyle modifications , ( d ) antiobesity agents , and ( e ) bariatric surgery [ 1 , 19 ] . for instance , a recent report showed that bariatric surgery resulted in significant and sustained remission and improvement of t2 dm and other metabolic factors in severely obese patients . indeed , some of the therapeutic strategies discussed above will be met with success in some patients while in others it may not . there remains a need for new and innovative alternative approaches to the management of diabetes and obesity . recently , angiopoietin - like proteins ( angptls ) have been suggested as targets for treatment of obesity . it was proposed that suppression of expression of angptl2 and increased expression of angptl6 may represent a therapeutic target for treatment of obesity . several studies have suggested that td may contribute to development of obesity , ir , and t2 dm and t therapy of men with td may ameliorate these conditions [ 2228 ] . men with td treated with t therapy experienced a positive effect on visceral obesity , as determined by reduction in body weight ( 2.66% ) , waist - hip ratio ( 3.96% ) , and body fat ( 5.65% ) when compared to the control group . this treatment also resulted in decreased fasting blood glucose and mean glycated haemoglobin ( hba1c ) . kapoor et al . also demonstrated that t therapy in men with t2 dm reduced visceral adiposity and reduced homoeostasis model assessment ( homa ) index , hba1c , and fasting glucose , suggesting improvement in insulin sensitivity and glycaemic control in men with td and t2 dm . jones et al . examined the effects of t therapy on ir , cvd risk , and symptoms of t deficiency in men with t2 dm and/or mets over a 12-month trial period . t therapy improved glycaemic control and reduced homa - ir as well as hba1c , suggesting a beneficial effect of t therapy in men with t2 dm . in a study by aversa et al . , in which the effects of t therapy on homoeostasis model assessment - estimated insulin resistance ( homa - ir ) , carotid intima media thickness ( cimt ) , and high - sensitivity c - reactive protein ( hscrp ) were investigated in men with td and metabolic syndrome ( mets ) , t therapy significantly improved homa - ir , cimt , and hscrp as compared to the placebo treated group . in another study in men with mets , kalinchenko et al . found significant decreases in weight , bmi , wc , and homa - ir . the effects of t therapy in 87 diabetic men with coronary artery disease ( cad ) were investigated for a period of 12 weeks . t treatment significantly reduced the number of anginal attacks , silent ischaemic episodes , and total ischaemic burden , as compared with the placebo group . total cholesterol ( tc ) , plasma triglycerides ( tg ) , and homa index were also significantly reduced in the t treated group compared to the placebo group , suggesting beneficial effects of t on t2 dm complications . in patients with chronic heart failure , t treatment significantly reduced insulin resistance measured by fasting insulin and homa - ir . in a recent meta - analysis , corona et al . showed that t therapy was associated with marked reduction in fasting blood glucose , homa - ir , tg , and wc with concomitant increase in high density lipoprotein - cholesterol ( hdl ) , suggesting that t therapy improves metabolic control and may ameliorate central obesity . several recent reports have shown that long - term t therapy in men with td has produced significant weight loss , reduction in wc and bmi , as well as marked and significant reduction in total cholesterol , low density lipoprotein - cholesterol ( ldl ) , tg , and increased hdl [ 23 , 25 , 34 ] . also , marked reductions were noted in fasting glucose , hba1c , the nonspecific inflammatory marker hscrp , and liver enzymes aspartate aminotransferase ( asa ) and alanine aminotransferase ( ala ) suggesting improvement in hyperglycemia and reduction in the inflammatory response [ 23 , 25 , 34 , 35 ] . the first controlled five - year study using t in men with mets showed significant decreases in weight , wc , bmi , hba1c , homa - ir , total cholesterol , ldl cholesterol , triglycerides , hscrp , systolic , and diastolic blood pressure , and an increase in hdl . in the current study , we present data on the long - term effects of t therapy in men with diabesity . this study represents a pooled subgroup analysis of obese hypogonadal men with t2 dm from two cumulative registry studies of men , aged between 41 and 73 years ( mean 61.17 6.18 ) , who were seeking urological consultation in two urologists ' offices for various medical conditions such as erectile dysfunction , decreased libido , questions about their t status , or a variety of urological complaints . all subjects included in this study had subnormal plasma total t levels ( mean : 8.9 1.99 ; range : 1.6311.79 nmol / l ) and at least mild symptoms of hypogonadism assessed by the aging males ' symptoms scale ( ams ) . all patients had been diagnosed with t2 dm prior to seeking urological consultation and treated accordingly by their family physicians with various standard treatment modalities . at their first visit , all men received brief general advice that it would be beneficial if they attempted to lose weight by a healthier diet consisting of more fruits and vegetables and less meat and increasing their physical activity by walking or using the bicycle instead of the car . all men received treatment with parenteral t undecanoate 1000 mg ( nebido , bayer pharma , berlin , germany ) , administered at baseline and 6 weeks and thereafter every 12 weeks for up to 72 months , as described previously [ 23 , 25 ] . measurements of anthropometric parameters ( height , weight , and waist circumference ) were performed and blood samples drawn at baseline and at the majority of visits prior to the next injection of testosterone . therefore , t levels , measured by standard laboratory measurement , were trough levels at the end of an injection interval . waist circumference ( wc ) was measured midpoint between the iliac crest and the lowest rib . since not every measurement was performed at every single visit , values were averaged per patient and year . due to the cumulative registry design of the study , new subjects are entered into the database once they have received one year of treatment with t. all 156 subjects were followed for at least one year , 146 for at least two years , 136 for three years , 114 for four years , 105 for five years , and 69 for six years . the declining number of patients reflects duration of treatment but not the dropout rates . on the contrary , adherence to treatment was excellent , and t was only discontinued in two men who were diagnosed with prostate cancer . for continuous variables , the mean , median , standard deviation , range , minimum , maximum , and sample size for the overall sample and various groups were reported at each time point . we tested the hypotheses regarding change in outcome scores across the study period by fitting a linear mixed effects model to the data . time ( to indicate follow - up interviews ) was included as fixed effect in the model . estimation and test of change in scores were determined by computing the differences in least square means at baseline versus the score at each follow - up interview . for the correlation study , pearson correlation was calculated between baseline changes in outcomes at various time points . table 1 provides the baseline characteristics for 156 obese , diabetic men with td ( mean age 61.17 6.18 years ) . table 1 also contrasts the endpoints achieved with t therapy for most of the parameters described in the baseline characteristics . all 156 men had t2 dm and dyslipidaemia , 153 men had hypertension , 37 men had a history of cad , and 19 men had previously had a myocardial infarction . table 2 also lists the concomitant medications related to t2 dm , hypertension , and dyslipidaemia reported by the patients at baseline . total t levels showed a significant rise from 8.9 1.99 nmol / l at the beginning of therapy to above 16 nmol / l within the first year of therapy , and such physiological levels remained constant at this level throughout the course of treatment , as reported previously [ 23 , 25 ] ( data not shown ) . figure 1 demonstrates the measured reduction in wc subsequent to t therapy in obese diabetic men with td . wc declined from 114 8.69 cm ( min 89 , max 148 ) to 102.52 7.93 cm ( min 82.25 , max 121 ) with a mean reduction of 11.56 0.34 cm over the entire course of treatment ( p < 0.0001 ) . the reduction in wc was statistically significant at the end of each year compared to the previous year over the first five years ( p < 0.0001 ) and had a statistical significance of p = 0.0021 at the end of six compared to five years . at the end of the observation period , figure 2 shows the effects of t therapy on the body weight of obese diabetic men with td over the course of 6 years of therapy . body weight decreased from 113.56 11.53 kg ( minimum : 87 , maximum : 141 ) to 97.18 9.04 kg ( min 80 , max 118.5 ) with a mean loss of 17.49 0.58 kg over the course of treatment . this decrease in body weight was statistically significant at the end of each year compared to the previous year over the first five years ( p < 0.0001 ) and had a statistical significance of p = 0.0041 at the end of six compared to five years . marked and significant decrease in percentage body weight was noted over the course of t therapy . over the entire 6-year observation period , after one year , patients had lost 3.1 0.37% of their initial weight , after two years , 6.82 0.37% , after three years , 9.55 0.38% , after four years , 11.78 0.4% , after five years , 13.56 0.41% , and after 6 years , 15.04 0.48% . consistent and progressive decline in bmi was observed over the entire course of treatment with a mean reduction of 5.59 0.18 kg / m ( p < 0.0001 ) ( figure 4 ) . bmi declined from 36.31 3.51 to 35.09 3.44 after one year , 33.99 3.4 after two years , 33.03 3.19 after three years , 32.29 2.97 after four years , 31.58 2.8 after five years , and 31.19 2.6 after 6 years . the decline in bmi is consistent with the observed reductions in wc and body weight . at the end of the observation period , 48 patients ( 30.8% ) were overweight and one patient ( 0.6% ) had achieved normal weight . t therapy of obese , diabetic men with td resulted in a significant gradual decrease in fasting blood glucose from 7.06 1.74 mmol / l ( 128.37 31.63 mg / dl ) to 5.59 0.94 mmol / l ( 101.55 17.02 mg / dl ) ( figure 5 ) . the decrease was significant after one year ( p < 0.0001 ) , further declined after two years ( p = 0.0178 versus 12 months ) , and then reached a plateau with another slight but statistically significant decrease at five years compared to four years ( p = 0.0246 ) . a decrease of 1.49 0.14 mmol / l ( 27.14 2.48 mg / dl ) over the course of six years treatment was noted . the decrease in fasting blood glucose was accompanied by a marked decrease in hba1c from 8.08 0.09 % to 6.14 0.71% with a mean change of 1.93 0.06% ( p < 0.0001 ) at the end of the observation period ( figure 6 ) . the decrease in hba1c was progressive and statistically significant after one year ( p < 0.0001 ) , between two years and one year ( p < 0.0001 ) , between three and two years ( p < 0.0001 ) , between four and three years ( p < 0.0001 ) , and between five and four years ( p = 0.0003 ) and approached significance between six and five years ( p = 0.0635 ) ( figure 6 ) . at baseline , 25 subjects ( 16% ) had a hba1c target level of 7.0% . at the end of the observation period , 123 men ( 79% ) 12 patients ( 8% ) had a hba1c level of 6.5% . at the end of the observation period , 92 men ( 59% ) t therapy of obese , diabetic men with td produced marked and sustained gradual decrease in systolic blood pressure from 157.03 15.46 mmhg to 134.61 10.21 mmhg ( p < 0.0001 ) over the course of 6 years of treatment . the mean decrease ( 23.15 0.83 mmhg ) was significant and progressive over the first three years and reached a plateau at this level over the remaining course of the 6 years of treatment . similar results were recorded with the diastolic blood pressure which decreased from 93.89 11.67 to 79 5.57 mmhg ( p < 0.0001 ) with a mean change of 15.07 0.8 mmhg over the course of treatment ( figure 8) . a gradual and progressive decrease was noted over the first three years of treatment and then blood pressure stabilized over the remaining years of treatment . another finding was a significant reduction of pulse pressure from 63.07 10.7 ( minimum : 35 , maximum : 102 ) to 55.61 8.62 ( minimum : 38 , maximum : 70 ) . this decrease was statistically significant each year compared to the previous year during the first three years after which it remained stable . as shown in figure 9 , t therapy improved lipid profiles as demonstrated with increase in high density lipoprotein cholesterol ( hdl - c ) by 35.03 5.11% ( figure 9(a ) ) , significant reductions in total cholesterol ( tc ) by 32.12 1.41% , low density lipoprotein cholesterol ( ldl - c ) by 25.93 1.63% , and triglycerides ( tg ) by 29.91 2% ( figure 9(b ) ) . the mean changes in lipid profiles were gradual and progressive and were significant at each year when compared to baseline levels , reaching plateaus between three and four years . the ratio of total cholesterol to hdl cholesterol improved from 6.02 2.97 to 3.05 0.78 . these changes reached a plateau after three years with further slight but not statistically significant decreases . t therapy produced a marked and significant decrease in the concentration of the nonspecific inflammatory biomarker c - reactive protein ( crp ) from 3.16 4.12 to 0.72 0.56 u / l ( p < 0.0001 with a plateau after 24 months ) and a significant ( p < 0.0001 ) mean decrease of 2.88 0.28 over the course of treatment ( figure 10(a ) ) . moreover , t therapy reduced the concentration of the liver enzyme aspartate transaminase ( ast ) from 35.55 13.28 to 23.94 8.77 u / l ( p < 0.0001 with a plateau after 24 months ) and a significant ( p < 0.0001 ) mean change of 12.01 1.33 u / l over the course of treatment . similarly , alanine transaminase ( alt ) concentration was reduced from 39.04 19.38 to 26.08 14.42 u / l ( p < 0.0001 ) with a plateau after 12 months and significant mean change of 12.46 1.83 u / l over the entire course of treatment , suggesting a reduction in liver fat content , a reduced inflammatory response , and improvement in liver function ( figure 10(b ) ) . according to most of the current guidelines for testosterone replacement therapy , levels of total t below 8 nmol / l require treatment whilst levels above this threshold are considered borderline . we therefore analysed whether there were any differences in response in those of our patients who had t levels < 8 the results suggest that obese diabetic men in the higher t category respond to t treatment equally well ( table 3 ) . in this long - term , cumulative , uncontrolled , and observational registry study from two independent sites , we investigated the effects of t therapy on anthropometric parameters , fasting blood glucose ( fbg ) , hba1c , systolic and diastolic blood pressure , lipid profiles , and inflammatory markers in 156 obese , diabetic men with td . t therapy restored physiological t levels within the first 12 months and t levels were maintained with t therapy throughout the entire 6-year period . of particular interest , our data demonstrate that t therapy in obese , diabetic men with td produced significant and marked wl in 100% of all obese , diabetic patients . the wl subsequent to t therapy was gradual , progressive , and sustainable for the course of 6 years of treatment . the wl was significant and was associated with considerable and marked reductions in wc and bmi , suggesting that t therapy in obese , diabetic men results in improvement in metabolic function and probably behavioral changes with increased energy and motivation and physical activity that are translated into changes in body composition , which are accompanied by increases in lean body mass and reduction in fat mass , as discussed previously [ 22 , 23 , 25 ] . over the entire observation period of 72 months , the longest follow - up reported to date , t therapy produced marked reductions in wc and bmi , consistent with the hypothesis that t is an anabolic hormone required for maintenance of muscle mass and regulation of adipogenesis . it is of interest to note that the magnitude of changes over time in the obese , diabetic men was marked , progressive , and significant when compared with baseline data . these findings are not surprising since it is known that t promotes myogenesis and inhibits adipogenesis and regulates carbohydrate , lipid , and protein metabolism . thus , the changes in body composition noted in this study are attributed to t regulation of the metabolic processes in muscle and adipose tissues as well as functional metabolism . it is important to point out that all these hypogonadal , obese men had been diagnosed with t2 dm and received standard treatment by their family physician prior to initiation of t therapy . the mean baseline for hba1c in this study was 8.08% and only 25 men had an hba1c 7.0% , suggesting an overall poor control prior to t therapy . interestingly , however , t therapy produced marked decrease in fasting blood glucose and hba1c , suggesting that t therapy ameliorates hyperglycemia and ir in subjects with t2 dm , consistent with previous reports . these findings are also congruent with those reported in the ipass study in which 1438 men with td were treated with t and followed up for up to 12 months suggesting similar reductions in glucose and hba1c , particularly in patients with a poor hba1c control . we also noted a meaningful reduction in systolic and diastolic blood pressure in response to t therapy . that t therapy improves it is possible that t modulates arterial blood pressure , through a variety of mechanisms , such as direct effects on the heart , the kidney , and the vessels , as well as the endothelium [ 38 , 39 ] . our data are consistent with previous work in obese , hypogonadal , and diabetic men in which blood pressure was shown to decrease favorably in response to therapy with an oral t formulation [ 29 , 40 ] . men with prostate cancer who were treated with androgen deprivation therapy ( adt ) showed increased arterial stiffness [ 41 , 42 ] . in our study elevated pulse pressure is associated with an increase in large artery stiffness and recognized as an independent risk factor for cvd . our results are consistent with a placebo - controlled study that demonstrated a reduction in arterial stiffness , measured by use of the augmentation index , following t therapy . crp , a nonspecific marker of inflammation , was markedly reduced over the course of t therapy in obese , diabetic men with td . since it has been shown that wl significantly reduces plasma c - reactive protein ( crp ) concentration , it is likely that the reduced weight reestablishes a new equilibrium with attenuated inflammatory responses and reduced crp levels . it has been suggested that crp concentration was significantly and directly associated with change in systolic blood pressure ( sbp ) and wc but inversely associated with hdl cholesterol . since t therapy improved both systolic blood pressure and reduced wc in this cohort of obese , diabetic men , it is not surprising that crp levels were significantly reduced . in addition , we noted a marked reduction in the activities of several liver enzymes , used as markers of nonalcoholic fatty liver disease ( nafld ) and liver function , suggesting that t therapy reduces liver fat content and attenuates the inflammatory response and improves various physiological functions . our findings are consistent with the work by hoyos et al . who showed a reduction in liver fat content assessed by diagnostic imaging . taken together , these findings strongly suggest that normalizing t levels in obese men with td ameliorates a host of mets components and reduces inflammation , thus reducing the risk of cardiometabolic diseases . it is of interest to note that , in this group of obese , diabetic men with td , t therapy markedly and significantly reduced total cholesterol ( tc ) levels and this diminution was very pronounced and sustained over the entire 6-year period of t treatment . further , we noted that long - term t therapy reduced ldl throughout the treatment period , and ldl levels were maintained at low levels throughout the course of treatment . the clinical implication of this observation is that reduction in ldl correlates with reduced cvd risk . further , t therapy not only reduced the levels of ldl and tc but also produced small yet significant increases in hdl levels . moreover , the ratio of total cholesterol to hdl cholesterol dropped from > 6 to < 3.5 . these findings suggest that t therapy of obese , diabetic men with td may reduce cvd risk and increase health benefits , such as improved lipid profiles . another important observation in this study was the marked and significant reduction in triglycerides ( tgs ) in response to t therapy in these obese , diabetic men with td . since visceral fat storage depends on accumulation of tgs , thus , in men with td , it is expected that increased lipid accumulation will contribute to obesity and t therapy is expected to produce reduction in body weight , wc , and bmi [ 23 , 25 ] . finally , a closer look at the per cent reduction in the lipids concentrations ( tc , ldl , and tgs ) indicated that the reductions approach 30% , a value similar to that attained by use of statins in men with dyslipidaemia . obesity is a public health threat and its prevalence continues to increase worldwide [ 44 , 47 ] . obesity is associated with numerous comorbidities including t2 dm , hypertension , stroke , and coronary artery disease . from a global health policy perspective , the increased prevalence of t2 dm and obesity epidemics represents a warning salvo for all healthcare systems . evidence is accumulating suggesting that wl improves insulin sensitivity and -cell function [ 49 , 50 ] . it has been suggested that a loss of approximately 57% of body weight reduces the risk of developing diabetes by roughly 58% [ 4 , 51 ] . the potential benefits of a moderate 510% wl in high risk patients with a cluster of atherothrombotic proinflammatory abnormalities associated with hypertriglyceridaemic waist have been proposed by desprs et al . . the authors suggested that visceral obesity leads to t2 dm , dyslipidaemia , and hypertension , which result in increased risk and development of cvd , and a 510% wl can be very beneficial for improving health status and this should be pursued by behavioral therapy , exercise , diet , and pharmacotherapy . the role of obesity in development of td , t2 dm , and inflammation is complex and involves a bidirectional and integrated variety of endocrine factors that results in increased ir , decreased t secretion , and increased inflammatory cytokines [ 5356 ] . increased levels of circulating free fatty acids and blood glucose result in triglyceride formation and storage , thus increasing visceral and subcutaneous fat mass . this further promotes additional release of free fatty acids into the circulation due to activation of lipases . the increased sugar and fat uptake by the liver impairs the ability of insulin to regulate gluconeogenesis and activate glycogen synthesis . a recent study by phillips and perry suggested that reduced inflammatory status increases the likelihood of metabolic health , particularly among obese subjects . . showed that treatment which reduced inflammation , as assessed by crp levels , was also associated with reductions in hba1c in diabetic individuals . most studies have shown that hba1c control is not sustainable [ 59 , 60 ] . however , we observed that t therapy produced significant and sustained reduction in hba1c , suggesting that normalizing t levels reduces inflammation and restores glycaemic control . the prevalence of t2 dm is attributed , in part , to the increased prevalence of obesity combined with aging [ 14 , 61 ] . the increased prevalence of obesity together with diabetes in the same individuals , henceforth referred to as diabesity , has also been reported , and individuals with diabesity exhibited higher risks of cvd [ 62 , 63 ] . these inflammatory cytokines contribute to the impairment of insulin action and increased ir . in a recent study of middle - aged individuals without glucose lowering medication the contributions of ir and hyperglycaemia to the development of subclinical atherosclerosis were attributed to a large extent to abdominal adiposity . these findings suggest that weight management and prevention of weight gain in adulthood are critical for management of ir , obesity , and cvd . casanueva et al . reported that abdominal obesity was strongly associated with cvd and diabetes , even in patients who may be considered lean based on bmi assessment . it is well recognized that adipose tissue is a unique endocrine organ which produces proinflammatory cytokines and free fatty acids that may promote development of ir and therefore will contribute to the development of atherosclerosis and cvd through hyperglycemia , inflammation , dyslipidaemia , and hypertension [ 6567 ] . we believe that t therapy may confer health benefits since it results in significant and sustained wl in individuals with abdominal adiposity , as suggested previously . in fact , some studies have suggested that wc may be a risk factor for all - cause mortality [ 69 , 70 ] , incident cvd , and diabetes [ 17 , 71 ] . tseng suggested that bmi and wc may be used as determinants of cad in men with t2 dm . since t therapy produces significant reductions in wc , we believe that t therapy may provide significant health benefits in obese and diabetic patients with td and may improve cardiometabolic function and reduce the risk of cvd . therapeutic approaches to treatment of obesity and diabetes include bariatric surgery , which were reported to be successful in management of this condition , producing rapid and durable weight loss , reductions in cvd events and overall mortality , and sustained remission of diabetes in most patients [ 73 , 74 ] . however , bariatric surgery is not indicated for all obese diabetic patients and only select patients undergo this procedure , and thus this may be considered to be a limitation , given the large number of obese / diabetic patients . several other therapeutic approaches have also been used as potential treatment for diabetes , including sulfonylureas , thiazolidinediones , metformin , and insulin . however , with the exception of metformin , many of these agents result in weight gain and may increase ir risk . alternative approaches also include the development of stable glucagon like peptide 1 ( glp-1 ) receptor agonists , which increase insulin secretion and suppress glucagon secretion and appetite . glp-1 is an effective agent in improving glycaemic control , when administered subcutaneously in patients with t2 dm [ 76 , 77 ] . also the use of dipeptidyl peptidase inhibitors ( dpp-4 ) to increase the intracellular level of endogenous glp-1 has been proposed . these approaches were considered advantageous in treating diabetes when compared with the thiazolidinediones or sulfonylureas which result in weight gain . t is well known to regulate a host of metabolic functions in liver , adipose tissue , muscles , coronary arteries , and the heart . an inverse relationship exists between t and obesity and td is associated with dyslipidaemia , atherosclerosis , cardiovascular diseases , mets , and diabetes ( for review cf . kelly et al . several studies have suggested an association between reduced t levels and visceral obesity and diabetes . visceral fat accumulation in men was positively associated with ir , hyperglycaemia , and c - peptide and is negatively associated with t levels . a significant association between td and bmi and wc was noted in 355 male patients with t2 dm ( mean age 58 years ) . these findings suggest that wc and bmi in diabetic patients are associated with increased obesity . a number of studies have shown that t therapy in diabetic men with td reduced body weight , wc , and body fat and reduced fasting blood glucose and hba1c [ 24 , 2733 ] . these findings support the data presented in this study and suggest that t therapy may be a novel therapeutic approach to the management of t2 dm and obesity in men with diabesity . it should be pointed out that a number of studies have evaluated several drugs approved for management of diabetes and obesity with some positive outcome , albeit the efficacy of these drugs in maintaining wl or sustained weight loss remains debatable . dyson summarized the findings from studies on lifestyle , diet , and behavioral interventions on wl and wl maintenance . based on a systematic review and meta - analysis , the authors suggested that all lifestyle interventions have a modest but significant effect on wl ranging between 1 and 4.9 kg in the overweight and obese subjects . the authors concluded that exercise alone results in a moderate effect but is more effective when combined with dietary interventions . it was strongly suggested that behavioral therapy is an effective approach when combined with diet and exercise . the diabetes prevention program ( dpp ) research group investigated the long - term safety and tolerability of metformin , along with wl , and change in wc during the diabetic prevention program ( dpp ) in a randomized double - blind clinical trial followed by a 7 - 8-year open - label extension . most importantly , the magnitude of wl during the 2-year period was dependent on patients ' adherence to treatment . during the open follow - up period , wl remained significantly greater in the metformin group than in the placebo group . in a double - blind clinical trial , smith et al . investigated the effects of lorcaserin together with diet and exercise and counseling in a large number of obese or overweight adults over a period of 52 weeks . after one year of treatment , approximately 47.5% of patients who received lorcaserin lost 5% or more of their body weight ( ~5.8 0.2 kg ) . interestingly , even in the treatment group , some weight gain was noted over the course of the second year of treatment . when treatment was discontinued , patients regained the weight lost over the course of the year . investigated the effects of oral phentermine and topiramate on wl in overweight or obese patients with several comorbidities such as hypertension , dyslipidaemia , diabetes , or abdominal obesity . the authors suggested that the combination of phentermine and topiramate , with office - based lifestyle interventions , might be a valuable treatment for obesity . in a subanalysis of the aforementioned study , the investigators could show reductions of 70.5% and 78.7% in the annualised incidence rate of t2 dm . investigated the effects of liraglutide over a two - year period and assessed the effects of this drug when coupled with diet and exercise on mean change in bw and wc in obese patients . the findings of this study indicated that liraglutide , together with diet and exercise , provides sustained wl over 2 years , with improvements in obesity associated metabolic and cardiovascular risk factors . chilton and colleagues analysed data comparing orlistat or lorcaserin to lifestyle modifications ( placebo ) with sibutramine , rimonabant , or metformin on changes in wc and dropout rates attributed to adverse events of these agents . the authors suggested that orlistat significantly reduced wc by 6.96 cm when compared to placebo at 6 and 12 months . lorcaserin also reduced wc and was more effective than that of other interventions at 12 months . approximately 6.5% of patients on orlistat and 5.4% of those on lorcaserin discontinued their treatment due to adverse events at 12 months . the authors suggested that orlistat should be combined with lifestyle interventions in the treatment of obesity . thus , while a number of drugs , combined with behavioral and lifestyle changes , are shown to have moderate effect on wl and maintenance of weight loss , our data suggest that t therapy in obese diabetic men has a profound effect on wl and wc and this is irrespective of diet and exercise or behavioral counseling . this is meaningful , since the wl and the reduction in wc were observed over a long period of followup . we believe that since obesity is associated with hypogonadism ( td ) and may , in fact , cause td , long - term t treatment in hypogonadal men results in continuous improvements in weight and wc and may be an effective tool for weight management in obese men . the reduction in wl and wc with t - undecanoate in more than 500 hypogonadal men [ 23 , 34 ] appears to be superior when compared to data published with other drugs , in combination with lifestyle and behavioural interventions . our study is the first to report 6-year data of t therapy in hypogonadal obese men with t2 dm , showing a progressive and sustainable reduction of hba1c levels . this result seems to be highly favorable when compared to any other reported interventions where maintaining glycaemic control over a prolonged period of time appears extremely difficult to achieve , even with a modern generation of t2 dm drugs [ 59 , 85 , 86 ] . moreover , the findings that a large proportion of patients reach hba1c targets appear to be unique in comparison to other studies reported in the literature . most testosterone studies in men with t2 dm and/or metabolic syndrome have been performed over a relatively short period of time . hackett et al . , in their 18-month study , found the marked decrease in hba1c during the last 12 months of their study , indicating that these effects may take some time to occur . as shown in the united kingdom prospective diabetes study ( ukpds ) , hba1c levels were not maintained over time even with intensive control treatment [ 59 , 85 ] . similarly , glycaemic control as assessed by fasting blood glucose and hba1c were not maintained over time with treatment with rosiglitazone , metformin , or glyburide monotherapy . these findings suggest that control of hyperglycemia is not maintained over time by these pharmacotherapeutic agents . in contrast , the data from our study showed that t therapy consistently lowered hba1c , and the reduced hba1c and fasting blood glucose were maintained at the lower level and we did not observe any return to higher levels during the 72 months of t treatment . these findings are further supported by a recent study in which t therapy in diabetic patients was shown to reduce mortality , which could be explained in part by the positive effects of t on cardiometabolic function . because , six years ago when this study was commenced , we did not anticipate changes in diabetic parameters in response to t therapy , comedication was only assessed at baseline . the initial mean hba1c of 8.08% and the small proportion of men within hba1c targets at baseline indicated that the hyperglycemia in these patients was not very well controlled . the lack of assessment of antidiabetic treatment on the outcome of t therapy may represent a potential limitation of this study . reported a marked reduction in hba1c as a result of t therapy , in addition to standard antidiabetic treatment . the reduction in hba1c in the cohort of poorly controlled patients was 0.41% by 6 weeks and was maintained at 18 and 30 weeks of treatment , respectively . in this well - controlled study , there were no changes in antidiabetic medications . hypertension and obesity are common comorbidities in patients with t2 dm . thus , any pharmacotherapy that not only ameliorates hyperglycemia , but also improves blood pressure and reduces adipogenesis would be of interest . the prevalence of hypertension is higher in patients with t2 dm and ~60% of patients diagnosed exhibit arterial hypertension . this is attributed to ( i ) hyperinsulinaemia , ( ii ) increased sympathetic tone , and ( iii ) increased renin - angiotensin - aldosterone system activity . our findings suggest that t therapy not only improves lipid profiles , but also improves hyperglycemia and blood pressure . this combined two - center , open - label observational study was not a randomized controlled study and therefore may limit the scope of interpretation of the presented findings . this may introduce unintended bias since many of the subjects are seeking medical treatment of various urological conditions . another potential limitation is that we used total t levels and not free t levels , in combination with signs and symptoms , to evaluate td ( hypogonadism ) . an additional limitation was that the patients in these registries had a number of different comorbidities . since our results were not foreseen when the study was designed and commenced six years ago , we did not assess the duration of t2 dm , neither was a continued assessment of potential changes in comedications performed . therefore , we did not have the possibility to assess whether there were any cases of remission of t2 dm ( defined as hba1c < 6.0% without antidiabetic medications for at least one year ) . another limitation is that we did not assess behavioral and lifestyle changes , that is , changes in nutritional and exercise habits . since weight loss had not been expected , we did not consider the use of questionnaires or other methods to measure dietary changes or changes in physical activity . in summary , the findings of this study demonstrate that long - term treatment with t - undecanoate in obese men with diabetes and td restored physiological t levels and produced marked and significant wl and reduced wc and bmi . further , t treatment significantly reduced fasting blood glucose and hba1c levels . of equal importance , t therapy significantly reduced total cholesterol , ldl cholesterol , and triglycerides and increased hdl cholesterol levels and improved systolic and diastolic blood pressure . t therapy also reduced the levels of inflammatory markers , suggesting reduction in the inflammation response . these findings strongly suggest that t therapy of obese , diabetic men improves glycaemic control and lipid profiles and may prove useful in reducing the risk of cvd . these findings further suggest that long - term t treatment of diabetic obese men with td may produce important clinical benefits in men with diabesity . one unique aspect of this study is that it followed up diabetic obese men with td for a period of 6 years , which is the longest reported duration of t treatment to date . we suggest that t therapy in obese diabetic men may prove useful in management of men with diabesity .

to investigate effects of long - term testosterone ( t ) therapy in obese men with t deficiency ( td ) and type 2 diabetes mellitus ( t2 dm ) , data were collected from two observational , prospective , and cumulative registry studies of 561 men with td receiving t therapy for up to 6 years . a subgroup of obese hypogonadal men with t2 dm was analyzed . weight , height , waist circumference ( wc ) , fasting blood glucose ( fbg ) , glycated haemoglobin ( hba1c ) blood pressure , lipid profile , c - reactive protein ( crp ) , and liver enzymes were measured . a total of 156 obese , diabetic men with t deficiency , aged 61.17 6.18 years , fulfilled selection criteria . subsequent to t therapy , wc decreased by 11.56 cm and weight declined by 17.49 kg ( 15.04% ) . fasting glucose declined from 7.06 1.74 to 5.59 0.94 mmol / l ( p < 0.0001 for all ) . hba1c decreased from 8.08 to 6.14% , with a mean change of 1.93% . systolic and diastolic blood pressure , lipid profiles including total cholesterol : hdl ratio , crp , and liver enzymes all improved ( p < 0.0001 ) . long - term t therapy for up to 6 years resulted in significant and sustained improvements in weight , t2 dm , and other cardiometabolic risk factors in obese , diabetic men with td and this therapy may play an important role in the management of obesity and diabetes ( diabesity ) in men with t deficiency .

1. Introduction 2. Methods and Procedures 3. Statistical Analyses 4. Results 5. Discussion

obesity is also associated with a host of other comorbidities including sleep apnoea , hypogonadism ( testosterone deficiency , td ) , dyslipidaemia , and hypertension . the prevalence of obesity and t2 dm in the usa is higher than reported in other parts of the world and obesity is now considered a disease condition to be treated as any other disease conditions . because increases in obesity are paralleled with increases in t2 dm , the diagnosis of obesity and t2 dm in the same individual presents clinical and therapeutic challenges to healthcare providers . for instance , a recent report showed that bariatric surgery resulted in significant and sustained remission and improvement of t2 dm and other metabolic factors in severely obese patients . several studies have suggested that td may contribute to development of obesity , ir , and t2 dm and t therapy of men with td may ameliorate these conditions [ 2228 ] . men with td treated with t therapy experienced a positive effect on visceral obesity , as determined by reduction in body weight ( 2.66% ) , waist - hip ratio ( 3.96% ) , and body fat ( 5.65% ) when compared to the control group . this treatment also resulted in decreased fasting blood glucose and mean glycated haemoglobin ( hba1c ) . also demonstrated that t therapy in men with t2 dm reduced visceral adiposity and reduced homoeostasis model assessment ( homa ) index , hba1c , and fasting glucose , suggesting improvement in insulin sensitivity and glycaemic control in men with td and t2 dm . examined the effects of t therapy on ir , cvd risk , and symptoms of t deficiency in men with t2 dm and/or mets over a 12-month trial period . t therapy improved glycaemic control and reduced homa - ir as well as hba1c , suggesting a beneficial effect of t therapy in men with t2 dm . , in which the effects of t therapy on homoeostasis model assessment - estimated insulin resistance ( homa - ir ) , carotid intima media thickness ( cimt ) , and high - sensitivity c - reactive protein ( hscrp ) were investigated in men with td and metabolic syndrome ( mets ) , t therapy significantly improved homa - ir , cimt , and hscrp as compared to the placebo treated group . the effects of t therapy in 87 diabetic men with coronary artery disease ( cad ) were investigated for a period of 12 weeks . total cholesterol ( tc ) , plasma triglycerides ( tg ) , and homa index were also significantly reduced in the t treated group compared to the placebo group , suggesting beneficial effects of t on t2 dm complications . showed that t therapy was associated with marked reduction in fasting blood glucose , homa - ir , tg , and wc with concomitant increase in high density lipoprotein - cholesterol ( hdl ) , suggesting that t therapy improves metabolic control and may ameliorate central obesity . several recent reports have shown that long - term t therapy in men with td has produced significant weight loss , reduction in wc and bmi , as well as marked and significant reduction in total cholesterol , low density lipoprotein - cholesterol ( ldl ) , tg , and increased hdl [ 23 , 25 , 34 ] . also , marked reductions were noted in fasting glucose , hba1c , the nonspecific inflammatory marker hscrp , and liver enzymes aspartate aminotransferase ( asa ) and alanine aminotransferase ( ala ) suggesting improvement in hyperglycemia and reduction in the inflammatory response [ 23 , 25 , 34 , 35 ] . the first controlled five - year study using t in men with mets showed significant decreases in weight , wc , bmi , hba1c , homa - ir , total cholesterol , ldl cholesterol , triglycerides , hscrp , systolic , and diastolic blood pressure , and an increase in hdl . in the current study , we present data on the long - term effects of t therapy in men with diabesity . this study represents a pooled subgroup analysis of obese hypogonadal men with t2 dm from two cumulative registry studies of men , aged between 41 and 73 years ( mean 61.17 6.18 ) , who were seeking urological consultation in two urologists ' offices for various medical conditions such as erectile dysfunction , decreased libido , questions about their t status , or a variety of urological complaints . measurements of anthropometric parameters ( height , weight , and waist circumference ) were performed and blood samples drawn at baseline and at the majority of visits prior to the next injection of testosterone . table 1 provides the baseline characteristics for 156 obese , diabetic men with td ( mean age 61.17 6.18 years ) . figure 1 demonstrates the measured reduction in wc subsequent to t therapy in obese diabetic men with td . wc declined from 114 8.69 cm ( min 89 , max 148 ) to 102.52 7.93 cm ( min 82.25 , max 121 ) with a mean reduction of 11.56 0.34 cm over the entire course of treatment ( p < 0.0001 ) . the reduction in wc was statistically significant at the end of each year compared to the previous year over the first five years ( p < 0.0001 ) and had a statistical significance of p = 0.0021 at the end of six compared to five years . at the end of the observation period , figure 2 shows the effects of t therapy on the body weight of obese diabetic men with td over the course of 6 years of therapy . body weight decreased from 113.56 11.53 kg ( minimum : 87 , maximum : 141 ) to 97.18 9.04 kg ( min 80 , max 118.5 ) with a mean loss of 17.49 0.58 kg over the course of treatment . this decrease in body weight was statistically significant at the end of each year compared to the previous year over the first five years ( p < 0.0001 ) and had a statistical significance of p = 0.0041 at the end of six compared to five years . over the entire 6-year observation period , after one year , patients had lost 3.1 0.37% of their initial weight , after two years , 6.82 0.37% , after three years , 9.55 0.38% , after four years , 11.78 0.4% , after five years , 13.56 0.41% , and after 6 years , 15.04 0.48% . consistent and progressive decline in bmi was observed over the entire course of treatment with a mean reduction of 5.59 0.18 kg / m ( p < 0.0001 ) ( figure 4 ) . bmi declined from 36.31 3.51 to 35.09 3.44 after one year , 33.99 3.4 after two years , 33.03 3.19 after three years , 32.29 2.97 after four years , 31.58 2.8 after five years , and 31.19 2.6 after 6 years . t therapy of obese , diabetic men with td resulted in a significant gradual decrease in fasting blood glucose from 7.06 1.74 mmol / l ( 128.37 31.63 mg / dl ) to 5.59 0.94 mmol / l ( 101.55 17.02 mg / dl ) ( figure 5 ) . the decrease was significant after one year ( p < 0.0001 ) , further declined after two years ( p = 0.0178 versus 12 months ) , and then reached a plateau with another slight but statistically significant decrease at five years compared to four years ( p = 0.0246 ) . the decrease in fasting blood glucose was accompanied by a marked decrease in hba1c from 8.08 0.09 % to 6.14 0.71% with a mean change of 1.93 0.06% ( p < 0.0001 ) at the end of the observation period ( figure 6 ) . the decrease in hba1c was progressive and statistically significant after one year ( p < 0.0001 ) , between two years and one year ( p < 0.0001 ) , between three and two years ( p < 0.0001 ) , between four and three years ( p < 0.0001 ) , and between five and four years ( p = 0.0003 ) and approached significance between six and five years ( p = 0.0635 ) ( figure 6 ) . at the end of the observation period , 92 men ( 59% ) t therapy of obese , diabetic men with td produced marked and sustained gradual decrease in systolic blood pressure from 157.03 15.46 mmhg to 134.61 10.21 mmhg ( p < 0.0001 ) over the course of 6 years of treatment . similar results were recorded with the diastolic blood pressure which decreased from 93.89 11.67 to 79 5.57 mmhg ( p < 0.0001 ) with a mean change of 15.07 0.8 mmhg over the course of treatment ( figure 8) . as shown in figure 9 , t therapy improved lipid profiles as demonstrated with increase in high density lipoprotein cholesterol ( hdl - c ) by 35.03 5.11% ( figure 9(a ) ) , significant reductions in total cholesterol ( tc ) by 32.12 1.41% , low density lipoprotein cholesterol ( ldl - c ) by 25.93 1.63% , and triglycerides ( tg ) by 29.91 2% ( figure 9(b ) ) . t therapy produced a marked and significant decrease in the concentration of the nonspecific inflammatory biomarker c - reactive protein ( crp ) from 3.16 4.12 to 0.72 0.56 u / l ( p < 0.0001 with a plateau after 24 months ) and a significant ( p < 0.0001 ) mean decrease of 2.88 0.28 over the course of treatment ( figure 10(a ) ) . moreover , t therapy reduced the concentration of the liver enzyme aspartate transaminase ( ast ) from 35.55 13.28 to 23.94 8.77 u / l ( p < 0.0001 with a plateau after 24 months ) and a significant ( p < 0.0001 ) mean change of 12.01 1.33 u / l over the course of treatment . similarly , alanine transaminase ( alt ) concentration was reduced from 39.04 19.38 to 26.08 14.42 u / l ( p < 0.0001 ) with a plateau after 12 months and significant mean change of 12.46 1.83 u / l over the entire course of treatment , suggesting a reduction in liver fat content , a reduced inflammatory response , and improvement in liver function ( figure 10(b ) ) . in this long - term , cumulative , uncontrolled , and observational registry study from two independent sites , we investigated the effects of t therapy on anthropometric parameters , fasting blood glucose ( fbg ) , hba1c , systolic and diastolic blood pressure , lipid profiles , and inflammatory markers in 156 obese , diabetic men with td . of particular interest , our data demonstrate that t therapy in obese , diabetic men with td produced significant and marked wl in 100% of all obese , diabetic patients . the wl subsequent to t therapy was gradual , progressive , and sustainable for the course of 6 years of treatment . the wl was significant and was associated with considerable and marked reductions in wc and bmi , suggesting that t therapy in obese , diabetic men results in improvement in metabolic function and probably behavioral changes with increased energy and motivation and physical activity that are translated into changes in body composition , which are accompanied by increases in lean body mass and reduction in fat mass , as discussed previously [ 22 , 23 , 25 ] . it is of interest to note that the magnitude of changes over time in the obese , diabetic men was marked , progressive , and significant when compared with baseline data . interestingly , however , t therapy produced marked decrease in fasting blood glucose and hba1c , suggesting that t therapy ameliorates hyperglycemia and ir in subjects with t2 dm , consistent with previous reports . these findings are also congruent with those reported in the ipass study in which 1438 men with td were treated with t and followed up for up to 12 months suggesting similar reductions in glucose and hba1c , particularly in patients with a poor hba1c control . we also noted a meaningful reduction in systolic and diastolic blood pressure in response to t therapy . that t therapy improves it is possible that t modulates arterial blood pressure , through a variety of mechanisms , such as direct effects on the heart , the kidney , and the vessels , as well as the endothelium [ 38 , 39 ] . our data are consistent with previous work in obese , hypogonadal , and diabetic men in which blood pressure was shown to decrease favorably in response to therapy with an oral t formulation [ 29 , 40 ] . crp , a nonspecific marker of inflammation , was markedly reduced over the course of t therapy in obese , diabetic men with td . since it has been shown that wl significantly reduces plasma c - reactive protein ( crp ) concentration , it is likely that the reduced weight reestablishes a new equilibrium with attenuated inflammatory responses and reduced crp levels . since t therapy improved both systolic blood pressure and reduced wc in this cohort of obese , diabetic men , it is not surprising that crp levels were significantly reduced . in addition , we noted a marked reduction in the activities of several liver enzymes , used as markers of nonalcoholic fatty liver disease ( nafld ) and liver function , suggesting that t therapy reduces liver fat content and attenuates the inflammatory response and improves various physiological functions . taken together , these findings strongly suggest that normalizing t levels in obese men with td ameliorates a host of mets components and reduces inflammation , thus reducing the risk of cardiometabolic diseases . it is of interest to note that , in this group of obese , diabetic men with td , t therapy markedly and significantly reduced total cholesterol ( tc ) levels and this diminution was very pronounced and sustained over the entire 6-year period of t treatment . further , we noted that long - term t therapy reduced ldl throughout the treatment period , and ldl levels were maintained at low levels throughout the course of treatment . these findings suggest that t therapy of obese , diabetic men with td may reduce cvd risk and increase health benefits , such as improved lipid profiles . another important observation in this study was the marked and significant reduction in triglycerides ( tgs ) in response to t therapy in these obese , diabetic men with td . since visceral fat storage depends on accumulation of tgs , thus , in men with td , it is expected that increased lipid accumulation will contribute to obesity and t therapy is expected to produce reduction in body weight , wc , and bmi [ 23 , 25 ] . finally , a closer look at the per cent reduction in the lipids concentrations ( tc , ldl , and tgs ) indicated that the reductions approach 30% , a value similar to that attained by use of statins in men with dyslipidaemia . the authors suggested that visceral obesity leads to t2 dm , dyslipidaemia , and hypertension , which result in increased risk and development of cvd , and a 510% wl can be very beneficial for improving health status and this should be pursued by behavioral therapy , exercise , diet , and pharmacotherapy . the role of obesity in development of td , t2 dm , and inflammation is complex and involves a bidirectional and integrated variety of endocrine factors that results in increased ir , decreased t secretion , and increased inflammatory cytokines [ 5356 ] . we believe that t therapy may confer health benefits since it results in significant and sustained wl in individuals with abdominal adiposity , as suggested previously . since t therapy produces significant reductions in wc , we believe that t therapy may provide significant health benefits in obese and diabetic patients with td and may improve cardiometabolic function and reduce the risk of cvd . therapeutic approaches to treatment of obesity and diabetes include bariatric surgery , which were reported to be successful in management of this condition , producing rapid and durable weight loss , reductions in cvd events and overall mortality , and sustained remission of diabetes in most patients [ 73 , 74 ] . visceral fat accumulation in men was positively associated with ir , hyperglycaemia , and c - peptide and is negatively associated with t levels . a number of studies have shown that t therapy in diabetic men with td reduced body weight , wc , and body fat and reduced fasting blood glucose and hba1c [ 24 , 2733 ] . these findings support the data presented in this study and suggest that t therapy may be a novel therapeutic approach to the management of t2 dm and obesity in men with diabesity . the diabetes prevention program ( dpp ) research group investigated the long - term safety and tolerability of metformin , along with wl , and change in wc during the diabetic prevention program ( dpp ) in a randomized double - blind clinical trial followed by a 7 - 8-year open - label extension . the findings of this study indicated that liraglutide , together with diet and exercise , provides sustained wl over 2 years , with improvements in obesity associated metabolic and cardiovascular risk factors . thus , while a number of drugs , combined with behavioral and lifestyle changes , are shown to have moderate effect on wl and maintenance of weight loss , our data suggest that t therapy in obese diabetic men has a profound effect on wl and wc and this is irrespective of diet and exercise or behavioral counseling . we believe that since obesity is associated with hypogonadism ( td ) and may , in fact , cause td , long - term t treatment in hypogonadal men results in continuous improvements in weight and wc and may be an effective tool for weight management in obese men . our study is the first to report 6-year data of t therapy in hypogonadal obese men with t2 dm , showing a progressive and sustainable reduction of hba1c levels . most testosterone studies in men with t2 dm and/or metabolic syndrome have been performed over a relatively short period of time . in summary , the findings of this study demonstrate that long - term treatment with t - undecanoate in obese men with diabetes and td restored physiological t levels and produced marked and significant wl and reduced wc and bmi . of equal importance , t therapy significantly reduced total cholesterol , ldl cholesterol , and triglycerides and increased hdl cholesterol levels and improved systolic and diastolic blood pressure . these findings strongly suggest that t therapy of obese , diabetic men improves glycaemic control and lipid profiles and may prove useful in reducing the risk of cvd . these findings further suggest that long - term t treatment of diabetic obese men with td may produce important clinical benefits in men with diabesity . one unique aspect of this study is that it followed up diabetic obese men with td for a period of 6 years , which is the longest reported duration of t treatment to date . we suggest that t therapy in obese diabetic men may prove useful in management of men with diabesity .

tobacco was traded from north america to the world about 500 years ago . since then , tobacco use by smoking cigarettes , cigars , and pipes , or by chewing , has wreaked havoc on mankind . nearly 1.3 billion people are active smokers worldwide , who also pose a threat of indirect exposure to even more nonsmokers through secondhand smoke ( shs , also known as environmental tobacco smoke , ets ) . based on the international agency for research on cancer ( iarc ) , cigarette smoking is associated with cancers in many organs / tissues such as lung , head , neck , and bladder . the lung is particularly vulnerable as 90% of lung cancer cases are caused by cigarette smoking . cigarette smoke causes other diseases as well , including pulmonary disorders , cardiovascular diseases and stroke , and developmental defects . there is also sufficient evidence in recent years that shs causes lung cancer . in us nonsmokers , shs is responsible for about 3,000 lung cancer deaths , 46,000 cardiac - related illnesses , and 430 sudden infant death syndrome ( sids ) per year . four types of smoke have been classified so far : ( 1 ) mainstream smoke ( mss ) , created by tobacco combustion at approximately 1,2001,600c , when smokers inhale the tobacco smoke from a burning cigarette , ( 2 ) sidestream smoke ( sss ) , emanating from the smouldering end of a lit cigarette at ~900c when no active smoking occurs while the smoker pauses before taking the next puff , ( 3 ) shs , a mixture of about 85% of sss and 15% of exhaled mss , and ( 4 ) thirdhand smoke ( ths ) , a newly emerged type , defined as residual tobacco smoke adsorbed onto indoor surfaces after active smoking has ceased , where the semivolatile and nonvolatile components undergo chemical transformation to produce new toxicants [ 68 ] . in the last 50 years , many studies have been performed to identify chemical toxicants in cigarette smoke , which may represent the most rich resource of exogenous human mutagens and carcinogens . mss contains more than 4,000 chemicals . among them , over 60 have been classified by iarc as carcinogens . these include 10 polycyclic aromatic hydrocarbons ( pahs ) , 6 hydrocarbons , 10 nitrosamines , 13 aromatic amines , 2 aldehydes , 3 phenolic compounds , 4 volatile hydrocarbons , 3 nitro compounds , 12 miscellaneous organic compounds , and 9 inorganic and metals compounds . this list contains some of the strong animal and/or human carcinogens , such as pahs , n - nitrosamines , and aromatic amines , all of which react with dna to form adducts [ 1113 ] . the most prevalent ones in the vapor phase are aldehydes , benzene , and butadiene . it should be emphasized that sss or shs also contains several thousand individual compounds as does mss , and most of the above - mentioned carcinogens are also present in sss / shs . since such a carcinogenic source , that is , cigarette smoke , is preventable , and dna adduct levels correlate with cigarette consumption , tobacco smoke provides a unique model for understanding the cause - effect or environment - gene relationship in smoking - related cancer development . however , the real assessment of such relationships is very difficult due to multiple reasons . in addition , cigarette smoke contains co - carcinogens and tumor promoters that are also crucial for tumorigenicity of smoke condensates [ 12 , 15 , 16 ] . although certain carcinogens in cigarette smoke , such as formaldehyde and ,-unsaturated aldehydes ( enals ) , directly react with dna to form covalent adducts , most of carcinogenic compounds are so - called procarcinogens that must be metabolically activated to form ultimate carcinogens . the well - studied microsomal cytochrome p450 ( cyp ) system activates many tobacco carcinogens such as pahs , n - nitrosamines , aromatic amines , and benzene [ 1820 ] . therefore , carcinogen metabolism is often a double - edged sword in that it not only detoxifies and excretes toxicants but may also convert them into harmful reactive species . the individual variation in metabolic activation such as genetic polymorphisms in carcinogen - metabolizing genes is an important determinant of dna adduct levels and is used to identify smokers with increased cancer risk [ 21 , 22 ] . cigarette smoke condensates were initially known to have mutagenic activity by the 1970s , and adducts were detected in cellular dna from smokers in the 1980s . since then , many tobacco carcinogen - derived adducts have been identified in vitro and in vivo , owing to the development of highly sensitive analytical detection methods , such as p - postlabeling and mass spectrometry ( ms ) [ 25 , 26 ] . it has been shown that cigarette smokers have higher levels of dna adducts than nonsmokers [ 12 , 13 , 27 , 28 ] . studies have also shown that current smokers have higher adduct levels compared with former smokers . with some exceptions of inconsistency , many epidemiologic and clinical studies have shown an association between the in vivo levels of dna adducts resulting from cigarette smoke and the occurrence of tobacco - related cancers in lung , head and neck , and bladder . there are numerous reviews specifically related to the relationships between tobacco carcinogen exposure , dna adduct formation , carcinogen / adduct mutagenic potential , and increased cancer risk related to smoking [ 13 , 19 , 26 , 27 , 2932 ] . tobacco carcinogens generate a broad spectrum of dna lesions ranging from sugar damage , apurinic / apyrimidinic ( ap ) sites , small modified bases ( e.g. , o - mg and 8-oxog ) , and bulky base adducts to more deleterious lesions such as dna crosslinks and strand breaks . the so - called bulky dna adducts are formed by the covalent binding of those chemical carcinogens with large size , such as pahs and aromatic amines , to various sites on dna bases . these adducts also include exocyclic dna bases such as the etheno , propano , and benzetheno adducts formed by respective bifunctional compounds [ 33 , 34 ] . these bulky adducts represent a major and important class of dna damage originating from exposure to cigarette smoke . one characteristic of these bulky adducts is that they tend to significantly disrupt the dna helical structure and block watson - crick base pairing [ 35 , 36 ] . they are usually highly mutagenic , as exemplified by the pah - dna adducts and exocyclic dna adducts [ 34 , 37 ] . some of them may not be repaired ( e.g. , benzo[c]phenanthrene n - da adducts ) or only poorly repaired ( e.g. , two dibenzo[a , l]pyrene - induced dna adduct ) , thus leading to their persistence in genomic dna . smokers with high levels of these bulky adducts have been shown to be associated with an increased risk of cancers [ 40 , 41 ] . in fact , most of the compelling data on the connection of dna adducts with cancer have been obtained with bulky dna adducts and their respective carcinogens . for example , pah- and acrolein - dna adducts are preferentially formed in the same mutational hotspots of p53 in the lung cancers of smokers [ 30 , 42 , 43 ] . there is also evidence that a high level of bulky dna adducts in tissues , such as those caused by pahs and vinyl chloride ( vc ) , is associated with an increased risk of tumor in humans and animals [ 40 , 4446 ] . it should be pointed out that tobacco smoke also produces reactive oxygen species ( ros ) and induces oxidative stress [ 11 , 47 ] . those lesions that arise directly from ros attack of a base ( e.g. , 8-oxog ) or deoxyribose ( e.g. , base propenals ) [ 48 , 49 ] could also play a role in tobacco carcinogenesis . there is evidence that only a single bpde - dna adduct can effectively block expression of a reporter gene . dna damage can either activate checkpoint signaling pathways leading to cell cycle arrest or induce cell apoptosis by recruitment of immunologic and inflammatory responses . more importantly , persistence of dna adducts such as those formed by tobacco carcinogens pahs and n - nitrosamines plays a central role in tobacco - induced carcinogenesis . these adducts not only represent a very early event by inducing specific genetic changes that are a prerequisite to the initiation of cancer , but also occur during the continuum of the carcinogenic process . mutations in the p53 gene are more commonly observed in lung cancers from smokers than nonsmokers [ 51 , 52 ] . exposure to smoking has been associated with activating mutations in proto - oncogenes ( e.g. , the ras gene family ) and inactivation of tumor suppressor genes ( e.g. , p53 and p16 ) in cancers [ 5356 ] . microarray - based analyses also reveal that cigarette smoking alters expression of many genes involved in other functions . in addition to point mutations , there are correlations between dna adduct levels and other somatic alterations , for example , loss of heterozygosity ( loh ) that may occur at the very early stages of tobacco carcinogenesis [ 58 , 59 ] . in addition , epigenetic changes such as abnormal promoter methylation of certain genes also occur more frequently in lung tumors from smokers than in never - smokers or may appear in healthy individuals who start to smoke [ 60 , 61 ] , highlighting the importance of both genetic and epigenetic changes in tobacco carcinogenesis . tobacco carcinogens can also contribute to tumorigenesis by interacting with proteins , rna , and lipids , in addition to dna . to avoid tobacco carcinogen - induced dna damage however , once such damage is formed , dna repair is the next major defense mechanism ( figure 1 ) . organisms from prokaryotes to mammals have evolved a number of repair pathways , including direct reversal , base excision repair ( ber ) , nucleotide excision repair ( ner ) , mismatch repair ( mms ) , and double - strand break ( dsb ) repair [ 6264 ] . in model systems , such as cultured cells and genetically manipulated organisms , ner is the major pathway for the repair of various duplex - distorting bulky dna lesions such as those induced by pahs . small alkylated and oxidized lesions , including those arising from endogenous sources , are excised by the ber pathway which also repairs certain single - ring exocyclic dna adducts . for certain alkylated bases and etheno adducts , they can be repaired through direct reversal carried out by specialized repair proteins . in general , the understanding of damage recognition and mechanism of repair is important to gain insight into the specific roles of tobacco dna adducts in the development of cancer and other chronic diseases since , at the end , the overall cellular repair capacity in response to exposure is critically related to the levels of dna adducts in the genome or mutations in genes . the role of individual variability in repair , for example , polymorphisms in repair genes , has been related to the increased cancer risk in smokers [ 22 , 65 , 66 ] . ultimately , it is the impaired or poor repair of dna adducts ( e.g. , those bulky adducts and oxidized bases with cytotoxicity and mutagenicity ) that is expected to be most important in the etiology of smoking - related cancer and other disorders . studies in the last decade have revealed that if a dna adduct is unrepaired or irreparable , cells may use translesion dna synthesis ( tls ) to bypass the adduct to ensure the continuum of dna replication [ 6769 ] . tls is performed by various specialized dna polymerases ( pols ) , mostly from the y - family , with the possibility of nucleotide misincorporation [ 70 , 71 ] . these enzymes possess an open and preformed active site , enabling accommodation of a broad spectrum of dna adducts with different structures . in studies reported in literature , error - prone incorporations opposite an adducted nucleotide appear to occur commonly or co - exist with error - free incorporations [ 7274 ] . however , in some other cases , tls pols perform error - free bypass on damaged dna templates such as the efficient and correct nucleotide incorporation at the acrolein adduct -ho - pdg by pol and subsequent extension of replication by pol . the fidelity of tls observed in these experiments tends to depend on the individual pol tested as well as the structure of the target adduct . in general , the primary roles of these pols and how they operate in the cell with regard to interacting with replication and repair machineries remain to be further understood . this paper will focus on the formation and repair of bulky / exocyclic dna adducts induced by the major tobacco carcinogens in relation to tobacco mutagenesis and carcinogenesis . for small base lesions induced by tobacco chemical carcinogens , see previous reviews by singer and shrivastav et al . . in general , the literature so far on the covered review topics has been extensive . therefore , only selected published data are used to illustrate the relevant areas , ideas , and concepts . i regret that this review does not permit acknowledgment of the many researchers who made the original findings in these important areas . since smokers are repetitively exposed to complex mixtures of genotoxic carcinogens , the collective formation of dna adducts is very complex as reflected by their chemical types and cellular levels . although this paper is focused on bulky dna adducts , other types of dna lesions by tobacco carcinogens may be equally or more important than bulky dna adducts for a given carcinogen or cancer type . dna adduct levels are normally analyzed in target tissues in order to elucidate the relationship between tobacco carcinogens and cancer development . these levels should reach steady state such that the number of newly formed adducts equals the number of adducts lost every day which are related to a number of factors including carcinogen reactivity , exposure doses , timing of exposure , metabolic processes , and dna repair capacity . understanding of dna adducts with regard to their formation , isolation , and identification can be critical in several ways : ( 1 ) to understand the mechanism of tobacco carcinogens ; for example , the analysis of formation of dna adduct at gene mutational hotspots [ 30 , 78 ] has provided important insight into the cancer etiology ; ( 2 ) to assess the biologically effective doses of tobacco carcinogens ; ( 3 ) to assess dna repair capacity ( drc ) towards the adducts [ 13 , 79 ] ; ( 4 ) to find biomarkers of tobacco genotoxicity and uptake / metabolism of specific carcinogens [ 12 , 13 ] . many types of dna adducts , including those formed by benzo[a]pyrene ( b[a]p ) , 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone ( nnk ) , n -nitrosonornicotine ( nnn ) , and 4-aminobiphenyl ( 4-abp ) , have been detected from tissues of smokers as well as nonsmokers exposed to shs [ 1113 , 8082 ] . the common tobacco carcinogens and related metabolites that give rise to bulky dna adducts are listed in figure 2 . in tissues , the typical adduct levels are at about 1 adduct in 10 - 10 normal bases [ 27 , 83 ] . in general , dna adduct levels as low as 1 in 1010 normal bases can be significant with definite biological consequences . although p - postlabeling and immunoassay have been extensively utilized for adduct analysis , the detection and identification of dna adducts at these or even lower levels have been greatly facilitated by the highly sensitive / specific and new types of techniques [ 11 , 12 , 25 , 26 ] such as hplc with fluorescence , mass spectrometry ( ms ) , and electrochemical detection , particularly the coupling of liquid chromatography ( lc ) to ms and electrospray ionization ( esi ) , that is , lc - esi - ms . the number of compounds / dna adducts in figure 2 is expected to grow when more of such studies are carried out . it should be emphasized that shs also contains all of the common carcinogenic compounds listed in figure 2 , albeit with varying concentrations . some of the significantly existing chemical carcinogens in shs are nnk , nnn , b[a]p , benz(a)anthracene , benzene , 1,3-butadiene , 4-abp , and 2-napthylamine . in addition to being directly formed by tobacco carcinogens , dna adducts can be generated through inflammation , particularly by ros and reactive nitrogen species ( rns ) . due to the direct surface exposure , cigarette smoking triggers an inflammatory response in human lung and causes chronic obstructive pulmonary disease ( copd ) , which has been shown to possess significant abnormalities in inflammatory pathways [ 8689 ] . smokers are known to have elevated levels of oxidative stress [ 11 , 47 ] , which is increasingly linked to cancer and neurological diseases [ 90 , 91 ] . cigarette smoke may induce oxidative stress by several mechanisms [ 11 , 47 ] : ( 1 ) it contains oxidizing compounds and ros ; ( 2 ) the ros - generating redox cycling by quinone - hydroquinone complex as well as pah quinones and their corresponding catechols ; ( 3 ) smoking may weaken the antioxidant defense system . the elevated oxidative stress in smokers is accompanied by lipid peroxidation ( lpo ) which results from reactions of reduced oxygen species with polyunsaturated fatty acids ( pufas ) . it is well documented that lpo produces enals , including acrolein , crotonaldehyde , and trans-4-hydroxy-2-nonenal ( hne ) [ 9294 ] . these compounds react with dna to produce etheno ( )-adducts as well as propano adducts [ 9294 ] . this explains why chronic inflammation in humans is accomplished by increased levels of such adducts . it should be noted that these adducts are also present in tissues of humans and untreated animals at very low levels as background lesions [ 9294 ] . to understand the chemistry between a carcinogen and dna bases is instrumental in revealing the molecular mechanism of mutagenicity and carcinogenicity of the carcinogen . a single carcinogen can cause several different types of dna damage , mainly due to the process of metabolism that can yield several or many reactive metabolites . all the carcinogens listed in figure 2 can form more than one type of dna adducts . for example , nnk can form both simple methylated and bulky pyridyloxobutyl ( pob ) adducts [ 80 , 84 ] . a single electrophilic carcinogen can form multiple adducts of the same nature by reacting with all four dna bases . for example , benzene metabolites , hydroquinone ( hq ) and para - benzoquinone ( p - bq ) , form exocyclic adducts on da , dc , and dg [ 9598 ] . bpde , by way of another example , can generate both dg and da adducts . predominantly bind to its 2-nh2 group , alkylating agents such as tobacco - specific nitrosamines ( tsnas ) mainly react at the n-7 or o position , and aromatic amines tend to bind to the 8-carbon position . all oxygen and nitrogen sites on dna bases are actually reactive with alkylating agents in vitro under physiological conditions . of the dna adducts formed by tobacco carcinogens , exocyclic adducts have been extensively studied for their chemistry of formation [ 34 , 99 ] . bifunctional electrophilic compounds such as hq and p - bq , acrolein , and vc metabolites , are all able to form exocyclic adducts . the common sites for forming an exocyclic ring are n-1 and n of da , n-3 and adducts may be promutagenic if formed at coding sites of the bases , including o , n-1 , and n of dg , n-1 and n of da , o , n-3 , and n of dc , and o and n-3 of t . structurally , exocyclic adducts are analogous but can differ in ring structure such as size ( e.g. , 5- versus 6-membered ) , number ( e.g. , one ring versus two rings ) , angularity ( e.g. , linear versus angular ) , substituents ' nature ( e.g. , oh versus ch2oh ) , and location ( e.g. , -ho - pdg versus -ho - pdg ) [ 34 , 102 ] . the structural features of specific adducts may define the specificity and efficiency of their repair , as discussed below , as well as their mutagenicity . in the last two decades or so , considerable progress has been made in understanding the specificity , mechanism of action , and in vivo importance of many repair enzymes and pathways . this has been greatly facilitated by major advances in discovery of new enzymes or novel activities , synthesis of site - directed damage - containing oligonucleotides , construction of damage - containing shuttle vectors and viral genomes for in vivo studies , determination of high - resolution structures of repair enzymes and damaged dna , development of gene mutant models , identification of protein interaction networks , gene analyses such as mutation spectrum mapping and single nucleotide polymorphisms ( snps ) , and by the latest studies using omic profiling technology . there are many excellent reviews specifically related to the complete process as well as specific repair pathways that restore dna to its normal state [ 6264 , 103108 ] . several major mechanisms have been shown to be involved in the repair of bulky dna adducts that can be induced by tobacco carcinogens , as discussed below in detail . it is important to determine which adducts are removed efficiently or poorly , as those adducts that persist may cause a greater long - term mutagenic potential . excision repair , whether it is base ( ber ) or nucleotide excision ( ner ) , has to be at least a two - step process in which the damage recognition and excision is followed by dna replication , whereas direct reversal , catalyzed by o - alkylguanine dna alkyltransferase ( agt , also known as mgmt ) or alkb homologs ( abhs ) , restores the normal base without excision . multiple repair mechanisms could be involved in the removal of various dna adducts produced by a single compound . as will be described below , the benzetheno adducts of hq / p - bq are substrates for ap endonuclease , and the hydroxyphenyl dg adduct formed by the same compounds is repaired by ner . in some cases , more than one enzyme or mechanism can act on the same adduct , which may serve as backups or operate with different functions in the cell . for example , 3 , n - ethenocytosine ( c ) is excised by three different dna glycosylases and repaired by two different repair pathways , ber and abh2 . although the mismatch repair ( mmr ) pathway appears not to be directly implicated yet as significantly as the above pathways in response to the bulky adducts , muts protein has been shown to bind to the propano dg and m1 g adducts , suggesting that muts can bind to exocyclic adducts and may trigger a mmr - mediated response . although certain repair data come from research using prokaryotic enzymes , this paper will concentrate on mammalian / human repair enzymes whenever possible . in principle , the analogous enzymes and general mechanisms exist in both prokaryotes and eukaryotes , and such conservation has provided a solid foundation for our understanding of mammalian repair . it should also be pointed out that much of the repair data concerning tobacco carcinogen - derived adducts was not directly obtained from tobacco - related studies , but rather based on reports focusing on chemical carcinogens per se . ner is the most versatile repair pathway in the cell and the primary mechanism for the removal of chemical carcinogen - induced bulky dna adducts that significantly distort the dna helix structure [ 64 , 107 , 110 , 111 ] . its pathway in eukaryotes consists of at least 30 gene products and can be reconstituted with purified key proteins in vitro [ 113 , 114 ] . mutations in some of these ner genes may lead to xeroderma pigmentosum ( xp ) , a genetic disorder with seven complementation groups ( from xpa to xpg ) , and a higher incidence of skin cancer . the steps in ner consist of sequential assembly of proteins that perform different functions : damage recognition by xpc - hr23b , opening of a denaturation bubble by tfiih , incision of the damaged strand by xpg and ercc1-xpf , displacement and excision of the lesion - containing oligonucleotide ( 2432 base long ) , repair synthesis by dna polymerase , and dna ligation by ligase iii . there are two subsets of the pathway : global genomic repair ( ggr ) and transcription - coupled repair ( tcr ) that differ in the mode of damage recognition and are regulated by differential cellular mechanisms [ 105 , 107 , 110 , 111 ] . ggr is involved in repair of dna lesions from the transcriptionally silent regions of the genome and the nontranscribed strand of the active genes . tcr preferentially repairs the distorting lesions on the transcribed strand in active genes in order to avoid a stalled rna polymerase ii . the mammalian ner activity appears to be mostly modulated by posttranslational modifications and by protein - protein interactions . ner activity can be measured in cell - free extracts by the cleavage of site - directed oligonucleotide containing an adduct or by the extent of dna repair synthesis in damaged plasmid dna . figure 3 shows the structures of important toxic and mutagenic bulky adducts as ner substrates which are formed by those major carcinogens in cigarette smoke , including pahs , acrolein , 4-abp , and benzene . in addition , intra- and interstrand crosslinks such as those generated by uv light and cisplatin are usually repaired by ner . these crosslinks could also be formed by bifunctional tobacco chemicals such as acrolein and crotonaldehyde . therefore , ner is a critical repair pathway for protecting against the tobacco carcinogen - induced mutagenesis and carcinogenesis . ( 1 ) formation and repair of pah - derived dna adductspahs are thought to be the major contributors to the etiology of smoke - induced cancers , particularly lung cancer . b[a]p was the first carcinogen to be found in cigarette smoke and has been extensively studied as a surrogate for pahs . it is present in cigarette smoke at low levels ( 1050 ng / cigarette ) but is higher than other pahs . in one well studied metabolic pathway mediated by successive p450/epoxide hydrolase / p450 , b[a]p yields active carcinogenic epoxides , mainly b[a]p-7,8-diol-9,10-epoxide ( bpde ) [ 18 , 118 ] . the diol epoxide exists as two diastereoisomers , anti- and syn - bpde , each of which can be resolved into ( + ) - and ( )-enantiomers . the isomer ( + ) -anti - bpde has the greatest tumorigenicity in vivo . the latter reacts at the n of dg to yield either trans- or cis - ring opening of the epoxide ring , forming ( + ) -trans- and ( + ) -cis - bpde- n - dg adducts [ 120 , 121 ] . similarly , ( )-anti - bpde forms ( )-trans- and ( )-cis - bpde - n - dg adducts . of these four stereoisomeric adducts , ( + ) -trans - bpde - n - dg is the most abundant , which is also the major adduct identified in vivo [ 122 , 123 ] and was detected in 45% of smokers ' lung . a second path for the activation of b[a]p involves p450-mediated activation to yield free cations that can induce unstable adducts leading to ap sites . a third metabolic pathway is through aldo - keto reductase superfamily - mediated oxidation of b[a]p-7,8-diol to catechol that enters into a redox cycle to form a reactive b[a]p-7,8-quinone ( bpq ) [ 126 , 127 ] . although a recent study did not support that bpq forms stable dna adducts in mice , there is evidence that this pathway operates in human lungs leading to ros - mediated genotoxicity such as causing g to t transversions that inactivate p53 [ 127 , 129 ] . so far , the relative importance of these pathways in cancer development remains to be determined.bpde-dna adducts are recognized and repaired by e. coli ner complex uvrabc nuclease and human ner [ 131133 ] . taking advantage of the stereochemistry involved in the formation of these bulky adducts , a number of studies addressed the effects of adduct conformation , base paring , and sequence context on dna repair . for example , using an in vitro repair system with oligonucleotides containing one of the four bpde - n - dg adducts described above hess et al . showed that the rates of human ner repair of these adducts are dependent on their different stereochemical configurations . the rates of excision were found to vary over 100-fold among these dg adducts , and the cis - adducts of dg are repaired more rapidly than the trans - adducts . it was later found that different conformations of these adducts are recognized differentially by the ner lesion recognition complex xpc - hr23b , which can be correlated with the relatively low repair of ( + ) -trans - bpde - n - dg . similar correlations were observed with uvrabc nuclease . to further show the importance of local dna conformation , the nature of the base opposite a bpde adduct is found to be critical in modulating the repair rates . as will be discussed below is section 2.5 , the processing of bpde - dna adducts by both uvrabc and human bpde forms n - da adducts in native dna as well , although relatively inefficiently [ 135 , 136 ] , which exhibit differential conformation and perturbation of dna duplex than the bpde - dg adducts . using cell extracts , human ner activity has been shown for the ( + ) - or ( )-trans - anti - bpde - da adducts [ 38 , 131].several early studies showed that repair of bpde - dna adducts occurs much faster in the transcribed strand than in the nontranscribed strand of hprt or p53 genes , indicating that these adducts are subject to tcr [ 137 , 138 ] . these adducts block human rna pol ii elongation on the transcribed strand , which could be a signal for initiating tcr , also in a stereochemistry- and sequence - dependent manner . a later work shows that common genetic variations in cockayne syndrome a ( csa ) and b ( csb ) proteins are associated with ner repair capacity of bpde - induced dna damage in smokers . , this strand preference in repair may contribute to the mutational property of the human lung cancer p53 gene in response to bpde exposure : repair of bpde adducts along the nontranscribed strand of p53 is consistently slower than repair in the transcribed strand , and repair at the major damage hotspots in the nontranscribed strand is 24 times slower than repair at other damage sites .dibenzo[a , l]pyrene ( db[a , l]p ) is another pah that has been found to be present in tobacco smoke particulates and is the most potent carcinogen of the pahs tested to date in rodent systems . similar to the b[a]p - derived adducts , the bulky adducts formed by ( )-anti - dbpde possess different structures and adopt different conformations . they are differentially repaired by ner in human cells with some being poorly removed , as shown by a recent study . the repair of dbpde - dna adducts by ner has been shown to be slower than the repair of bpde - dna adducts . in general , the poor repair by ner of dbpde - dna adducts , at least some of them , may account for the high carcinogenicity of the parent compound . pahs are thought to be the major contributors to the etiology of smoke - induced cancers , particularly lung cancer . b[a]p was the first carcinogen to be found in cigarette smoke and has been extensively studied as a surrogate for pahs . it is present in cigarette smoke at low levels ( 1050 ng / cigarette ) but is higher than other pahs . in one well studied metabolic pathway mediated by successive p450/epoxide hydrolase / p450 , b[a]p yields active carcinogenic epoxides , mainly b[a]p-7,8-diol-9,10-epoxide ( bpde ) [ 18 , 118 ] . the diol epoxide exists as two diastereoisomers , anti- and syn - bpde , each of which can be resolved into ( + ) - and ( )-enantiomers . the isomer ( + ) -anti - bpde has the greatest tumorigenicity in vivo . the latter reacts at the n of dg to yield either trans- or cis - ring opening of the epoxide ring , forming ( + ) -trans- and ( + ) -cis - bpde- n - dg adducts [ 120 , 121 ] . similarly , ( )-anti - bpde forms ( )-trans- and ( )-cis - bpde - n - dg adducts . of these four stereoisomeric adducts , ( + ) -trans - bpde - n - dg is the most abundant , which is also the major adduct identified in vivo [ 122 , 123 ] and was detected in 45% of smokers ' lung . a second path for the activation of b[a]p involves p450-mediated activation to yield free cations that can induce unstable adducts leading to ap sites . a third metabolic pathway is through aldo - keto reductase superfamily - mediated oxidation of b[a]p-7,8-diol to catechol that enters into a redox cycle to form a reactive b[a]p-7,8-quinone ( bpq ) [ 126 , 127 ] . although a recent study did not support that bpq forms stable dna adducts in mice , there is evidence that this pathway operates in human lungs leading to ros - mediated genotoxicity such as causing g to t transversions that inactivate p53 [ 127 , 129 ] . so far , the relative importance of these pathways in cancer development remains to be determined . bpde - dna adducts are recognized and repaired by e. coli ner complex uvrabc nuclease and human ner [ 131133 ] . taking advantage of the stereochemistry involved in the formation of these bulky adducts , a number of studies addressed the effects of adduct conformation , base paring , and sequence context on dna repair . for example , using an in vitro repair system with oligonucleotides containing one of the four bpde - n - dg adducts described above hess et al . showed that the rates of human ner repair of these adducts are dependent on their different stereochemical configurations . the rates of excision were found to vary over 100-fold among these dg adducts , and the cis - adducts of dg are repaired more rapidly than the trans - adducts . it was later found that different conformations of these adducts are recognized differentially by the ner lesion recognition complex xpc - hr23b , which can be correlated with the relatively low repair of ( + ) -trans - bpde - n - dg . similar correlations were observed with uvrabc nuclease . to further show the importance of local dna conformation , the nature of the base opposite a bpde adduct is found to be critical in modulating the repair rates . as will be discussed below is section 2.5 , the processing of bpde - dna adducts by both uvrabc and human bpde forms n - da adducts in native dna as well , although relatively inefficiently [ 135 , 136 ] , which exhibit differential conformation and perturbation of dna duplex than the bpde - dg adducts . using cell extracts , human ner activity has been shown for the ( + ) - or ( )-trans - anti - bpde - da adducts [ 38 , 131 ] . several early studies showed that repair of bpde - dna adducts occurs much faster in the transcribed strand than in the nontranscribed strand of hprt or p53 genes , indicating that these adducts are subject to tcr [ 137 , 138 ] . these adducts block human rna pol ii elongation on the transcribed strand , which could be a signal for initiating tcr , also in a stereochemistry- and sequence - dependent manner . a later work shows that common genetic variations in cockayne syndrome a ( csa ) and b ( csb ) proteins are associated with ner repair capacity of bpde - induced dna damage in smokers . , this strand preference in repair may contribute to the mutational property of the human lung cancer p53 gene in response to bpde exposure : repair of bpde adducts along the nontranscribed strand of p53 is consistently slower than repair in the transcribed strand , and repair at the major damage hotspots in the nontranscribed strand is 24 times slower than repair at other damage sites . dibenzo[a , l]pyrene ( db[a , l]p ) is another pah that has been found to be present in tobacco smoke particulates and is the most potent carcinogen of the pahs tested to date in rodent systems . similar to the b[a]p - derived adducts , the bulky adducts formed by ( )-anti - dbpde possess different structures and adopt different conformations . they are differentially repaired by ner in human cells with some being poorly removed , as shown by a recent study . the repair of dbpde - dna adducts by ner has been shown to be slower than the repair of bpde - dna adducts . in general , the poor repair by ner of dbpde - dna adducts , at least some of them , may account for the high carcinogenicity of the parent compound . ( 2 ) formation and repair of dna adducts of aromatic amine 4-abpchemicals in this class such as 4-abp bind to dna bases mainly at c-8 position . adducts can also be formed at n- and o- of dg and n- of da . 4-abp forms dna adducts after n - hydroxylation by p450 to the mutagenic metabolite n - hydroxy-4-aminobiphenyl ( n - oh-4-abp ) . n-(deoxyguanosin-8-yl)-4-aminobiphenyl ( dg - c8-abp ) ( figure 2 ) is the major adduct of 4-abp , and the minor adduct is n-(deoxyadenosin-8-yl)-4-aminobiphenyl ( da - c8-abp ) . the major adduct has been detected in the human cells after exposure to n - oh-4-abp . this adduct was also identified from dna of the bladder biopsy samples from smokers and is quantitatively related to smoking status . dg - c8-abp adducts have been identified from human bladder cancer tissues [ 147 , 148 ] . moreover , higher levels of dna adducts correlated with more invasive tumors ( higher tumor grades ) . the unique binding pattern of 4-abp in the p53 gene , that is , the p53 mutational hotspots in bladder cancer at several codons are also the preferential sites for 4-abp adduct formation , links 4-abp to the etiology of bladder cancer .although the detailed molecular mechanism of the repair of 4-abp - dna adducts is not clear , dna fragments modified with n - oh-4-abp were shown to be incised by e. coli ner complex , the uvrabc nuclease . an early study investigated the rate of disappearance of dg - c8-abp in human transitional cell carcinomas of the bladder and showed that the majority of the adducts can be removed within 48 hours after treatment with 4-abp . another study showed that dg - abp was repaired rapidly while da - abp persisted in human uroepithelial cells . there is evidence of the human ner pathway involvement in the repair of these adducts , as the host cell reactivation ( hcr ) assays performed in ner - deficient cells showed reduced repair of dna lesions from plasmid treated with 4-abp . in addition , it was shown that loss of function of the p53 gene in human bladder epithelial cancer cells reduces the efficiency of repair of dg - c8-abp , suggesting that p53 may modulate its repair in target cells [ 151 , 153 ] . the relationship between deficient dna repair of 4-abp - dna adducts and increased bladder cancer risk was supported by the findings that such repair capacity was significantly lower in bladder cancer cases than in controls , and ever - smokers with low dna repair capacity exhibited a 6-fold increased risk compared with never smokers with normal repair capacity . chemicals in this class such as 4-abp bind to dna bases mainly at c-8 position . adducts can also be formed at n- and o- of dg and n- of da . 4-abp forms dna adducts after n - hydroxylation by p450 to the mutagenic metabolite n - hydroxy-4-aminobiphenyl ( n - oh-4-abp ) . n-(deoxyguanosin-8-yl)-4-aminobiphenyl ( dg - c8-abp ) ( figure 2 ) is the major adduct of 4-abp , and the minor adduct is n-(deoxyadenosin-8-yl)-4-aminobiphenyl ( da - c8-abp ) . the major adduct has been detected in the human cells after exposure to n - oh-4-abp . this adduct was also identified from dna of the bladder biopsy samples from smokers and is quantitatively related to smoking status . dg - c8-abp adducts have been identified from human bladder cancer tissues [ 147 , 148 ] . moreover , higher levels of dna adducts correlated with more invasive tumors ( higher tumor grades ) . the unique binding pattern of 4-abp in the p53 gene , that is , the p53 mutational hotspots in bladder cancer at several codons are also the preferential sites for 4-abp adduct formation , links 4-abp to the etiology of bladder cancer . although the detailed molecular mechanism of the repair of 4-abp - dna adducts is not clear , dna fragments modified with n - oh-4-abp were shown to be incised by e. coli ner complex , the uvrabc nuclease . an early study investigated the rate of disappearance of dg - c8-abp in human transitional cell carcinomas of the bladder and showed that the majority of the adducts can be removed within 48 hours after treatment with 4-abp . another study showed that dg - abp was repaired rapidly while da - abp persisted in human uroepithelial cells . there is evidence of the human ner pathway involvement in the repair of these adducts , as the host cell reactivation ( hcr ) assays performed in ner - deficient cells showed reduced repair of dna lesions from plasmid treated with 4-abp . in addition , it was shown that loss of function of the p53 gene in human bladder epithelial cancer cells reduces the efficiency of repair of dg - c8-abp , suggesting that p53 may modulate its repair in target cells [ 151 , 153 ] . the relationship between deficient dna repair of 4-abp - dna adducts and increased bladder cancer risk was supported by the findings that such repair capacity was significantly lower in bladder cancer cases than in controls , and ever - smokers with low dna repair capacity exhibited a 6-fold increased risk compared with never smokers with normal repair capacity . ( 3 ) formation and repair of propano adducts of ,-unsaturated aldehydes ( enals)enals can arise from both cigarette smoking and endogenous lpo . acrolein is the simplest enal and is a model chemical for this class of carcinogens . acrolein is one of the most abundant compounds in mss ( 60100 g / cigarette ) and is also present in sss at high concentrations . acrolein forms exocyclic adducts on dna bases , predominantly 1,n - dg adducts [ 155 , 156 ] . the principal adduct is -hydroxypropano-2-deoxyguanosine ( -oh - pdg ) that exists as a mixture of c8-oh epimers ( figure 3 ) , and the other adduct is -hydroxypropano-2-deoxyguanosine ( -oh - pdg ) . the mutagenicity of -oh - pdg is well established , while the mutagenicity of -oh - pdg has been reported with mixed results [ 158 , 159 ] . both adducts were recently found in human lungs using lc - esi - ms / ms . acrolein - dna adducts have been detected in the tissues of cigarette smokers with significantly higher levels than those of nonsmokers [ 161 , 162 ] . likewise , both crotonaldehyde and hne also form stereoisomeric propano dg adducts , and increased crotonaldehyde - dg adduct levels were observed in smokers . hne adducts have also been detected in rodent and human tissues [ 163 , 164 ] . the mutagenic potential of these dg adducts were recently summarized by minko et al . . -ho - pdg is a substrate for e. coli uvrabc nuclease [ 43 , 165 ] . in humans , there is also biochemical evidence that the hne - dna adducts are repaired by uvrabc and mammalian ner in cell - free extracts [ 92 , 166 ] . a recent study revealed that ner and recombination , but not mmr , are involved in repair of hne - treated phage dna replicating in e. coli . moreover , the repair rates were shown to be affected by the adduct stereochemistry when four hne - dg isomers were tested . interestingly , although ber is able to excise the 1,n-g adduct , it appears to have no in vitro activity towards the structurally analogous adducts -oh - pdg , -oh - pdg , and pdg and no in vivo protective role in a mutagenesis assay based on the vector containing a -oh - pdg .recent findings also pointed to the role of highly accurate tls in protecting cells from the potential genotoxicity of the acrolein - dna adducts [ 158 , 165 ] . previous in vivo site - specific mutagenicity studies have shown an efficient error - free bypass of the -ho - pdg adduct [ 165 , 170 ] . work from e. coli indicated that ner , recombination repair , and error - free tls are all involved in the cellular response to this major acrolein - dg adduct . acrolein is the simplest enal and is a model chemical for this class of carcinogens . acrolein is one of the most abundant compounds in mss ( 60100 g / cigarette ) and is also present in sss at high concentrations . acrolein forms exocyclic adducts on dna bases , predominantly 1,n - dg adducts [ 155 , 156 ] . the principal adduct is -hydroxypropano-2-deoxyguanosine ( -oh - pdg ) that exists as a mixture of c8-oh epimers ( figure 3 ) , and the other adduct is -hydroxypropano-2-deoxyguanosine ( -oh - pdg ) . the mutagenicity of -oh - pdg is well established , while the mutagenicity of -oh - pdg has been reported with mixed results [ 158 , 159 ] . both adducts were recently found in human lungs using lc - esi - ms / ms . acrolein - dna adducts have been detected in the tissues of cigarette smokers with significantly higher levels than those of nonsmokers [ 161 , 162 ] . likewise , both crotonaldehyde and hne also form stereoisomeric propano dg adducts , and increased crotonaldehyde - dg adduct levels were observed in smokers . hne adducts have also been detected in rodent and human tissues [ 163 , 164 ] . the mutagenic potential of these dg adducts were recently summarized by minko et al . . -ho - pdg is a substrate for e. coli uvrabc nuclease [ 43 , 165 ] . in humans , there is also biochemical evidence that the hne - dna adducts are repaired by uvrabc and mammalian ner in cell - free extracts [ 92 , 166 ] . a recent study revealed that ner and recombination , but not mmr , are involved in repair of hne - treated phage dna replicating in e. coli . moreover , the repair rates were shown to be affected by the adduct stereochemistry when four hne - dg isomers were tested . interestingly , although ber is able to excise the 1,n-g adduct , it appears to have no in vitro activity towards the structurally analogous adducts -oh - pdg , -oh - pdg , and pdg and no in vivo protective role in a mutagenesis assay based on the vector containing a -oh - pdg . recent findings also pointed to the role of highly accurate tls in protecting cells from the potential genotoxicity of the acrolein - dna adducts [ 158 , 165 ] . previous in vivo site - specific mutagenicity studies have shown an efficient error - free bypass of the -ho - pdg adduct [ 165 , 170 ] . work from e. coli indicated that ner , recombination repair , and error - free tls are all involved in the cellular response to this major acrolein - dg adduct . ( 4 ) formation and repair of in vivo hq-/p - bq - induced hydroxyphenyl adductsbenzene is a well - established human carcinogen and is associated with an increased risk of leukemia . it is a significant volatile compound in the vapor phase ( 1248 g / cigarette ) . in one major metabolic pathway , benzene is converted by p450 to benzene oxide which is further converted to phenol , catechol ( cat ) and various derivatives [ 20 , 172 ] ( figure 4 ) . one biologically important stable metabolite is p - bq , an oxidation product of hq [ 20 , 172 ] . a number of bulky dna adducts have been detected in vitro and in vivo when exposed to hq or p - bq [ 9598 , 173177 ] . reaction of p - bq or hq with dna in vitro has been shown to result in the formation of two ring exocyclic benzetheno adducts on dc , da , and dg [ 9598 ] . these adducts are highly mutagenic as tested in vitro with human pols involved in tls and in yeast by site - directed mutagenesis . the bodell group has found that the dna adducts formed in animals after benzene administration are identical to those produced in cells treated with hq , suggesting that hq is the main benzene metabolite causing adduct formation in vivo . by p - postlabeling , the principal dna adduct caused by hq or p - bq corresponds to n-(4-hydroxyphenyl)-2-dg(n-4-hoph - dg ) [ 173 , 177 ] . exocyclic adducts were also detected in vitro from reactions of trans , trans - muconaldehyde ( muc ) , a reactive ring - opened diene dialdehyde formed from a minor metabolic route [ 179 , 180 ] . it is still unclear as to what role the above covalent dna adducts may play in benzene - induced carcinogenesis , since benzene also induces other types of dna damage as well as chromosomal damage . for example , oxidized bases such as 8-oxog can be caused through the quinone / hydroquinone redox cycling ( also see section 2.1 ) . benzene also generates dna strand breaks [ 181 , 182 ] through direct attack by ros or unstable dna adducts . as shown in figure 4 , catechol o - quinones can react with dna by 1,4-michael addition to yield major n3a and n7 g adducts which are unstable and generate ap sites .we recently reported the repair of n-4-hoph - dg e. coli uvrabc nuclease . the specificity of such repair was also compared with those of dna glycosylases and damage - specific endonucleases of e. coli both of which were found to have no detectable activity toward this adduct . we also showed that p - bq - modified plasmid is efficiently cleaved by uvrabc , indicating the involvement of ner in repair of benzene - derived dna damage . the role of ner in the repair of hq / p - bq - induced dna damage was also suggested in another mutagenesis study using hq- or p - bq - treated plasmid containing the supf reporter gene in ner - deficient ( xpa ) human cells . note that hq- and p - bq - derived exocyclic adducts are repaired by a different mechanism called nucleotide incision repair ( nir ) , as discussed below . in general , although benzene metabolites show relatively low dna binding activity in vivo , their induced dna damage and repair seem to be complex . benzene is a well - established human carcinogen and is associated with an increased risk of leukemia . it is a significant volatile compound in the vapor phase ( 1248 g / cigarette ) . in one major metabolic pathway , benzene is converted by p450 to benzene oxide which is further converted to phenol , catechol ( cat ) and various derivatives [ 20 , 172 ] ( figure 4 ) . one biologically important stable metabolite is p - bq , an oxidation product of hq [ 20 , 172 ] . a number of bulky dna adducts have been detected in vitro and in vivo when exposed to hq or p - bq [ 9598 , 173177 ] . reaction of p - bq or hq with dna in vitro has been shown to result in the formation of two ring exocyclic benzetheno adducts on dc , da , and dg [ 9598 ] . these adducts are highly mutagenic as tested in vitro with human pols involved in tls and in yeast by site - directed mutagenesis . the bodell group has found that the dna adducts formed in animals after benzene administration are identical to those produced in cells treated with hq , suggesting that hq is the main benzene metabolite causing adduct formation in vivo . by p - postlabeling , the principal dna adduct caused by hq or p - bq corresponds to n-(4-hydroxyphenyl)-2-dg(n-4-hoph - dg ) [ 173 , 177 ] . exocyclic adducts were also detected in vitro from reactions of trans , trans - muconaldehyde ( muc ) , a reactive ring - opened diene dialdehyde formed from a minor metabolic route [ 179 , 180 ] . it is still unclear as to what role the above covalent dna adducts may play in benzene - induced carcinogenesis , since benzene also induces other types of dna damage as well as chromosomal damage . for example , oxidized bases such as 8-oxog can be caused through the quinone / hydroquinone redox cycling ( also see section 2.1 ) . benzene also generates dna strand breaks [ 181 , 182 ] through direct attack by ros or unstable dna adducts . as shown in figure 4 , catechol o - quinones can react with dna by 1,4-michael addition to yield major n3a and n7 g adducts which are unstable and generate ap sites . the specificity of such repair was also compared with those of dna glycosylases and damage - specific endonucleases of e. coli both of which were found to have no detectable activity toward this adduct . we also showed that p - bq - modified plasmid is efficiently cleaved by uvrabc , indicating the involvement of ner in repair of benzene - derived dna damage . the role of ner in the repair of hq / p - bq - induced dna damage was also suggested in another mutagenesis study using hq- or p - bq - treated plasmid containing the supf reporter gene in ner - deficient ( xpa ) human cells . note that hq- and p - bq - derived exocyclic adducts are repaired by a different mechanism called nucleotide incision repair ( nir ) , as discussed below . in general , although benzene metabolites show relatively low dna binding activity in vivo , their induced dna damage and repair seem to be complex . ber is the primary repair mechanism for the removal of small dna lesions such as alkylated , oxidized , and deaminated bases from endogenous sources or environmental carcinogens [ 64 , 187191 ] . the steps of the ber pathway have been well characterized : it is initiated by a damage - specific dna glycosylase that recognizes a modified base and cleaves the n - glycosylic bond between the base and the sugar moiety . glycosylases can be divided into monofunctional , for example , alkylpurine - dna glycosylase ( aag , also mpg , apng , and anpg ) and thymine - dna glycosylase ( tdg ) , and bifunctional ( with an ap lyase activity ) , for example , ogg1 , endonuclease iii homolog 1 ( nth1 ) , and endonuclease viii - like glycosylases ( neils ) . each dna glycosylase has its unique specificity , but overlapping activities are common among various dna glycosylases that may have different structures and/or catalytic mechanisms . after glycosylase , the resulting ap site is processed by 5 ap endonuclease , ap lyase , and dna polymerase activities to cleave the ap site , trim strand break intermediates , and catalyze repair synthesis . a dna ligase finally completes the process by sealing the remaining nick . the basic ber mechanism described above is complicated by the identification of subpathways ( i.e. , the short - patch and long - patch ber ) in mammalian systems . there is also a network of protein - protein interactions involving numerous proteins inside and outside of ber , which is thought to play a key role in coordination of ber components as well as in regulation of cellular ber functions [ 193195 ] . evidence has emerged to support that ber deficiency is an important contributing factor of cancer susceptibility , as shown in both animal models and human studies . surveying the activities of known glycosylases indicates that many of them are able to excise tobacco carcinogen - induced dna adducts , including the common alkylated and oxidized bases and some exocyclic adducts . the tobacco carcinogen - derived exocyclic dna adducts listed in figure 5 are known substrates for respective glycosylases as described below . it should be noted that a crucial role of ber is to repair an ap site which is mutagenic because of its noncoding nature [ 197 , 198 ] . as stated above , certain tobacco carcinogens generate unstable dna adducts that are an important source of the ap sites in the genome . ( 1 ) formation and repair of etheno dna adductsetheno ( ) adducts are the most extensively studied exocyclic adducts [ 34 , 199 ] which are formed by the attack of bifunctional aldehydes or epoxides at a nitrogen of the base , followed by dehydration and ring closure . cigarette smoke is a significant source for these adducts as shown by the urinary levels of c and 1,n-g in smokers . these adducts could be formed by vc in cigarette smoke ( 530 ng / cigarette ) as well as lpo products . vc is processed by p450 yield unstable chloroethylene oxide ( ceo ) , which quickly converts to chloroacetaldehyde ( caa ) [ 202 , 203 ] . both ceo and caa can form -adducts . in experimental animals and in humans exposed to vc , caa has been studied extensively in terms of forming -adducts , [ 18 , 99 ] , and the quantitative relationships in double - stranded dna treated with caa are as follows : 3,n-c 1,n-a > n,3-g 1,n-g . the mutagenic properties of these adducts have been established , and there is evidence that -adducts may be responsible for ras and p53 mutations in liver tumors of vc - exposed humans .studies on repair of exocyclic adducts have largely been focused on ber , except for propano - dg adducts that are repaired by ner . -adducts are repaired by ber , initiated mainly by two human dna glycosylases : aag and tdg . in e. coli , they are repaired by functional homologs of aag and tdg : alka ( ma - dna glycosylase ii ) and mismatch uracil - dna glycosylase ( mug ) , respectively . excision of ahuman aag excises this adduct from double - stranded [ 205 , 206 ] and single - stranded dna . the crystal structure of human aag bound to dna containing an a has been solved [ 208 , 209 ] . aag is also the major activity against a in vivo , as shown in aag knockout mice [ 210 , 211 ] . increased mutations were observed in the hprt gene , and levels of a were significantly higher and persisted longer in dna from aag mice than those from wild - type mice when treated with vinyl carbamate [ 210 , 211 ] . moreover a and c accumulated to higher levels in aag mice following stimulation of colonic inflammation , indicating that the repair of such adducts formed by lpo is important for protection against chronic inflammation - induced ros and carcinogenesis . as described below , the alkb / abh pathway is also involved in the repair of a and c adducts . excision of cwe and saparbaev et al . independently found that the c activity resides in both human tdg and e. coli mug proteins [ 214 , 215 ] . the main biological role of tdg appears to remove thymine from a t : g mismatch resulting from the deamination of 5-methylcytosine ( 5-mc ) in a cpg site , which could be involved in active dna demethylation when in combination with a deaminase that converts 5-mc to t leading to a t : g mismatch . in vitro assays have shown that the activity of tdg is most efficient when t : g or c : g is in a cpg sequence context [ 217 , 218 ] . recently , the crystal structure of tdg ( the catalytic domain ) complexed with dna containing an ap site was reported . other studies have also shown low excision of c by human single - strand - selective monofunctional uracil - dna glycosylase ( smug1 ) and methyl - cpg binding domain protein ( mbd4 or med1 ) . excision of g adductsin rodents , n,3-g represents the predominant -adduct and is readily induced in hepatic nonparenchymal cells by vc , the target cells for this compound . there is a correlation between the levels of this adduct and the incidence of vc - induced angiosarcoma . both in vitro and animal studies showed that the human removal of n,3-g is slow . it was also shown that repair capacity would be different in various cell types in liver in that the expression of aag mrna was induced in the hepatocytes of rat exposed to vc , while the nonparenchymal cells had only 20% of the aag mrna of hepatocytes , indicating that the target cells for vc had much lower expression of this glycosylase . it should be noted that n,3-g is also an endogenous adduct arising from lpo . 1,n-g , an isomer of n,3-g , is a substrate for both e. coli mug and human aag as tested in vitro [ 207 , 226 ] . excision of hydroxymethyl -adductswe recently studied in vitro repair of two exocyclic adducts formed by acrolein metabolite glycidaldehyde ( gda ) , a potent mutagen and animal carcinogen . 7-(hydroxymethyl)-1,n - ethenoadenine ( 7-hm-a ) , the main adduct , can be found in skin cells of mice treated topically with gda . these analogs are expected to be as promutagenic as the corresponding -adducts , and 8-hm-c has been shown to miscode when tested with mammalian dna pols . while 8-hm-c is excised by e. coli mug and human tdg , these excision activities were from half - to a few - fold lower than the corresponding activities , which could be attributed to the extra ch2oh group on the -ring . etheno ( ) adducts are the most extensively studied exocyclic adducts [ 34 , 199 ] which are formed by the attack of bifunctional aldehydes or epoxides at a nitrogen of the base , followed by dehydration and ring closure . cigarette smoke is a significant source for these adducts as shown by the urinary levels of c and 1,n-g in smokers . these adducts could be formed by vc in cigarette smoke ( 530 ng / cigarette ) as well as lpo products . vc is processed by p450 yield unstable chloroethylene oxide ( ceo ) , which quickly converts to chloroacetaldehyde ( caa ) [ 202 , 203 ] . both ceo and caa can form -adducts . in experimental animals and in humans exposed to vc , caa has been studied extensively in terms of forming -adducts , [ 18 , 99 ] , and the quantitative relationships in double - stranded dna treated with caa are as follows : 3,n-c 1,n-a > n,3-g 1,n-g . the mutagenic properties of these adducts have been established , and there is evidence that -adducts may be responsible for ras and p53 mutations in liver tumors of vc - exposed humans . studies on repair of exocyclic adducts have largely been focused on ber , except for propano - dg adducts that are repaired by ner . -adducts are repaired by ber , initiated mainly by two human dna glycosylases : aag and tdg . in e. coli , they are repaired by functional homologs of aag and tdg : alka ( ma - dna glycosylase ii ) and mismatch uracil - dna glycosylase ( mug ) , respectively . excision of ahuman aag excises this adduct from double - stranded [ 205 , 206 ] and single - stranded dna . the crystal structure of human aag bound to dna containing an a has been solved [ 208 , 209 ] . aag is also the major activity against a in vivo , as shown in aag knockout mice [ 210 , 211 ] . increased mutations were observed in the hprt gene , and levels of a were significantly higher and persisted longer in dna from aag mice than those from wild - type mice when treated with vinyl carbamate [ 210 , 211 ] . moreover a and c accumulated to higher levels in aag mice following stimulation of colonic inflammation , indicating that the repair of such adducts formed by lpo is important for protection against chronic inflammation - induced ros and carcinogenesis . as described below , the alkb / abh pathway is also involved in the repair of a and c adducts . excision of cwe and saparbaev et al . independently found that the c activity resides in both human tdg and e. coli mug proteins [ 214 , 215 ] . the main biological role of tdg appears to remove thymine from a t : g mismatch resulting from the deamination of 5-methylcytosine ( 5-mc ) in a cpg site , which could be involved in active dna demethylation when in combination with a deaminase that converts 5-mc to t leading to a t : g mismatch . in vitro assays have shown that the activity of tdg is most efficient when t : g or c : g is in a cpg sequence context [ 217 , 218 ] . recently , the crystal structure of tdg ( the catalytic domain ) complexed with dna containing an ap site was reported . other studies have also shown low excision of c by human single - strand - selective monofunctional uracil - dna glycosylase ( smug1 ) and methyl - cpg binding domain protein ( mbd4 or med1 ) . excision of g adductsin rodents , n,3-g represents the predominant -adduct and is readily induced in hepatic nonparenchymal cells by vc , the target cells for this compound . there is a correlation between the levels of this adduct and the incidence of vc - induced angiosarcoma . both in vitro and animal studies showed that the human removal of n,3-g is slow . it was also shown that repair capacity would be different in various cell types in liver in that the expression of aag mrna was induced in the hepatocytes of rat exposed to vc , while the nonparenchymal cells had only 20% of the aag mrna of hepatocytes , indicating that the target cells for vc had much lower expression of this glycosylase . it should be noted that n,3-g is also an endogenous adduct arising from lpo . 1,n-g , an isomer of n,3-g , is a substrate for both e. coli mug and human aag as tested in vitro [ 207 , 226 ] . excision of hydroxymethyl -adductswe recently studied in vitro repair of two exocyclic adducts formed by acrolein metabolite glycidaldehyde ( gda ) , a potent mutagen and animal carcinogen . 7-(hydroxymethyl)-1,n - ethenoadenine ( 7-hm-a ) , the main adduct , can be found in skin cells of mice treated topically with gda . these analogs are expected to be as promutagenic as the corresponding -adducts , and 8-hm-c has been shown to miscode when tested with mammalian dna pols . while 8-hm-c is excised by e. coli mug and human tdg , these excision activities were from half - to a few - fold lower than the corresponding activities , which could be attributed to the extra ch2oh group on the -ring . human aag excises this adduct from double - stranded [ 205 , 206 ] and single - stranded dna . the crystal structure of human aag bound to dna containing an a has been solved [ 208 , 209 ] . aag is also the major activity against a in vivo , as shown in aag knockout mice [ 210 , 211 ] . increased mutations were observed in the hprt gene , and levels of a were significantly higher and persisted longer in dna from aag mice than those from wild - type mice when treated with vinyl carbamate [ 210 , 211 ] . moreover a and c accumulated to higher levels in aag mice following stimulation of colonic inflammation , indicating that the repair of such adducts formed by lpo is important for protection against chronic inflammation - induced ros and carcinogenesis . as described below , the alkb / abh pathway is also involved in the repair of a and c adducts . we and saparbaev et al . independently found that the c activity resides in both human tdg and e. coli mug proteins [ 214 , 215 ] . the main biological role of tdg appears to remove thymine from a t : g mismatch resulting from the deamination of 5-methylcytosine ( 5-mc ) in a cpg site , which could be involved in active dna demethylation when in combination with a deaminase that converts 5-mc to t leading to a t : g mismatch . in vitro assays have shown that the activity of tdg is most efficient when t : g or c : g is in a cpg sequence context [ 217 , 218 ] . recently , the crystal structure of tdg ( the catalytic domain ) complexed with dna containing an ap site was reported . other studies have also shown low excision of c by human single - strand - selective monofunctional uracil - dna glycosylase ( smug1 ) and methyl - cpg binding domain protein ( mbd4 or med1 ) . in rodents , n,3-g represents the predominant -adduct and is readily induced in hepatic nonparenchymal cells by vc , the target cells for this compound . there is a correlation between the levels of this adduct and the incidence of vc - induced angiosarcoma . both in vitro and animal studies showed that the human removal of n,3-g is slow . it was also shown that repair capacity would be different in various cell types in liver in that the expression of aag mrna was induced in the hepatocytes of rat exposed to vc , while the nonparenchymal cells had only 20% of the aag mrna of hepatocytes , indicating that the target cells for vc had much lower expression of this glycosylase . it should be noted that n,3-g is also an endogenous adduct arising from lpo . 1,n-g , an isomer of n,3-g , is a substrate for both e. coli mug and human aag as tested in vitro [ 207 , 226 ] . we recently studied in vitro repair of two exocyclic adducts formed by acrolein metabolite glycidaldehyde ( gda ) , a potent mutagen and animal carcinogen . 7-(hydroxymethyl)-1,n - ethenoadenine ( 7-hm-a ) , the main adduct , can be found in skin cells of mice treated topically with gda . these analogs are expected to be as promutagenic as the corresponding -adducts , and 8-hm-c has been shown to miscode when tested with mammalian dna pols . while 8-hm-c is excised by e. coli mug and human tdg , these excision activities were from half - to a few - fold lower than the corresponding activities , which could be attributed to the extra ch2oh group on the -ring . ( 1 ) formation and repair of pyridyloxobutyl ( pob)-dna adducts of tsnastobacco - specific nitrosamines ( tsnas ) are exclusively found in cigarette smoke and are formed through n - nitrosation of nicotine during tobacco curing and processing . common tsnas found in cigarette smoke particles include nnk , nnn , and n - nitrosoanatabine ( nab ) . in addition , nnk- and nnn - generated reactive intermediates form bulky pob - dna adducts , including the 7- and o - positions of dg and the o - position of dc and t . four of them have been recently characterized and detected in nnk- or nnn - treated animals . one of them , o-[4-(3-pyridyl)-4-oxobut-1-yl]-2-dg ( o - pob - dg ) [ 237239 ] ( figure 2 ) , will be discussed here , which has been shown to be mutagenic in both e. coli and human cells using a site - specific mutagenesis assay and is considered a critical lesion in nnk / nnn carcinogenesis . higher levels of adducts are found in lung and tracheobrunchial tissues of smokers than in nonsmokers , by the detection of 4-hydroxy-1-(3-pyridyl)-1-butanone ( hpb ) , a product from acid hydrolysis of the pob - dna adducts . most recently , 1-(n - methyl - n - nitrosamino)-1-(3-pyridinyl)-4-butanal ( nna ) was identified from thirdhand smoke ( ths ) as the major product resulting from the reaction of nicotine with nitrous acid ( hono ) , along with nnk and nnn ( see section 3.2).both o - mg and o - pob - dg adducts have been shown to be substrates for agt [ 106 , 237 , 242 ] . however , the repair of the bulky pob adducts has been much less studied compared to o - mg . agt primarily repairs o - alkylguanine adducts and protects against mutagenicity of respective alkylating agents . repair occurs by transfer of the alkyl group at the o position of g to a cysteine residue at its active site of the protein , which results in a protein conformational change that signals for its degradation . agt reaction is stoichiometric with o - mg acting as a suicide substrate ; therefore , the cellular repair capacity is limited by the constitutive levels of agt that can be also depleted under overdose of alkylating agents . o - pob - dg has been shown to be repaired by agt both in vitro and in vivo . this adduct is efficiently repaired by mammalian agts but poorly repaired by bacterial counterparts , adac and ogt [ 235 , 244 ] . since both o - mg and o - pob - dg may have implications in nnk - induced carcinogenesis , the relative repair rates of these two adducts by agt should be an important factor in determining the levels and biological importance of these two lesions . studies by mijal et al . demonstrated that human agt showed an ~2-fold preference for the removal of o - mg over o - pob - dg , rodent agts exhibited the same rate , and the bacterial proteins reacted poorly with o - pob - dg . these data indicate the high importance of protein structure with respect to substrate efficiency . in conclusion , agt is expected to be critical in the repair of o - alkylguanine adducts formed by tobacco - derived n - nitrosamines . it should be noted that cytotoxicity and mutagenesis studies suggest an ner involvement in the removal of nnk - derived dna damage [ 236 , 245 ] . a very weak but time - dependent in vitro ner activity was also detected using oligonucleotide containing an o - pob - dg and reconstituted human excision nuclease . tobacco - specific nitrosamines ( tsnas ) are exclusively found in cigarette smoke and are formed through n - nitrosation of nicotine during tobacco curing and processing . common tsnas found in cigarette smoke particles include nnk , nnn , and n - nitrosoanatabine ( nab ) . in addition , nnk- and nnn - generated reactive intermediates form bulky pob - dna adducts , including the 7- and o - positions of dg and the o - position of dc and t . four of them have been recently characterized and detected in nnk- or nnn - treated animals . one of them , o-[4-(3-pyridyl)-4-oxobut-1-yl]-2-dg ( o - pob - dg ) [ 237239 ] ( figure 2 ) , will be discussed here , which has been shown to be mutagenic in both e. coli and human cells using a site - specific mutagenesis assay and is considered a critical lesion in nnk / nnn carcinogenesis . higher levels of adducts are found in lung and tracheobrunchial tissues of smokers than in nonsmokers , by the detection of 4-hydroxy-1-(3-pyridyl)-1-butanone ( hpb ) , a product from acid hydrolysis of the pob - dna adducts . most recently , 1-(n - methyl - n - nitrosamino)-1-(3-pyridinyl)-4-butanal ( nna ) was identified from thirdhand smoke ( ths ) as the major product resulting from the reaction of nicotine with nitrous acid ( hono ) , along with nnk and nnn ( see section 3.2 ) . both o - mg and o - pob - dg adducts have been shown to be substrates for agt [ 106 , 237 , 242 ] . however , the repair of the bulky pob adducts has been much less studied compared to o - mg . agt primarily repairs o - alkylguanine adducts and protects against mutagenicity of respective alkylating agents . repair occurs by transfer of the alkyl group at the o position of g to a cysteine residue at its active site of the protein , which results in a protein conformational change that signals for its degradation . agt reaction is stoichiometric with o - mg acting as a suicide substrate ; therefore , the cellular repair capacity is limited by the constitutive levels of agt that can be also depleted under overdose of alkylating agents . o - pob - dg has been shown to be repaired by agt both in vitro and in vivo . this adduct is efficiently repaired by mammalian agts but poorly repaired by bacterial counterparts , adac and ogt [ 235 , 244 ] . since both o - mg and o - pob - dg may have implications in nnk - induced carcinogenesis , the relative repair rates of these two adducts by agt should be an important factor in determining the levels and biological importance of these two lesions . studies by mijal et al . demonstrated that human agt showed an ~2-fold preference for the removal of o - mg over o - pob - dg , rodent agts exhibited the same rate , and the bacterial proteins reacted poorly with o - pob - dg . these data indicate the high importance of protein structure with respect to substrate efficiency . in conclusion , agt is expected to be critical in the repair of o - alkylguanine adducts formed by tobacco - derived n - nitrosamines . it should be noted that cytotoxicity and mutagenesis studies suggest an ner involvement in the removal of nnk - derived dna damage [ 236 , 245 ] . a very weak but time - dependent in vitro ner activity was also detected using oligonucleotide containing an o - pob - dg and reconstituted human excision nuclease . ( 2 ) repair of etheno adducts by alkb homologsit was reported in 2005 that e. coli alkb protein and its human homolog , abh3 , repair a and c in vitro [ 246 , 247 ] . later , abh2 was shown to exhibit robust activity for a and is the principal dioxygenase for removal of a in vivo as shown by abh2 mouse studies . further experiments showed that abh2 , but not abh3 , is able to complement the e. coli alkb mutant which is defective in the repair of -adducts . alkb is a member of the superfamily of iron-/-ketoglutarate - dependent dioxygenases . using bioinformatics , eight mammalian homologs of alkb , abh1 to abh8 , the direct reversal mechanism for their action involves a unique iron - mediated reaction with cofactor -ketoglutarate that could epoxidize the exocyclic double bond of the -adducts [ 246 , 247 ] . the epoxide generated can be hydrolyzed to form the lesion - free base and glyoxal . in addition to a and c , these proteins also repair other methylated / ethylated bases [ 250 , 251 ] . they can act on both single- and double - stranded dna substrates and may play different / complementary roles to the glycosylases as mentioned above . in general , single - strand specificity suggests repair of lesions in single - stranded dna regions that are transiently generated during replication and transcription.given that dna glycosylases also act on -adducts ( see section 2.3.2(1 ) ) , at least two repair pathways may act on these adducts in vivo . this may explain why the incidences of carcinomas were similar between wild - type and aag - knockout mice treated with vinyl carbamate . genetic studies using alka - proficient and -deficient cells show that alkb is important for counteracting the mutagenicity of the -adducts . a study comparing the repair efficiency of alkb versus alka in e. coli shows that alka seems to be the more important enzyme in response to exposure to caa . similar data were obtained from knockout mice , which showed that the abh2 activity is not sufficient for the removal of spontaneously produced a adducts in aag mouse liver , whereas mouse aag activity is sufficient to repair spontaneously produced a lesions in abh2 mouse liver . these results suggest that both aag and abh2/3 proteins can play a role in the cellular response to the exposure of tobacco carcinogens that generate these -adducts . it was reported in 2005 that e. coli alkb protein and its human homolog , abh3 , repair a and c in vitro [ 246 , 247 ] . later , abh2 was shown to exhibit robust activity for a and is the principal dioxygenase for removal of a in vivo as shown by abh2 mouse studies . further experiments showed that abh2 , but not abh3 , is able to complement the e. coli alkb mutant which is defective in the repair of -adducts . alkb is a member of the superfamily of iron-/-ketoglutarate - dependent dioxygenases . using bioinformatics , eight mammalian homologs of alkb , abh1 to abh8 , the direct reversal mechanism for their action involves a unique iron - mediated reaction with cofactor -ketoglutarate that could epoxidize the exocyclic double bond of the -adducts [ 246 , 247 ] . the epoxide generated can be hydrolyzed to form the lesion - free base and glyoxal . in addition to a and c , these proteins also repair other methylated / ethylated bases [ 250 , 251 ] . they can act on both single- and double - stranded dna substrates and may play different / complementary roles to the glycosylases as mentioned above . in general , single - strand specificity suggests repair of lesions in single - stranded dna regions that are transiently generated during replication and transcription . given that dna glycosylases also act on -adducts ( see section 2.3.2(1 ) ) , at least two repair pathways may act on these adducts in vivo . this may explain why the incidences of carcinomas were similar between wild - type and aag - knockout mice treated with vinyl carbamate . genetic studies using alka - proficient and -deficient cells show that alkb is important for counteracting the mutagenicity of the -adducts . a study comparing the repair efficiency of alkb versus alka in e. coli shows that alka seems to be the more important enzyme in response to exposure to caa . similar data were obtained from knockout mice , which showed that the abh2 activity is not sufficient for the removal of spontaneously produced a adducts in aag mouse liver , whereas mouse aag activity is sufficient to repair spontaneously produced a lesions in abh2 mouse liver . these results suggest that both aag and abh2/3 proteins can play a role in the cellular response to the exposure of tobacco carcinogens that generate these -adducts . the exocyclic benzetheno p - bq adducts are bulkier than the -adducts , with an additional five - membered ring and a hydroxy group . as might be expected , such bulky adducts hinder replication in vitro and in vivo and cause frameshift deletions and base mispairing . in the past years , we have studied repair of three major in vitro adducts formed by hq and p - bq ( designated as pbq adducts ) , 1,n - pbq - da , 3,n - pbq - dc , and 1,n - pbq - dg ( figure 6 ) . our initial study discovered that these adducts are recognized by the major human ap endonuclease ( ape1 , also known as hap1 , apex , and ref-1 ) as well as e. coli exonuclease iii and endonuclease iv [ 253 , 254 ] . mechanistic studies showed that human ape1 hydrolyzes the phosphodiester bond 5 next to the adduct , leaving the p - bq derivative on the 5-terminal of the 3 fragment as a dangling base [ 253 , 255 , 256 ] . this mode of incision was later named nucleotide incision pathway [ 257 , 258 ] , which also acts on several oxidized dna bases . while the ap site is the preferred substrate for ape1 , cleavage of the pbq - dc adduct requires the same catalytic center as the ap site as shown from mutant ape1 proteins . molecular dynamics simulations suggest that ape1 utilizes a reaction mechanism for phosphodiester bond cleavage of dna containing pbq - dc similar to that reported for the ap site . given that these adducts have not been reported to be present in vivo , the biological role of these adducts as well their repair by nir awaits further investigation . the repair mechanisms for representative bulky dna adducts discussed above are summarized in figure 7 . it should be emphasized that most of the repair studies in the past have applied a single compound for modification or exposure , and results from such studies can not be simply extrapolated to real exposures . however , the information on repair specificity and efficiency from such studies provides the framework for further evaluation of potential relationships among repair deficiencies , carcinogen mutagenicity , and human susceptibility to cigarette smoke . also , as stated above , tobacco carcinogens are able to generate other specific dna lesions in addition to bulky dna adducts . the importance of bulky dna adducts relative to other types of dna lesions needs further investigation , and in any case , a combined action from different types of dna / chromosomal damage , as demonstrated in figure 4 for benzene ' biological effects , is expected to be the basis of genotoxicity conferred by many tobacco carcinogens . a crucial question in repair is how repair proteins recognize dna adducts , since repair specificity has both biochemical and biological implications . a related question is what are the factors responsible for good and poor repair ? to date , we have learned a great deal with regard to what structural factors of adducts and repair proteins determine the specificity and rate of repair , mainly based on biochemical data atomic resolution structures of adducted dna and repair proteins [ 64 , 102 , 260 , 261 ] . the study of how a dna adduct affects the structure of dna and how it interacts with its repair protein is essential in order to develop a theory for both why only some adducts are repaired and the specific mode of repair . when surveying the multiplicity of dna substrates for ner , the primary repair pathway for many bulky dna adducts , one basic question is how ner recognizes these chemically diverse substrates through the common structural features of the recognition unit xpc - hr23b . moreover , what is the basis for the fact that the repair rates of different bulky adducts by ner can vary by several orders of magnitude [ 111 , 262 ] ? it should be recognized that , as described above , the complexity of these adducts is enormous , including those adducts that are formed by compounds with stereochemical properties ( e.g. , bpde , acrolein , and hne ) . recognition and repair rates of such stereoisomeric adducts generally reflect or depend on adduct conformation . in the case of bpde - n - dg adducts , both their removal by human ner and patterns of helix opening by xpc - hr23b are stereochemistry dependent . recently , crystal structures of ner proteins , that is , bacterial uvrb and yeast rad4 ( human xpc homolog ) , complexed with damaged dna , have been described , both of which suggest a mode of action involving strand separation and nucleotide flipping for the bulky dna lesions . as for ber , a number of high - resolution structures of glycosylases , including those complexed with dna lesions , have provided a valuable insight into adduct selection as well as mechanisms of base flipping and catalysis [ 260 , 261 , 265 ] . in addition , molecular modeling studies such as molecular dynamics simulations have offered additional information about the structural features of dna substrates and their interactions with repair enzymes [ 36 , 266 ] . some common features of recognition by dna glycosylases can be summarized based on the reported structural studies : ( 1 ) adduct shape , hydrogen - bonding potential , and electric charge distribution are key for recognition ; ( 2 ) base unstacking is present at the lesion site ; ( 3 ) the target nucleotide has to be flipped out of the dna duplex and fit in the active site of a glycosylase . similarly , ap endonucleases flip an ap site out into their active sites , as shown by the co - crystal structures [ 259 , 267 ] . for instance , ber excises the 5-membered unsaturated exocyclic g but not the 6-membered propano - dg [ 34 , 157 ] . also , why can lesions with largely diverse structures be processed by the same protein , as seen by human ape1 acting on both ap site and pbq - dc [ 253 , 255 , 256 ] ? in general , the key to the specificity of recognition has been known to be not only the primary adduct structure , but the localized effect of each adduct on dna structure as well as on thermodynamics , and , moreover , the structure and function of the repair protein . since most repair enzymes act on adducts in double - stranded dna , each adduct may cause differing distortion and flexibility as a result of factors such as being in the major or minor groove , syn or anti , planar or angular , adjacent base pair tilting , propeller twist , and helical twist . ultimately , it is hoped that we can predict repair specificity / efficiency as well as identify structural hallmarks of mutagenic lesions in the genome , using appropriate computational and/or screening approaches . this can only be done after a large amount of structural and theoretical data have been acquired , which relate adduct structural features with outcomes of repair and mutagenesis . the sequence - dependent repair infers that local dna structures adjacent to an adduced nucleotide are important determinants of repair specificity and efficiency . the study of the role of sequence in base modification was started mainly in the 1980s in terms of sequence selectivity for mutational events which were generally induced by environmental agents . extensive work on relating sequence - dependent adduct formation and mutation has been done using chemical modification ( e.g. , bpde ) of genomic dna , followed by determination of the mutation pattern and spectra . these data were among the first used to substantiate the concept of hotspots ; that is , for a given reagent , there was site specificity for dna modification . an example is that in vivo , only the second guanine residue in codon 12 ( gga ) of the h - ras gene was modified by an alkylating agent , leading to g : c to a : t mutations , which is consistent with other studies demonstrating the sequence - dependent formation of o - alkylguanine in dna [ 270 , 271 ] . in addition to preferential dna adduct formation at specific sites , poor repair is another major determinant of mutational hotspots . many studies have highlighted the importance of sequence context in influencing the rate and extent of repair . examples among the tobacco carcinogen - induced bulky dna adducts are bpde - dna adducts [ 134 , 138 , 272274 ] , pob - dna adducts , a , and c , whose repair efficiencies could vary over manyfold when present in different neighbor sequences . these examples involve repair systems including at least ner , ber , nir , and agt . a review by singer and hang commented on many enzymes in these pathways with regard to the role of adduct , neighbor bases , and repair rate . also , donigan and sweasy recently summarized the known sequence context - specific activities of several glycosylases and polymerase in ber . it can be concluded now that sequence - dependent repair tends to be predominant , instead of being a random phenomenon . mechanistically , the structural factors that modulate sequence - dependent repair have been well studied with certain adducts such as bpde - dna adducts [ 274 , 277 ] . an nmr study using a sequence containing a natural gg mutational hotspot showed that the presence of the major bpde - derived dg adduct at one of the two neighboring g positions resulted in significantly different local structural distortions , especially bending or kinking at the adduct position and destabilization of watson - crick hydrogen bonding of the flanking base pairs . using the same sequences , kropachev et al . demonstrated that such hydrogen bonding destabilization elicits the most significant ner response , while the flexible kink is less important in such interaction . it is also apparent that the chemical nature of a dna adduct itself can affect the effect of neighbor sequences . for example , the repair of a pob adduct by agt is more strongly influenced by its neighbor bases than that of the smaller methylated base substrate . regardless of the nature of the specific structural differences discussed above , current evidence also supports that thermodynamic stability of lesion - containing oligonucleotides plays an important role in sequence - dependent repair [ 268 , 274 , 278 ] . in the case of bpde - dna adducts , the degree of local thermodynamic destabilization was related to the degree of recognition of duplex sequences containing a bulky adduct by the ner machinery [ 262 , 274 , 279 ] . both of our studies on sequence - dependent repair of a and ap site also demonstrated a role of thermodynamic properties in influencing double - strandedness of the substrates and repair their efficiency . recent studies have discovered a strong coincidence of mutational hotspots in human lung cancers and the sites of preferential binding of bpde [ 42 , 281 ] and acrolein in the p53 gene . the overall prevalence of p53 mutations is higher in cigarette smokers than in nonsmokers [ 51 , 52 ] . a number of hotspots have been found along p53 in lung cancer which are generally g to t transversions . it has been reported that in pah- or acrolein - treated cells the same positions of their mutation hotspots are also major hotspots for mutations observed in human lung cancers from smokers , strongly suggesting a role of dna adducts in etiology of these cancers . as discussed above , poor / slow repair of dna adducts at these sites may be a major factor for their occurrence and persistence at these mutational hotspots . to further support this nation several groups in the mid-1990s examined the in vivo repair rates along a gene fragment using the ligation - mediated pcr technique . as for bpde adducts in human hprt and p53 genes , wei et al . found that repair rates can differ markedly from site to site over a time period , as measured by the percentage of adduct remaining . moreover , very slow repair was observed at certain positions that are frequently mutated after bpde treatment . these studies clearly indicate a correlation between inefficient dna repair and the occurrence of mutation hotspots . finally , in addition to site - specific preferential formation of dna adducts and sequence context of dna repair , the biological selection of induced mutations is also considered important for the hotspot phenomenon , which gives cells with specific mutation(s ) a growth advantage and results in dominant mutations in cancer cells [ 283 , 284 ] . it has long been recognized that only a small percent of cigarette smokers develop cancer , for example , ~11%24% of smokers develop lung cancer , which suggested that interindividual variability in key cellular processes is crucial in response to tobacco carcinogens . many molecular and epidemiological studies have been revealing a multifactorial nature of such variability [ 286289 ] . one top aim of the target cancer prevention programs is to identify smokers and nonsmokers exposed to shs with higher susceptibility . the interindividual differences discussed below will be focused on those genotypes and phenotypes involving dna repair capacity ( drc ) in relation to the mutagenicity and carcinogenicity of tobacco - induced bulky adducts . considerable progress has been made towards a better understanding of the association between individual tobacco carcinogens and tumor development in specific organs / tissues , as exemplified by the following cases : nnk and pah are potent lung carcinogens ; aromatic amines such as 4-abp are the main cause of bladder cancer in smokers ; benzene induces acute myelogenous leukemia ( aml ) . whether reduced or deficient drc for tobacco carcinogen - derived dna damage is associated with somatic mutation and susceptibility to cancer has been a subject of investigation . a commonly used approach is to measure the repair or levels of specific dna adducts which serve as an intermediate end - point of genotoxicity . decreased repair activities for bulky dna adducts have been observed in cells / tissues of cancer patients . for example , epidemiologic studies using the hcr assay showed that low cellular drc in response to bpde - induced dna damage is associated with increased risk of lung , head , and neck cancers [ 65 , 286 , 290 ] . biochemical studies also showed that reduced repair of a and c adducts was present in lung adenocarcinomas . in principle , the phenotypes of dna repair must be characterized for mutagenic adducts and any newly identified adducts in smokers and nonsmokers exposed to shs . a common one is polymorphisms in relevant dna repair genes , such as those identified in the ner , ber , and agt pathways [ 289 , 292 , 293 ] . it has been shown that cellular levels of dna adducts such as those arising from bpde exposure can be affected in some of those genetic variants [ 132 , 294 ] . in the last decade , although mixed or discrepant results have been reported , positive results on numerous polymorphisms in dna repair genes , along with those in metabolic genes , have been revealed and are continuously being found in the context of cigarette smoking and cancer [ 22 , 295 , 296 ] . it seems that polymorphisms in tobacco metabolism and/or repair should lead to differences in both local carcinogen levels and/or dna adduct levels in vivo . another mechanism that can cause the loss of dna repair capacity is loh ; for instance , loh at the human 8-oxog - dna glycosylase ( ogg1 ) gene locus is a frequent event in lung cancer , which would increase the mutational load from 8-oxog due to ros in smokers . drc could also be influenced by nongenetic factors that cause a phenotypic reduction / ablation of repair activities . examples of such factors include those that are disease related , for example , ner deficiency in xp and cockayne syndrome patients [ 64 , 140 ] , and those that are repair - inhibition based . there are at least 30 metals in cigarette smoke , including arsenic , cadmium , nickel , and chromium , which have been shown to inhibit various dna repair enzymes and pathways [ 298302 ] . for instance , both arsenic and nickel compounds interfere with the repair of bpde - dna adducts [ 298 , 299 ] . cadmium and chromium ( vi ) are also known as effective dna repair inhibitors and play a similar role in influencing adduct levels in smokers [ 303 , 304 ] . therefore , coexposure to these heavy metals by smokers may enhance the mutagenic potential of genotoxic tobacco carcinogens . in addition to metals , certain tobacco carcinogens themselves have been found to inhibit dna repair , as exemplified by acrolein inhibiting ner repair of bpde - dna adducts . similar to metals , the coexistence of these chemicals in cigarette smoke is also expected to lead to more persistent or severe dna damage as a result of suppressed dna repair . in addition to tissue differences in bioactivation of tobacco carcinogens [ 306 , 307 ] , recent studies in rodents suggest that the drc in a given organ / tissue / cell type may play a role in organoselectivity of tobacco carcinogens . for example , both agt and ner activities are related to the interorgan differences in response to nnk treatment , which could be an important factor in determining organ - specific susceptibility to nnk - induced carcinogenesis . dna adduct levels in the bone marrow of benzene - treated mice are significantly higher than those in liver , as shown by tissue distribution studies . interestingly , although no repair capacity has been tested for a particular benzene adduct in vivo , when treated with alkylating agents , the drc of primary human hematopoietic cd34 cells from bone marrow was significantly lower than more differentiated cd34 cells of the same donor . in general , the tissue- or cell type - specific responses to tobacco exposure and their mechanisms are not well understood and await more extensive investigation . the bulky dna adducts discussed here are only a small list of representatives derived from tobacco carcinogens . we need not only more information on these important bulky adducts , but also more knowledge of those other adducts that are still not understood . ( 1 ) there are many other chemicals in cigarette smoke that have not yet been evaluated for dna adduct - forming ability . for instance ( 2 ) chemicals may be neglected only because of their lower concentrations in cigarette smoke . for example , some pahs exist in cigarette smoke at low amounts relative to b[a]p . ( 3 ) the research attention tends to be more focused on the effect of the major adducts of a chemical carcinogen . however , in some cases , the minor adducts are substantially more mutagenic than the major ones . for example , the biological response of n - nitrosamines could be correlated with minor forms of dna damage . however , the repair specificity and kinetics of these dna lesions are still unknown . ( 4 ) many bulky adducts have been identified in vitro under physiological conditions but have not been detected yet in vivo . examples include a number of exocyclic adducts formed by benzene metabolites ( figure 4 ) . although many in vitro studies have been focused on the hq- and p - bq - derived benzetheno adducts and muc - dna adducts , no conclusion can yet be drawn on whether they are formed in cells / tissues . ( 5 ) in some cases , bulky dna adducts are detected from the tissues of exposed humans or animals using p - postlabeling , but their chemical structures have not been elucidated . these adducts were generally described in the litrature as aromatic or hydrophobic adducts . ( 6 ) endogenous dna adducts can be formed as a result of cigarette smoke , which is currently attributed to the formation of lpo products [ 9294 ] . further research is needed to learn the dose - response relationship with regard to the formation of these adducts as well as their relative importance in smoking - induced carcinogenesis . the complexity of such analysis is that the same dna adducts , for example , the -dna adducts , are generated by both tobacco carcinogens and endogenously formed compounds , even though they can be chemically different . ( 7 ) although the majority of studies on adducts and cancer have focused on stable ones , many adducts formed by tobacco carcinogens are chemically unstable , for example , the benzene - derived cat-4-n3a and cat-4-n7 g , and the potential effects of such adducts versus stable adducts are largely unknown . ( 8) many dna adducts have been detected , some in cells / tissues , but not yet characterized with respect to their repair . an example is that the epoxides of 1,3-butadiene [ 314 , 315 ] , a tobacco carcinogen in both mss and sss , cause the formation of n-1-(2,3,4-trihydroxybutyl ) adenine adduct in human samples , but its repair is unknown . ( 9 ) sometimes cellular repair has been detected using repair - deficient cells / animals in combination with mutagenesis assays , but detailed biochemical properties of such repair have not been elucidated . in other cases , repair studies have only been performed in vitro , such as the p - bq - derived benzetheno adducts [ 253 , 255 ] . although animal studies have limitations with significant differences from humans , many important experiments on the formation and repair of carcinogenic adducts can only be performed in appropriate animals . the major difference is that in vivo , enzymes may be inducible and become saturated at the carcinogen dose used , and in the genome , there can be preferential repair as well as organ and cell variations and there may be cooperative repair mechanisms which have not been well understood . this is only a partial list of the reasons why repair specificity and efficiency discovered in vitro have to be validated in vivo . to investigate the relationship between cigarette smoking and dna damage , understanding of the chemical and biophysical properties of various forms of tobacco smoke is also critical . for instance , although a great deal is known about the chemistry and toxicity of mss and shs , little is known about the identity and molecular toxicology of toxicants produced de novo in ths [ 6 , 7 ] . however , it has been well established that indoor surfaces significantly adsorb semi- and nonvolatile shs compounds , for example , nicotine , 3-ethenylpyridine , naphthalene , cresols , and phenol which are slowly reemitted into the air [ 317320 ] . moreover , compounds sorbed onto a surface can undergo chemical transformations by reacting with common reactive atmospheric species . recent indoor chemistry studies have elegantly revealed that nicotine reacts with ozone ( o3 ) to yield aldehydes and possibly myosmine and with nitrous acid ( hono ) to form nna , nnn , and nnk ( figure 8) . nna was identified as the major product , which is absent in freshly emitted tobacco smoke but found in in vitro reaction of nicotine with nano2 . nna has a mutagenic activity similar to that of nnn , but its tumorigenic activity in animals was not conclusive [ 323 , 324 ] . so far , nothing has been tested for its potential to form dna adducts , but it is expected to be reactive with dna due to its aldehyde group . therefore , it would be important to assess its intake and biological properties . based on their chemical structure , all of the by - products shown in figure 8 are anticipated or have already been known to form dna adducts . as stated earlier , sss and shs , the precursors of ths , contain thousands of chemicals , including nicotine and well - defined carcinogens , partitioned between the gas and particulate phases . the secondary analysis by schick 's group of animal experimental data from the past documents of philip morris available at university of california , san francisco ( ucsf ) concluded that the mature and aged sss is several - folds more toxic than the fresh sss . such evidence constitutes compelling and sufficient rationale to determine the chemical composition of unique toxicants produced in ths de novo and to detect new / different types of dna adducts formed by aged sss . in conclusion , this is a new and important research area with regard to developing strategies and methods to identify the chemical components shs aging and ths and to assess currently unknown genotoxic potential and biomarker availability through dna adduct studies . understanding the mechanisms of formation and repair of bulky dna adducts is critical for analysis and management of tobacco - induced mutagenesis and carcinogenesis . it is hoped that continued studies will provide more information about the structural and biological implications of specific bulky dna adducts and the broader range of their effects during the pathogenesis . future efforts should be made to identify and characterize novel compounds and adducts , such as those produced in aged shs and ths , to identify those highly mutagenic adducts that are refractory to dna repair , to find adducts as reliable biomarkers for measuring exposure , especially for shs and ths , to explore new potential biological functions of adducts , such as their interactions with cellular signaling networks , impact on stem cells of target tissues , and roles in epigenetic changes , and to find effective ways to inhibit dna adduct formation in target organs . innovative study designs along with more comprehensive approaches ( e.g. , systems biology ) and new technology development ( e.g. , high - throughput analysis ) will be important for achieving these goals . ultimately , a better knowledge of the mechanisms by which the chemical carcinogen exposure increases cancer risk in smokers and individuals exposed to shs / ths could lead to new strategies for cancer prevention and could serve as the experimental evidence for framing and enforcing tobacco control policies in order to minimize health hazards and protect vulnerable populations .

dna adducts play a central role in chemical carcinogenesis . the analysis of formation and repair of smoking - related dna adducts remains particularly challenging as both smokers and nonsmokers exposed to smoke are repetitively under attack from complex mixtures of carcinogens such as polycyclic aromatic hydrocarbons and n - nitrosamines . the bulky dna adducts , which usually have complex structure , are particularly important because of their biological relevance . several known cellular dna repair pathways have been known to operate in human cells on specific types of bulky dna adducts , for example , nucleotide excision repair , base excision repair , and direct reversal involving o6-alkylguanine dna alkyltransferase or alkb homologs . understanding the mechanisms of adduct formation and repair processes is critical for the assessment of cancer risk resulting from exposure to cigarette smoke , and ultimately for developing strategies of cancer prevention . this paper highlights the recent progress made in the areas concerning formation and repair of bulky dna adducts in the context of tobacco carcinogen - associated genotoxic and carcinogenic effects .

1. Introduction 2. Formation and Repair of Bulky DNA Adducts by Tobacco Carcinogens 3. From the Known to the Unknown: Future and Perspective

in the last 50 years , many studies have been performed to identify chemical toxicants in cigarette smoke , which may represent the most rich resource of exogenous human mutagens and carcinogens . this list contains some of the strong animal and/or human carcinogens , such as pahs , n - nitrosamines , and aromatic amines , all of which react with dna to form adducts [ 1113 ] . since such a carcinogenic source , that is , cigarette smoke , is preventable , and dna adduct levels correlate with cigarette consumption , tobacco smoke provides a unique model for understanding the cause - effect or environment - gene relationship in smoking - related cancer development . the well - studied microsomal cytochrome p450 ( cyp ) system activates many tobacco carcinogens such as pahs , n - nitrosamines , aromatic amines , and benzene [ 1820 ] . cigarette smoke condensates were initially known to have mutagenic activity by the 1970s , and adducts were detected in cellular dna from smokers in the 1980s . since then , many tobacco carcinogen - derived adducts have been identified in vitro and in vivo , owing to the development of highly sensitive analytical detection methods , such as p - postlabeling and mass spectrometry ( ms ) [ 25 , 26 ] . with some exceptions of inconsistency , many epidemiologic and clinical studies have shown an association between the in vivo levels of dna adducts resulting from cigarette smoke and the occurrence of tobacco - related cancers in lung , head and neck , and bladder . there are numerous reviews specifically related to the relationships between tobacco carcinogen exposure , dna adduct formation , carcinogen / adduct mutagenic potential , and increased cancer risk related to smoking [ 13 , 19 , 26 , 27 , 2932 ] . these bulky adducts represent a major and important class of dna damage originating from exposure to cigarette smoke . for example , pah- and acrolein - dna adducts are preferentially formed in the same mutational hotspots of p53 in the lung cancers of smokers [ 30 , 42 , 43 ] . there is also evidence that a high level of bulky dna adducts in tissues , such as those caused by pahs and vinyl chloride ( vc ) , is associated with an increased risk of tumor in humans and animals [ 40 , 4446 ] . more importantly , persistence of dna adducts such as those formed by tobacco carcinogens pahs and n - nitrosamines plays a central role in tobacco - induced carcinogenesis . in addition to point mutations , there are correlations between dna adduct levels and other somatic alterations , for example , loss of heterozygosity ( loh ) that may occur at the very early stages of tobacco carcinogenesis [ 58 , 59 ] . organisms from prokaryotes to mammals have evolved a number of repair pathways , including direct reversal , base excision repair ( ber ) , nucleotide excision repair ( ner ) , mismatch repair ( mms ) , and double - strand break ( dsb ) repair [ 6264 ] . in model systems , such as cultured cells and genetically manipulated organisms , ner is the major pathway for the repair of various duplex - distorting bulky dna lesions such as those induced by pahs . in general , the understanding of damage recognition and mechanism of repair is important to gain insight into the specific roles of tobacco dna adducts in the development of cancer and other chronic diseases since , at the end , the overall cellular repair capacity in response to exposure is critically related to the levels of dna adducts in the genome or mutations in genes . the role of individual variability in repair , for example , polymorphisms in repair genes , has been related to the increased cancer risk in smokers [ 22 , 65 , 66 ] . , those bulky adducts and oxidized bases with cytotoxicity and mutagenicity ) that is expected to be most important in the etiology of smoking - related cancer and other disorders . this paper will focus on the formation and repair of bulky / exocyclic dna adducts induced by the major tobacco carcinogens in relation to tobacco mutagenesis and carcinogenesis . since smokers are repetitively exposed to complex mixtures of genotoxic carcinogens , the collective formation of dna adducts is very complex as reflected by their chemical types and cellular levels . although this paper is focused on bulky dna adducts , other types of dna lesions by tobacco carcinogens may be equally or more important than bulky dna adducts for a given carcinogen or cancer type . understanding of dna adducts with regard to their formation , isolation , and identification can be critical in several ways : ( 1 ) to understand the mechanism of tobacco carcinogens ; for example , the analysis of formation of dna adduct at gene mutational hotspots [ 30 , 78 ] has provided important insight into the cancer etiology ; ( 2 ) to assess the biologically effective doses of tobacco carcinogens ; ( 3 ) to assess dna repair capacity ( drc ) towards the adducts [ 13 , 79 ] ; ( 4 ) to find biomarkers of tobacco genotoxicity and uptake / metabolism of specific carcinogens [ 12 , 13 ] . many types of dna adducts , including those formed by benzo[a]pyrene ( b[a]p ) , 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone ( nnk ) , n -nitrosonornicotine ( nnn ) , and 4-aminobiphenyl ( 4-abp ) , have been detected from tissues of smokers as well as nonsmokers exposed to shs [ 1113 , 8082 ] . although p - postlabeling and immunoassay have been extensively utilized for adduct analysis , the detection and identification of dna adducts at these or even lower levels have been greatly facilitated by the highly sensitive / specific and new types of techniques [ 11 , 12 , 25 , 26 ] such as hplc with fluorescence , mass spectrometry ( ms ) , and electrochemical detection , particularly the coupling of liquid chromatography ( lc ) to ms and electrospray ionization ( esi ) , that is , lc - esi - ms . in the last two decades or so , considerable progress has been made in understanding the specificity , mechanism of action , and in vivo importance of many repair enzymes and pathways . several major mechanisms have been shown to be involved in the repair of bulky dna adducts that can be induced by tobacco carcinogens , as discussed below in detail . excision repair , whether it is base ( ber ) or nucleotide excision ( ner ) , has to be at least a two - step process in which the damage recognition and excision is followed by dna replication , whereas direct reversal , catalyzed by o - alkylguanine dna alkyltransferase ( agt , also known as mgmt ) or alkb homologs ( abhs ) , restores the normal base without excision . for example , 3 , n - ethenocytosine ( c ) is excised by three different dna glycosylases and repaired by two different repair pathways , ber and abh2 . ner is the most versatile repair pathway in the cell and the primary mechanism for the removal of chemical carcinogen - induced bulky dna adducts that significantly distort the dna helix structure [ 64 , 107 , 110 , 111 ] . although a recent study did not support that bpq forms stable dna adducts in mice , there is evidence that this pathway operates in human lungs leading to ros - mediated genotoxicity such as causing g to t transversions that inactivate p53 [ 127 , 129 ] . taking advantage of the stereochemistry involved in the formation of these bulky adducts , a number of studies addressed the effects of adduct conformation , base paring , and sequence context on dna repair . as will be discussed below is section 2.5 , the processing of bpde - dna adducts by both uvrabc and human bpde forms n - da adducts in native dna as well , although relatively inefficiently [ 135 , 136 ] , which exhibit differential conformation and perturbation of dna duplex than the bpde - dg adducts . using cell extracts , human ner activity has been shown for the ( + ) - or ( )-trans - anti - bpde - da adducts [ 38 , 131].several early studies showed that repair of bpde - dna adducts occurs much faster in the transcribed strand than in the nontranscribed strand of hprt or p53 genes , indicating that these adducts are subject to tcr [ 137 , 138 ] . , this strand preference in repair may contribute to the mutational property of the human lung cancer p53 gene in response to bpde exposure : repair of bpde adducts along the nontranscribed strand of p53 is consistently slower than repair in the transcribed strand , and repair at the major damage hotspots in the nontranscribed strand is 24 times slower than repair at other damage sites .dibenzo[a , l]pyrene ( db[a , l]p ) is another pah that has been found to be present in tobacco smoke particulates and is the most potent carcinogen of the pahs tested to date in rodent systems . although a recent study did not support that bpq forms stable dna adducts in mice , there is evidence that this pathway operates in human lungs leading to ros - mediated genotoxicity such as causing g to t transversions that inactivate p53 [ 127 , 129 ] . taking advantage of the stereochemistry involved in the formation of these bulky adducts , a number of studies addressed the effects of adduct conformation , base paring , and sequence context on dna repair . as will be discussed below is section 2.5 , the processing of bpde - dna adducts by both uvrabc and human bpde forms n - da adducts in native dna as well , although relatively inefficiently [ 135 , 136 ] , which exhibit differential conformation and perturbation of dna duplex than the bpde - dg adducts . , this strand preference in repair may contribute to the mutational property of the human lung cancer p53 gene in response to bpde exposure : repair of bpde adducts along the nontranscribed strand of p53 is consistently slower than repair in the transcribed strand , and repair at the major damage hotspots in the nontranscribed strand is 24 times slower than repair at other damage sites . ( 2 ) formation and repair of dna adducts of aromatic amine 4-abpchemicals in this class such as 4-abp bind to dna bases mainly at c-8 position . the major adduct has been detected in the human cells after exposure to n - oh-4-abp . the unique binding pattern of 4-abp in the p53 gene , that is , the p53 mutational hotspots in bladder cancer at several codons are also the preferential sites for 4-abp adduct formation , links 4-abp to the etiology of bladder cancer .although the detailed molecular mechanism of the repair of 4-abp - dna adducts is not clear , dna fragments modified with n - oh-4-abp were shown to be incised by e. coli ner complex , the uvrabc nuclease . the relationship between deficient dna repair of 4-abp - dna adducts and increased bladder cancer risk was supported by the findings that such repair capacity was significantly lower in bladder cancer cases than in controls , and ever - smokers with low dna repair capacity exhibited a 6-fold increased risk compared with never smokers with normal repair capacity . the major adduct has been detected in the human cells after exposure to n - oh-4-abp . the relationship between deficient dna repair of 4-abp - dna adducts and increased bladder cancer risk was supported by the findings that such repair capacity was significantly lower in bladder cancer cases than in controls , and ever - smokers with low dna repair capacity exhibited a 6-fold increased risk compared with never smokers with normal repair capacity . a number of bulky dna adducts have been detected in vitro and in vivo when exposed to hq or p - bq [ 9598 , 173177 ] . a number of bulky dna adducts have been detected in vitro and in vivo when exposed to hq or p - bq [ 9598 , 173177 ] . surveying the activities of known glycosylases indicates that many of them are able to excise tobacco carcinogen - induced dna adducts , including the common alkylated and oxidized bases and some exocyclic adducts . ( 1 ) formation and repair of pyridyloxobutyl ( pob)-dna adducts of tsnastobacco - specific nitrosamines ( tsnas ) are exclusively found in cigarette smoke and are formed through n - nitrosation of nicotine during tobacco curing and processing . common tsnas found in cigarette smoke particles include nnk , nnn , and n - nitrosoanatabine ( nab ) . in conclusion , agt is expected to be critical in the repair of o - alkylguanine adducts formed by tobacco - derived n - nitrosamines . common tsnas found in cigarette smoke particles include nnk , nnn , and n - nitrosoanatabine ( nab ) . in conclusion , agt is expected to be critical in the repair of o - alkylguanine adducts formed by tobacco - derived n - nitrosamines . these results suggest that both aag and abh2/3 proteins can play a role in the cellular response to the exposure of tobacco carcinogens that generate these -adducts . these results suggest that both aag and abh2/3 proteins can play a role in the cellular response to the exposure of tobacco carcinogens that generate these -adducts . the importance of bulky dna adducts relative to other types of dna lesions needs further investigation , and in any case , a combined action from different types of dna / chromosomal damage , as demonstrated in figure 4 for benzene ' biological effects , is expected to be the basis of genotoxicity conferred by many tobacco carcinogens . in general , the key to the specificity of recognition has been known to be not only the primary adduct structure , but the localized effect of each adduct on dna structure as well as on thermodynamics , and , moreover , the structure and function of the repair protein . since most repair enzymes act on adducts in double - stranded dna , each adduct may cause differing distortion and flexibility as a result of factors such as being in the major or minor groove , syn or anti , planar or angular , adjacent base pair tilting , propeller twist , and helical twist . examples among the tobacco carcinogen - induced bulky dna adducts are bpde - dna adducts [ 134 , 138 , 272274 ] , pob - dna adducts , a , and c , whose repair efficiencies could vary over manyfold when present in different neighbor sequences . it has been reported that in pah- or acrolein - treated cells the same positions of their mutation hotspots are also major hotspots for mutations observed in human lung cancers from smokers , strongly suggesting a role of dna adducts in etiology of these cancers . finally , in addition to site - specific preferential formation of dna adducts and sequence context of dna repair , the biological selection of induced mutations is also considered important for the hotspot phenomenon , which gives cells with specific mutation(s ) a growth advantage and results in dominant mutations in cancer cells [ 283 , 284 ] . it has long been recognized that only a small percent of cigarette smokers develop cancer , for example , ~11%24% of smokers develop lung cancer , which suggested that interindividual variability in key cellular processes is crucial in response to tobacco carcinogens . one top aim of the target cancer prevention programs is to identify smokers and nonsmokers exposed to shs with higher susceptibility . decreased repair activities for bulky dna adducts have been observed in cells / tissues of cancer patients . in principle , the phenotypes of dna repair must be characterized for mutagenic adducts and any newly identified adducts in smokers and nonsmokers exposed to shs . a common one is polymorphisms in relevant dna repair genes , such as those identified in the ner , ber , and agt pathways [ 289 , 292 , 293 ] . in the last decade , although mixed or discrepant results have been reported , positive results on numerous polymorphisms in dna repair genes , along with those in metabolic genes , have been revealed and are continuously being found in the context of cigarette smoking and cancer [ 22 , 295 , 296 ] . examples of such factors include those that are disease related , for example , ner deficiency in xp and cockayne syndrome patients [ 64 , 140 ] , and those that are repair - inhibition based . there are at least 30 metals in cigarette smoke , including arsenic , cadmium , nickel , and chromium , which have been shown to inhibit various dna repair enzymes and pathways [ 298302 ] . in addition to metals , certain tobacco carcinogens themselves have been found to inhibit dna repair , as exemplified by acrolein inhibiting ner repair of bpde - dna adducts . for example , the biological response of n - nitrosamines could be correlated with minor forms of dna damage . ( 6 ) endogenous dna adducts can be formed as a result of cigarette smoke , which is currently attributed to the formation of lpo products [ 9294 ] . the complexity of such analysis is that the same dna adducts , for example , the -dna adducts , are generated by both tobacco carcinogens and endogenously formed compounds , even though they can be chemically different . however , it has been well established that indoor surfaces significantly adsorb semi- and nonvolatile shs compounds , for example , nicotine , 3-ethenylpyridine , naphthalene , cresols , and phenol which are slowly reemitted into the air [ 317320 ] . based on their chemical structure , all of the by - products shown in figure 8 are anticipated or have already been known to form dna adducts . understanding the mechanisms of formation and repair of bulky dna adducts is critical for analysis and management of tobacco - induced mutagenesis and carcinogenesis . future efforts should be made to identify and characterize novel compounds and adducts , such as those produced in aged shs and ths , to identify those highly mutagenic adducts that are refractory to dna repair , to find adducts as reliable biomarkers for measuring exposure , especially for shs and ths , to explore new potential biological functions of adducts , such as their interactions with cellular signaling networks , impact on stem cells of target tissues , and roles in epigenetic changes , and to find effective ways to inhibit dna adduct formation in target organs . ultimately , a better knowledge of the mechanisms by which the chemical carcinogen exposure increases cancer risk in smokers and individuals exposed to shs / ths could lead to new strategies for cancer prevention and could serve as the experimental evidence for framing and enforcing tobacco control policies in order to minimize health hazards and protect vulnerable populations .

thrombin is the central protease in the cascade of blood coagulation proteases . structurally , thrombin consists of a double -barrel core surrounded by connecting loops and helices . genetic analysis of the clotting factor genes demonstrates that the clotting proteases of the chymotrypsinogen superfamily have evolved as a result of several gene duplications , exon shuffling , and intron sliding events . prothrombin has a unique exon organization and is thought to be the ancestral gene of the clotting factor family . the extended active site loops in thrombin are thought to have arisen from insertions in the serine protease that evolved to impart greater specificity . thrombin is produced in a low - activity zymogen form that requires proteolytic cleavage to attain full activity . this cleavage event results in small overall changes to the molecular architecture but results in a large change in dynamics wherein one -barrel becomes more dynamic and the other becomes less dynamic . the result is a more perfectly formed active site for rapid proteolytic cleavage activity . despite the highly specific nature of thrombin activity , in association with allosteric modulators , in addition , allostery is key to thrombin regulation , and misregulation can lead to bleeding disorders or thrombosis . although , traditionally , allostery was defined as occurring among subunits in a multisubunit system such as hemoglobin , the phenomenon of altered activity resulting from binding of a regulatory molecule on the opposite side of a monomeric enzyme is now also recognized as a form of allostery . several experimental and computational approaches have hinted that the solution structure of thrombin is a broad and malleable dynamic ensemble . h / d exchange mass spectrometry showed that d - phe - pro - arg chloromethylketone ( ppack ) occupation of the active site not only protected the active site loops but also propagated to decreased exchange in several regions of the protein distant from the active site . thrombin has two binding sites distal to the active site ; exosite 1 is where thrombomodulin binds , and exosite 2 is where heparin binds . isothermal titration calorimetry ( itc ) experiments showed alteration of thermodynamic parameters of ligands binding to thrombin exosites when the active site was occupied . binding of active site ligands altered the balance of enthalpic and entropic contributions to binding of exosite 1 ligands and vice versa . such thermodynamic compensation phenomena are more likely if the allosteric mechanism is entropic rather than enthalpic , suggesting that differences in the dynamic properties of the system affect the ligand binding mechanism . indeed , x - ray crystallography shows no significant changes in the thrombin structure upon ligand binding , providing further evidence that it may exist as a malleable dynamic ensemble . nmr studies and md simulations remain the most direct approaches to investigating protein dynamics . a recent nmr / md study on ppack - thrombin revealed a large degree of dynamic motions , particularly in the active site loops , spanning time scales from picoseconds to milliseconds . a computational exploration revealed a strong dynamic component in the allosteric regulation of thrombin by tm . in the work presented here , we combine conventional md , nmr - calibrated accelerated md ( amd ) , and analysis of the residual local frustration to further explore the dynamics of thrombin , with particular interest in changes that occur upon active site ligation . the combined approach allows us to analyze a broad range of motional time scales . atomic coordinates for thrombin were obtained from the protein data bank 1.9 x - ray crystal structure [ pdb i d : 1ppb ] . both systems were placed at the center of a periodically repeating box , and the simulation cell size was defined such that the distance between the edge of the simulation box and the surface of the solute was at least 12 . all simulations were performed in explicit solvent , and three cl counterions were introduced to obtain cell neutrality . six 20 ns conventional md simulations were performed using a different random seed generator for the maxwellian distribution of atomic velocities following standard energy minimization and equilibration procedures . electrostatic interactions were treated using the particle mesh ewald ( pme ) method with a direct space sum limit of 10 . the ff99sb force field was used for the solute residues , and the tip3p water force field was employed for the solvent molecules . in the case of ppack - thrombin simulations , the conventional md simulations were analyzed in the community analysis and also constituted the starting point for the amd simulations . these simulations also provided the average ( unbiased ) dihedral angle energy , v0(dih) and total energy v0(tot) values used to define the acceleration parameters in the amd simulations described below . allosteric networks were characterized using a community network analysis approach previously applied to investigate allostery in trna / protein complexes and other protein systems . this approach constructs a dynamic contact map consisting of a network graph in which each residue is treated as a two residues are deemed to be in contact throughout the majority of the simulation . clusters of residues ) of highly intraconnected but loosely interconnected nodes using the girvan central to this method is the calculation of edge betweenness , the number of shortest paths that cross an edge . the edge betweenness is calculated for all edges , and the edge with the greatest betweenness is removed . this process is repeated , and a modularity score is tracked to identify the division that results in the optimal community structure . amd simulations were performed as described previously using an in - house modified version of the amber 10 code . amd approach is used , in which two acceleration potentials are applied simultaneously to the system ; the first acceleration potential is applied to the torsional terms only , and a second , weaker acceleration is applied across the entire potential . this dual boost amd protocol represents a unified approach facilitating the efficient sampling of both the torsional degrees of freedom and slow diffusive motions in the solute . in total , six independent dual boost amd simulations were performed for 10 000 000 steps ( the equivalent of 20 ns of md ) for each system . the physical conditions , force fields , and all other simulation parameters employed were identical to those described for the conventional md simulations . the specific acceleration parameters used in this study were [ eb(dih ) v0(dih) ] = [ 4 kcal / mol no . of solute residues ] and (dih ) = [ 0.8 kcal / mol no . v0(tot) ] = (tot ) = [ 0.16 kcal / mol no . of atoms in the simulation cell ] for the background total acceleration . these acceleration parameters had been previously identified as the optimal acceleration parameters for the reproduction of experimental rdcs in ppack - thrombin , accessing configurational dynamics on time scales up to 10s100s of microseconds . for each amd simulation , a corrected canonical ensemble was obtained by reweighting each point in the configuration space on the modified potential by the strength of the boltzmann factor of the bias energy , exp[v(rt(i ) ) ] at that particular point , and the bias potential block averaging method was employed to remove statistical noise errors . the internal dynamics present in the different amd simulations of apo - thrombin and ppack - thrombin were assessed by calculating order parameters , s , from the free - energy weighted amd ensembles . members of each ensemble were superimposed onto the backbone atoms ( n , c , c ) of all heavy chain residues for the appropriate average structure , and order parameters , s were calculated aswhere i are the cartesian coordinates of the normalized internuclear vector of interest . others have shown that s values calculated from standard md simulations in this way were in excellent agreement with experimental s values calculated using the lipari the order parameters presented here are averaged over all six amd trajectories for each system . residue - by - residue cross - correlations for the free - energy - weighted amd ensembles were calculated using the generalized cross - correlation approach applied to all backbone c atomic coordinates based on the mutual information method developed by the grubmller group using the g_correlation module in gromacs 3.3.3 . an algorithm for determining residual local frustration , that is , whether a contact between amino acid residues is energetically optimized or not in the folded state , was developed by the wolynes and komives groups some time ago . residue interactions by systematically perturbing the identity of individual residues and evaluating the resulting total energy change . for the work presented here index , in which the decoy set involves randomizing not just the identities but also the distance and densities of the interacting amino acids i , j. this scheme effectively evaluates the native pair with respect to a set of structural decoys that might be encountered in the folding process . after constructing a histogram of the energy of the decoys and comparing the distribution to the native energy , cutoffs energetically favorable contacts between residues are minimally frustrated , whereas highly frustrated contacts are energetically unfavorable in the native state . depictions of the contacts on structural models typically show minimally frustrated contacts in green and highly frustrated contacts in red . the average of the frustration scores over all of the contacts made by a particular residue are also plotted in a per - residue format . a webserver is now available for performing these computations . for the work presented here , the minimally and highly frustrated contacts are depicted on the lowest - energy structure from the boltzmann - reweighted ensemble of structures from the amd simulations . to compute the average per - residue frustration , we clustered the boltzmann reweighted ensemble and averaged the frustration scores of all contacts made by each residue in the representative structure from the three most populated clusters . it is interesting to note that the residual local frustration varied between members of the ensemble , and the error bars on the residual local frustration plots represent one standard deviation . note : thrombin has several numbering schemes ; the chymotrypsin numbering scheme is used in the text because it is used in pdb files . this numbering scheme creates problems for the data presentation in this paper ; therefore , sequential numbering ( of the light chain or a - chain followed by the heavy chain ) is used in the plots , and sequential residue numbers are given in parentheses throughout the paper for reference . we performed a set of six independent 20 ns conventional md simulations for both apo - thrombin and ppack - thrombin . during the equilibration procedure , a rather large conformational transition in the active site loops was observed for apo - thrombin that involved a reorientation of both the -loop ( 178195 ) and the na binding loop ( 264271 ) , forming a more open active site pocket . a community network analysis approach was applied to identify groups of residues undergoing correlated motions in ppack - thrombin and apo - thrombin . representative community network analyses obtained from conventional md simulations are shown in figure 1 . the flow of information in the physical network of the protein was measured by the edge betweenness , defined as the number of shortest paths that pass through the edge in the network , and is a direct measure of the strength of intercommunity communication within the network ( black lines in figure 1a and b ) . ppack ligation causes consolidation of the community structure including the two active site communities most proximal to the ppack binding site ( green and brown , figure 1 ) . the community that includes part of the na binding loop in apo - thrombin ( figure 1c , brown ) consolidates with the active site serine195ct ( 241 ) , the 70s loop ( 98113 ) , residues 191194ct of the -loop ( 237240 ) , and the n - terminal residues 1719ct ( 3840 ) in the ppack - liganded form ( brown , figure 1d ) . ppack ligation also acts to consolidate the n - terminal -barrel , which is formed by two separate communities in apo - thrombin and forms a large community that also contains the 30s loop ( 5562 ) and part of the 60s loop ( 8294 ) ( orange , figure 1d ) . in summary , the community analysis revealed consolidation of the na binding site , the base of the -loop , and the n - terminus of the heavy chain into one community and most of the active site loops into a second community upon ppack binding . these two communities , which are strongly connected in ppack - thrombin , unite the residues required for proteolytic catalysis . the a - chain community also becomes more strongly connected to the active site and the community containing the 70s loop . on the basis of the substantial consolidation observed in the community analysis upon ppack binding , we set out to examine whether there were concomitant changes in dynamics . community analysis of apo - thrombin ( a , c ) and ppack - thrombin ( b , d ) . the two - dimensional view of the communities in panels ( a ) and ( b ) depicts the relative size of the communities ( based on the number of residues ) as colored circles of varying sizes with the thickness of the connecting lines representing the relative interconnectivity among communities . panels ( c ) and ( d ) are structural representations of communities superimposed on pdb 1ppb . residual dipolar couplings ( rdcs ) , which report on an ensemble average over all orientations of the magnetic dipole interaction vector up to the chemical shift coalescence limit , provide useful experimental data for determining the ensemble of structures that best represents the dynamic properties of a protein . when the experimentally derived rdcs are compared to rdcs that are back - calculated from ensembles of structures generated from amd simulations , the acceleration level that provides the most realistic representative structural ensemble can be identified . we previously demonstrated that the rdcs measured on ppack - thrombin did not agree well with the available crystal structures ( r = 0.72 ) . 80 ) when the rdcs were back - calculated from an ensemble of structures obtained by conventional md . however , remarkable agreement was obtained between the experimental rdcs and the rdcs back - calculated from the ensemble of structures obtained from an amd simulation at the optimal acceleration level ( r = 0.92 ) . the ensembles obtained from such amd simulations of both ppack - thrombin and apo - thrombin are shown in figure 2 , with the loops colored according to the scheme shown below the structure . ensemble of the 10 representative structures of the rmsd clusters from the boltzmann reweighted structures from the rdc - calibrated amd for apo - thrombin ( left ) and ppack - thrombin ( right ) . the loops are colored according to the schematic under the structures . for ppack - thrombin , order parameters ( s ) from conventional md simulations agreed extremely well with those measured by nmr relaxation experiments that are limited to the picosecond however , the fact that ensembles obtained from amd were required for good agreement with the rdc measurements suggested that motions on longer time scales are contributing to the solution structure . therefore , order parameters for the n h bond vectors were calculated from the amd ensembles ( samd2 ) . a comparison of samd2 for apo - thrombin to those obtained previously for ppack - thrombin is shown in figure 3a . most of the active site loops in both forms are highly flexible , yet a marked decrease in flexibility is observed upon active site ligation with ppack ( figure 3b ) . as expected , ppack ligation caused significant ordering ( s > 0.1 ) of residues that directly contact the ppack arg side chain . the loops that surround the active site also experience significant ordering , including the 60s loop ( 8294 ) , the -loop , and the 180s loop ( 225239 ) . some regions distal to the ppack also showed significant ordering upon ppack ligation , including the a - chain , the 30s loop , residue 221ct ( 269 ) of the sodium binding loop , and residues in the c - terminal helix ( figure 3c ) . ( a ) order parameters calculated from amd simulations of apo - thrombin ( red ) and ppack - thrombin ( black ) . ( b ) differences in order parameters between the forms : s = sppack2 sapo2 from amd order parameters . ( c ) those residues with a s > + 0.1 are marked with red spheres on the structure of thrombin ( pdb code 1ppb ) , indicating stabilization upon active site occupation by ppack . the schematic of important surface loops is provided above the graph . to identify residues undergoing correlated motions on longer time scales , amd simulations were performed that were optimized based on previous work comparing experimental rdcs to those back calculated from amd simulations carried out at different acceleration levels . the analysis of apo - thrombin revealed correlated motions between the active site loops , exosite 1 , and other distal sites . in particular , the entire -loop appears to undergo strongly correlated motions with the a - chain residues 1h1dct and 1214cct ( 15 and 2025 ) , the catalytic triad , h57ct , d102ct , s195ct ( 79 , 135 , 241 ) , the 60s loop , the 70s loop , the 90s loop ( 127133 ) , the surface strand under the 70s loop ( 145151 ) , the 170s loop ( 204219 ) , and the 180s loop ( figure 4 , lower triangle ) . whereas these correlated motions appeared to involve the entire -loop in apo - thrombin , only the tip of the -loop ( residues 146149ect ( 182190 ) ) appears to be undergoing the same set of motions in ppack - thrombin ( figure 4 , upper triangle ) . in apo - thrombin , the 170s and , to a lesser extent , the 180s loop , which are strongly correlated to the -loop , are also correlated with the a - chain and catalytic residues . upon ppack , the 30s loop and the 60s loop are weakly correlated , but the 30s loop is not correlated to the 70s and 90s loops . correlated motions between the 30s and 60s loops are stronger in ppack - thrombin , and these extend to the 70s and 90s loops ( figure 4 ) . analysis of correlated motions performed on the amd trajectories of ppack - thrombin ( top triangle ) and apo - thrombin ( bottom triangle ) . the motions range from 0.0 ( no correlation , white ) to 1.0 ( completely correlated , black ) . the schematic diagram indicating the location of surface loops is inserted above the correlated motions plot . the black boxes indicate correlations of the 170s and 180s loops with the a - chain and surface loops that are stronger in apo - thrombin than in ppack - thrombin . the blue boxes indicate correlations of the 30s loop with the 60s , 70s , and 90s loops that are stronger in ppack thrombin than in apo - thrombin . we applied a previously derived algorithm to identify the residual local frustration in representative structures from rmsd clusters of the boltzmann reweighted amd simulation results . according to the principle of minimal frustration , contacts made in the folded native state should be minimally frustrated , meaning that they are energetically favorable . we previously showed that while most contacts made in the native state are , indeed , minimally frustrated , some 1015% of contacts are energetically unfavorable ( i.e. , highly frustrated ) in the native state . these highly frustrated contacts map to functional sites and are thought to have been preserved in evolution . both apo- and ppack - thrombin show regions of high frustration in many of the surface loops ( figure 5 ) . to discover whether regions of high frustration also map to dynamic regions , we compared the average residual frustration across representative structures from the three most populated rmsd clusters derived from the amd simulations to the order parameters . the order parameters derived from conventional md simulations ( sns2 ) agree very well with order parameters derived from nmr relaxation experiments on thrombin and reveal the disorder resulting from motions in the nanosecond time regime . the order parameters derived from the rdc - calibrated amd simulations ( samd2 ) reveal the disorder resulting from motions on longer time scales . the sns2 did not correspond well to the regions of high residual frustration ; however , the correspondence with the samd2 is remarkable ( figure 6 ) . these results highlight that regions of high residual frustration may have evolved to allow slow time scale motions to occur with relative energetic ease , as previously suggested by wolynes and colleagues . analysis of residual local frustration in the lowest - energy structure from the rdc - calibrated amd ensemble of apo - thrombin ( left ) and ppack - thrombin ( right ) . the contacts that are minimally frustrated are shown in green , and the contacts that are highly frustrated are shown in red . thin lines represent water - mediated contacts . the active site catalytic residues are shown in magenta . comparison of the order parameters reflecting nanosecond time scale motions versus longer time scale motions ( samd2 ) , with the average per residue fraction of highly frustrated contacts for the three lowest - energy structures from the amd simulation . ( a ) the sns ( gray ) and samd ( red ) for apo - thrombin are compared to the average fraction of highly frustrated contacts ( cyan ) . ( b ) the sns2 ( gray ) and samd2 ( black ) for ppack - thrombin are compared to the average fraction of highly frustrated contacts ( blue ) . we used a combination of community network analysis , rdc - calibrated amd simulations , and analysis of residual frustration to explore the dynamic ensemble of thrombin in solution from the nanosecond to the microsecond time regime . comparative analysis of apo - thrombin and the active - site - ligated thrombin systems identified differences in the dynamic fluctuations and changes in correlated motions upon active site ligation . analysis of thrombin complexed with the relatively small substrate analogue ppack showed a substantial rearrangement of the community structure . ppack binding consolidated the two active site communities most proximal to the ppack binding site as well as the na binding loop with the active site serine , the 70s loop , part of the -loop , and part of the a - chain . interestingly , this consolidation resulted in more residual local frustration near the active site ( figure 5 ) . the generalized cross - correlation analysis predicted that many of the thrombin surface loops are undergoing correlated motions ( figure 4 ) . the analysis of residual local frustration revealed that all of these loops are highly frustrated in the native structure of thrombin ( figure 5 ) . the striking correlation between the long timescale dynamics and the residual local frustration suggests that evolution has selected for energetically unfavorable contacts within the surface loops in order to facilitate larger amplitude slower motions . upon ppack binding , many of the loops retain dynamics and also remain highly frustrated . the surface loops exhibiting correlated motions are not necessarily structurally proximal , hinting at how allosteric regulation by binding of ligands at sites distal to the active site might occur . upon ppack binding , the active site community consolidates with the 70s loop where fibrinogen binds to stimulate its own cleavage and where thrombomodulin binds to alter substrate specificity . indeed , previous experiments have demonstrated a thermodynamic coupling between the 70s loop and the active site . another loop that is distal to the active site but that differs between apo- and ppack - thrombin is the -loop . residues leading up to the -loop are highly frustrated and highly dynamic in apo - thrombin but become much less dynamic in the ppack - bound form , with only the tip of the -loop remaining dynamic . although the strongly correlated motions in apo - thrombin between the -loop and the 170s loop almost completely disappear upon ppack ligation , correlations are strengthened between the tip of the loop and nearly every other loop in thrombin , including the 30s loop , the 60s loop , the 70s loop , the -strand connecting the 60s and 70s loops , and the 90s loop . thus , we can speculate that the reduced dynamics result from the binding of ppack , causing these regions to sample a more restricted conformational space , and that this reduced sampling leads to stronger correlated motions extending between the active site and the surface loops involved in allosteric regulation . the order parameter analysis revealed , as expected , that active site occupation decreases the microsecond motions in the surface loops ( figure 3 ) , consistent with amide h / d exchange experiments that showed that binding of ppack dampened exchange in all of the surface loops including those distal to the active site . millisecond time scale could explain how active site occupation lowers the entropic penalty for binding an exosite 1 allosteric modulator . thermodynamic measurements of binding show that the allosteric pathway between these two sites operates in both directions and changes in order parameters in the thrombin surface loops upon ppack binding to the active site are similar to those seen upon tm56 binding to exosite 1 . the serine protease architecture is that of a double -barrel fold in which the -strands are connected by surface loops . the active site is between the two -barrels , each of which has several -strands connected by the surface loops . such a -strand architecture provides a direct connection between distal surface loops , allowing transmission of allosteric information by subtle redistribution of the dynamic ensemble . such local unfolding or cracking was predicted some time ago to be the fundamental mechanism of allostery , and residual local frustration is indeed found at such sites . it will be interesting to see if dynamic allostery is present in other proteins with mostly -strand structures similar to thrombin .

thrombin is the central protease in the cascade of blood coagulation proteases . the structure of thrombin consists of a double -barrel core surrounded by connecting loops and helices . compared to chymotrypsin , thrombin has more extended loops that are thought to have arisen from insertions in the serine protease that evolved to impart greater specificity . previous experiments showed thermodynamic coupling between ligand binding at the active site and distal exosites . we present a combined approach of molecular dynamics ( md ) , accelerated molecular dynamics ( amd ) , and analysis of the residual local frustration of apo - thrombin and active - site - bound ( ppack - thrombin ) . community analysis of the md ensembles identified changes upon active site occupation in groups of residues linked through correlated motions and physical contacts . amd simulations , calibrated on measured residual dipolar couplings , reveal that upon active site ligation , correlated loop motions are quenched , but new ones connecting the active site with distal sites where allosteric regulators bind emerge . residual local frustration analysis reveals a striking correlation between frustrated contacts and regions undergoing slow time scale dynamics . the results elucidate a motional network that probably evolved through retention of frustrated contacts to provide facile conversion between ensembles of states .

Introduction Methods Results Discussion

thrombin is the central protease in the cascade of blood coagulation proteases . structurally , thrombin consists of a double -barrel core surrounded by connecting loops and helices . genetic analysis of the clotting factor genes demonstrates that the clotting proteases of the chymotrypsinogen superfamily have evolved as a result of several gene duplications , exon shuffling , and intron sliding events . prothrombin has a unique exon organization and is thought to be the ancestral gene of the clotting factor family . the extended active site loops in thrombin are thought to have arisen from insertions in the serine protease that evolved to impart greater specificity . several experimental and computational approaches have hinted that the solution structure of thrombin is a broad and malleable dynamic ensemble . h / d exchange mass spectrometry showed that d - phe - pro - arg chloromethylketone ( ppack ) occupation of the active site not only protected the active site loops but also propagated to decreased exchange in several regions of the protein distant from the active site . thrombin has two binding sites distal to the active site ; exosite 1 is where thrombomodulin binds , and exosite 2 is where heparin binds . isothermal titration calorimetry ( itc ) experiments showed alteration of thermodynamic parameters of ligands binding to thrombin exosites when the active site was occupied . such thermodynamic compensation phenomena are more likely if the allosteric mechanism is entropic rather than enthalpic , suggesting that differences in the dynamic properties of the system affect the ligand binding mechanism . a recent nmr / md study on ppack - thrombin revealed a large degree of dynamic motions , particularly in the active site loops , spanning time scales from picoseconds to milliseconds . a computational exploration revealed a strong dynamic component in the allosteric regulation of thrombin by tm . in the work presented here , we combine conventional md , nmr - calibrated accelerated md ( amd ) , and analysis of the residual local frustration to further explore the dynamics of thrombin , with particular interest in changes that occur upon active site ligation . both systems were placed at the center of a periodically repeating box , and the simulation cell size was defined such that the distance between the edge of the simulation box and the surface of the solute was at least 12 . in the case of ppack - thrombin simulations , the conventional md simulations were analyzed in the community analysis and also constituted the starting point for the amd simulations . these simulations also provided the average ( unbiased ) dihedral angle energy , v0(dih) and total energy v0(tot) values used to define the acceleration parameters in the amd simulations described below . this approach constructs a dynamic contact map consisting of a network graph in which each residue is treated as a two residues are deemed to be in contact throughout the majority of the simulation . clusters of residues ) of highly intraconnected but loosely interconnected nodes using the girvan central to this method is the calculation of edge betweenness , the number of shortest paths that cross an edge . in total , six independent dual boost amd simulations were performed for 10 000 000 steps ( the equivalent of 20 ns of md ) for each system . for each amd simulation , a corrected canonical ensemble was obtained by reweighting each point in the configuration space on the modified potential by the strength of the boltzmann factor of the bias energy , exp[v(rt(i ) ) ] at that particular point , and the bias potential block averaging method was employed to remove statistical noise errors . the internal dynamics present in the different amd simulations of apo - thrombin and ppack - thrombin were assessed by calculating order parameters , s , from the free - energy weighted amd ensembles . members of each ensemble were superimposed onto the backbone atoms ( n , c , c ) of all heavy chain residues for the appropriate average structure , and order parameters , s were calculated aswhere i are the cartesian coordinates of the normalized internuclear vector of interest . an algorithm for determining residual local frustration , that is , whether a contact between amino acid residues is energetically optimized or not in the folded state , was developed by the wolynes and komives groups some time ago . for the work presented here index , in which the decoy set involves randomizing not just the identities but also the distance and densities of the interacting amino acids i , j. this scheme effectively evaluates the native pair with respect to a set of structural decoys that might be encountered in the folding process . after constructing a histogram of the energy of the decoys and comparing the distribution to the native energy , cutoffs energetically favorable contacts between residues are minimally frustrated , whereas highly frustrated contacts are energetically unfavorable in the native state . for the work presented here , the minimally and highly frustrated contacts are depicted on the lowest - energy structure from the boltzmann - reweighted ensemble of structures from the amd simulations . it is interesting to note that the residual local frustration varied between members of the ensemble , and the error bars on the residual local frustration plots represent one standard deviation . this numbering scheme creates problems for the data presentation in this paper ; therefore , sequential numbering ( of the light chain or a - chain followed by the heavy chain ) is used in the plots , and sequential residue numbers are given in parentheses throughout the paper for reference . we performed a set of six independent 20 ns conventional md simulations for both apo - thrombin and ppack - thrombin . during the equilibration procedure , a rather large conformational transition in the active site loops was observed for apo - thrombin that involved a reorientation of both the -loop ( 178195 ) and the na binding loop ( 264271 ) , forming a more open active site pocket . a community network analysis approach was applied to identify groups of residues undergoing correlated motions in ppack - thrombin and apo - thrombin . the flow of information in the physical network of the protein was measured by the edge betweenness , defined as the number of shortest paths that pass through the edge in the network , and is a direct measure of the strength of intercommunity communication within the network ( black lines in figure 1a and b ) . the community that includes part of the na binding loop in apo - thrombin ( figure 1c , brown ) consolidates with the active site serine195ct ( 241 ) , the 70s loop ( 98113 ) , residues 191194ct of the -loop ( 237240 ) , and the n - terminal residues 1719ct ( 3840 ) in the ppack - liganded form ( brown , figure 1d ) . ppack ligation also acts to consolidate the n - terminal -barrel , which is formed by two separate communities in apo - thrombin and forms a large community that also contains the 30s loop ( 5562 ) and part of the 60s loop ( 8294 ) ( orange , figure 1d ) . in summary , the community analysis revealed consolidation of the na binding site , the base of the -loop , and the n - terminus of the heavy chain into one community and most of the active site loops into a second community upon ppack binding . the a - chain community also becomes more strongly connected to the active site and the community containing the 70s loop . on the basis of the substantial consolidation observed in the community analysis upon ppack binding , we set out to examine whether there were concomitant changes in dynamics . community analysis of apo - thrombin ( a , c ) and ppack - thrombin ( b , d ) . the two - dimensional view of the communities in panels ( a ) and ( b ) depicts the relative size of the communities ( based on the number of residues ) as colored circles of varying sizes with the thickness of the connecting lines representing the relative interconnectivity among communities . residual dipolar couplings ( rdcs ) , which report on an ensemble average over all orientations of the magnetic dipole interaction vector up to the chemical shift coalescence limit , provide useful experimental data for determining the ensemble of structures that best represents the dynamic properties of a protein . when the experimentally derived rdcs are compared to rdcs that are back - calculated from ensembles of structures generated from amd simulations , the acceleration level that provides the most realistic representative structural ensemble can be identified . the ensembles obtained from such amd simulations of both ppack - thrombin and apo - thrombin are shown in figure 2 , with the loops colored according to the scheme shown below the structure . ensemble of the 10 representative structures of the rmsd clusters from the boltzmann reweighted structures from the rdc - calibrated amd for apo - thrombin ( left ) and ppack - thrombin ( right ) . for ppack - thrombin , order parameters ( s ) from conventional md simulations agreed extremely well with those measured by nmr relaxation experiments that are limited to the picosecond however , the fact that ensembles obtained from amd were required for good agreement with the rdc measurements suggested that motions on longer time scales are contributing to the solution structure . a comparison of samd2 for apo - thrombin to those obtained previously for ppack - thrombin is shown in figure 3a . most of the active site loops in both forms are highly flexible , yet a marked decrease in flexibility is observed upon active site ligation with ppack ( figure 3b ) . the loops that surround the active site also experience significant ordering , including the 60s loop ( 8294 ) , the -loop , and the 180s loop ( 225239 ) . some regions distal to the ppack also showed significant ordering upon ppack ligation , including the a - chain , the 30s loop , residue 221ct ( 269 ) of the sodium binding loop , and residues in the c - terminal helix ( figure 3c ) . ( a ) order parameters calculated from amd simulations of apo - thrombin ( red ) and ppack - thrombin ( black ) . ( c ) those residues with a s > + 0.1 are marked with red spheres on the structure of thrombin ( pdb code 1ppb ) , indicating stabilization upon active site occupation by ppack . the analysis of apo - thrombin revealed correlated motions between the active site loops , exosite 1 , and other distal sites . in particular , the entire -loop appears to undergo strongly correlated motions with the a - chain residues 1h1dct and 1214cct ( 15 and 2025 ) , the catalytic triad , h57ct , d102ct , s195ct ( 79 , 135 , 241 ) , the 60s loop , the 70s loop , the 90s loop ( 127133 ) , the surface strand under the 70s loop ( 145151 ) , the 170s loop ( 204219 ) , and the 180s loop ( figure 4 , lower triangle ) . whereas these correlated motions appeared to involve the entire -loop in apo - thrombin , only the tip of the -loop ( residues 146149ect ( 182190 ) ) appears to be undergoing the same set of motions in ppack - thrombin ( figure 4 , upper triangle ) . in apo - thrombin , the 170s and , to a lesser extent , the 180s loop , which are strongly correlated to the -loop , are also correlated with the a - chain and catalytic residues . correlated motions between the 30s and 60s loops are stronger in ppack - thrombin , and these extend to the 70s and 90s loops ( figure 4 ) . analysis of correlated motions performed on the amd trajectories of ppack - thrombin ( top triangle ) and apo - thrombin ( bottom triangle ) . the black boxes indicate correlations of the 170s and 180s loops with the a - chain and surface loops that are stronger in apo - thrombin than in ppack - thrombin . the blue boxes indicate correlations of the 30s loop with the 60s , 70s , and 90s loops that are stronger in ppack thrombin than in apo - thrombin . we applied a previously derived algorithm to identify the residual local frustration in representative structures from rmsd clusters of the boltzmann reweighted amd simulation results . these highly frustrated contacts map to functional sites and are thought to have been preserved in evolution . both apo- and ppack - thrombin show regions of high frustration in many of the surface loops ( figure 5 ) . the order parameters derived from conventional md simulations ( sns2 ) agree very well with order parameters derived from nmr relaxation experiments on thrombin and reveal the disorder resulting from motions in the nanosecond time regime . these results highlight that regions of high residual frustration may have evolved to allow slow time scale motions to occur with relative energetic ease , as previously suggested by wolynes and colleagues . analysis of residual local frustration in the lowest - energy structure from the rdc - calibrated amd ensemble of apo - thrombin ( left ) and ppack - thrombin ( right ) . the contacts that are minimally frustrated are shown in green , and the contacts that are highly frustrated are shown in red . the active site catalytic residues are shown in magenta . comparison of the order parameters reflecting nanosecond time scale motions versus longer time scale motions ( samd2 ) , with the average per residue fraction of highly frustrated contacts for the three lowest - energy structures from the amd simulation . ( a ) the sns ( gray ) and samd ( red ) for apo - thrombin are compared to the average fraction of highly frustrated contacts ( cyan ) . ( b ) the sns2 ( gray ) and samd2 ( black ) for ppack - thrombin are compared to the average fraction of highly frustrated contacts ( blue ) . we used a combination of community network analysis , rdc - calibrated amd simulations , and analysis of residual frustration to explore the dynamic ensemble of thrombin in solution from the nanosecond to the microsecond time regime . comparative analysis of apo - thrombin and the active - site - ligated thrombin systems identified differences in the dynamic fluctuations and changes in correlated motions upon active site ligation . analysis of thrombin complexed with the relatively small substrate analogue ppack showed a substantial rearrangement of the community structure . ppack binding consolidated the two active site communities most proximal to the ppack binding site as well as the na binding loop with the active site serine , the 70s loop , part of the -loop , and part of the a - chain . interestingly , this consolidation resulted in more residual local frustration near the active site ( figure 5 ) . the analysis of residual local frustration revealed that all of these loops are highly frustrated in the native structure of thrombin ( figure 5 ) . the striking correlation between the long timescale dynamics and the residual local frustration suggests that evolution has selected for energetically unfavorable contacts within the surface loops in order to facilitate larger amplitude slower motions . the surface loops exhibiting correlated motions are not necessarily structurally proximal , hinting at how allosteric regulation by binding of ligands at sites distal to the active site might occur . indeed , previous experiments have demonstrated a thermodynamic coupling between the 70s loop and the active site . another loop that is distal to the active site but that differs between apo- and ppack - thrombin is the -loop . residues leading up to the -loop are highly frustrated and highly dynamic in apo - thrombin but become much less dynamic in the ppack - bound form , with only the tip of the -loop remaining dynamic . although the strongly correlated motions in apo - thrombin between the -loop and the 170s loop almost completely disappear upon ppack ligation , correlations are strengthened between the tip of the loop and nearly every other loop in thrombin , including the 30s loop , the 60s loop , the 70s loop , the -strand connecting the 60s and 70s loops , and the 90s loop . thus , we can speculate that the reduced dynamics result from the binding of ppack , causing these regions to sample a more restricted conformational space , and that this reduced sampling leads to stronger correlated motions extending between the active site and the surface loops involved in allosteric regulation . the order parameter analysis revealed , as expected , that active site occupation decreases the microsecond motions in the surface loops ( figure 3 ) , consistent with amide h / d exchange experiments that showed that binding of ppack dampened exchange in all of the surface loops including those distal to the active site . millisecond time scale could explain how active site occupation lowers the entropic penalty for binding an exosite 1 allosteric modulator . thermodynamic measurements of binding show that the allosteric pathway between these two sites operates in both directions and changes in order parameters in the thrombin surface loops upon ppack binding to the active site are similar to those seen upon tm56 binding to exosite 1 . the serine protease architecture is that of a double -barrel fold in which the -strands are connected by surface loops . such local unfolding or cracking was predicted some time ago to be the fundamental mechanism of allostery , and residual local frustration is indeed found at such sites .

it is associated with foot ulceration and is the leading cause of nontrauma foot amputations . approved treatments to slow progression or prevent diabetic peripheral neuropathy have failed in clinical trials . while intensive insulin therapy to maintain near - normal blood glucose can delay onset of symptoms in type 1 diabetes it is not effective in preventing the development of neuropathy or slowing the progression in type 2 diabetes [ 2 , 3 ] . consumption of fish oil was shown to benefit those with cardiovascular diseases like congestive heart failure and has shown promise in type 2 diabetes , inflammatory diseases , stroke , depression , and anxiety disorders [ 48 ] . the ingestion of n-3 polyunsaturated fatty acids , eicosapentaenoic acid ( epa ) , and docosahexaenoic acid ( dha ) , those concentrated in fish oil , may be acted upon by lipoxygenases , acetylated cyclooxygenase , or cytochrome p450 enzymes producing serum elevations of various eicosanoid derivatives including resolvins of e and d series [ 5 , 9 ] . our current understanding of lipid derived health benefits implicates proresolving lipid mediators , such as resolvin d1 , as the major curative entity produced from the metabolism of epa and dha . in fact , results from many studies indicate that resolvins play a major endogenous role in the resolution of inflammation [ 11 , 12 ] . there are very few side effects of fish oil consumption , individuals complain of fishy taste or breath , and no adverse events were reported in a large multicenter clinical trial . our group has shown that enrichment of diet with fish oil can improve outcome measures of type 1 and type 2 diabetes in sprague - dawley rats [ 14 , 15 ] . further , we have shown that treatment of c57bl/6j mice with fish oil or 1 ng / g body weight of resolvin d1 ameliorates symptoms of peripheral neuropathy in a type 2 diabetic model . salsalate is a prodrug of salicylate and its primary mechanism of action is as a nonsteroidal anti - inflammatory that functions by inhibiting the synthesis of prostaglandins by way of inactivation of cyclooxygenase enzymes . experiments have established that salsalate , a nonacetylated salicylate , is insoluble in gastric juice and moves to the small intestine where it is hydrolyzed into two salicylic acid molecules . in clinical trials the main side effects of salsalate were tinnitus , headache , dizziness , and gastrointestinal discomfort , which occurred at higher doses and subside following withdrawal of treatment [ 1720 ] . salsalate produces fewer gastrointestinal bleedings when compared to acetylsalicylic acid due to less mucosal prostaglandin inhibition . in this study we have used a streptozotocin induced type 1 diabetes mouse model , which we previously characterized , to assess the effectiveness of salsalate , menhaden oil , a fish oil enriched with n-3 fatty acids epa and dha , the combination of salsalate and menhaden oil , or the dha derivative resolvin d1 in treating endpoints of diabetic peripheral neuropathy . c57bl/6j mice were purchased from jackson laboratories and housed in a certified animal care facility with food and water available ad libitum . experiments were conducted in accordance with international standards on animal welfare complying with institutional and national institutes of health animal use guidelines ( acurf protocol 1212258 ) . twelve - week - old c57bl/6j mice were divided into six groups , one vehicle treated control group and five groups treated with streptozotocin as previously described [ 22 , 23 ] . mice with a blood glucose level exceeding 300 mg / dl ( 16.7 mm ) were considered diabetic ( aviva accu - chek , roche , mannheim , germany ) . following 8 weeks of untreated diabetes animals received 12 weeks of treatment by way of diet supplementation or a daily injection of resolvin d1 . the targeted n for each group was 18 with an expectation of having 12 mice per group at the end of the study . the mice not having blood glucose greater than 300 mg / dl prior to initiation of treatment were excluded from the study . the caloric composition of the standard diet ( harlan teklad , # 7001 , madison , wi ) or diet enriched in menhaden oil ( research diets , new brunswick , nj ) is provided in table 1 . the fatty acid composition of the standard and menhaden oil enriched diets is presented in table 2 and was found to be similar to what we previously reported . control and one group of diabetic mice , the untreated diabetic group , received standard diet throughout the experiment . three groups of diabetic mice received diet containing either menhaden oil ( 25% kcal fat , in diet ) , salsalate ( 250 mg / kg , in diet ) , or the combination of both menhaden oil and salsalate . salsalate was mixed into the meal form of the standard or menhaden oil enriched diets and pelleted . the final group of diabetic mice was fed a standard diet and received a daily injection of resolvin d1 ( 1 ng / g body weight , i.p . ) . thermal nociceptive response was assessed in mice one week prior to euthanasia using the hargreaves method with an iitc life science inc . device ( woodland hills , ca , model 390 g ) as previously described [ 3 , 23 , 24 ] . five recordings were made with a 5-minute delay between recordings , the initial recording was discarded , and the remaining four were averaged and presented in seconds and served as the thermal nociceptive response latency . , abbott laboratories , north chicago , il ) and motor and sensory nerve conduction velocities were assessed as in previous experiments [ 3 , 23 , 24 ] . motor nerve conduction velocity was calculated by using the stimulus artifact of the evoked potential , subtracting the latency measurement ( in milliseconds ) from the sciatic notch from the latency measurement of the achilles tendon and dividing the difference by the distance between the two stimulating electrodes ( measured in millimeters ) . sensory nerve conduction velocity equaled the distance between stimulating and recording electrodes over the latency to initial peak negative deflection . the rostock cornea module for the heidelberg retina tomograph ( heidelberg engineering , vista , ca ) was used for in vivo assessment of subepithelial nerves in the mouse cornea as described previously [ 3 , 23 ] . anesthetized mice were fitted to a stereotaxic mouse head holder ( model 921-e , david kopf instruments , tujunga , ca ) and secured to a platform that allows for three - dimensional adjustments . genteal eye lubricant gel ( alcon , fort worth , tx ) was applied to the lens and advanced forward to make contact with the mouse cornea epithelium . at least three nonoverlapping images of the subepithelial nerves were acquired per mouse and assessed for nerve length . corneal nerve fiber length has proven to be the best morphological parameter in diagnosing diabetic neuropathy showing the lowest coefficient of variation [ 25 , 26 ] . corneal nerve fiber length is represented as a mean value of the nerve lengths measured from the images and expressed in mm / mm . during dissection the animal 's cornea was isolated for immunofluorescent analysis as previously described [ 3 , 23 ] . the cornea was dissected around the scleral - limbal region and fixed for 30 minutes in zamboni 's fixative . corneas were blocked in phosphate - buffered saline ( pbs ) containing 0.2% triton x-100 , 2% goat serum , and 1% bovine serum albumin for 2 hours followed by overnight incubation with neuronal class -iii tubulin mouse monoclonal antibody at 1 : 1000 in 4c ( covance , dedham , ma ) . following three 15-minute washes with pbs containing 0.2% triton x-100 , goat anti - mouse igg2a alexa 488 secondary antibody was applied for 2 hours at room temperature ( invitrogen , eugene , or ) . three more 15-minute washes in pbs containing 0.2% triton x-100 preceded the mounting of the cornea ( epithelium facing up ) to a microscope slide in prolong gold ( life technologies , carlsbad , ca ) . a cover slip was placed on the cornea and sealed with clear fingernail polish . using zeiss lsm710 confocal microscope running zen software ( carl zeiss , oberkochen , germany ) multiple images were collected to assess different nerve parameters in an attempt to more fully characterize the diseased cornea . firstly , a 20x objective ( plan - apochromat 20x/0.8 ) was used to make 8 8 tile scan z - stack ( 3400 m 3400 m 30 m ; 4096 pixels 4096 pixels 30 pixels ) images of the entire mouse cornea that were further processed to make a maximum projection intensity image for analysis . these images were analyzed for the amount of surface area covered by nerves using imaris version 7.6.4 x64 software ( bitplane , zurich , switzerland ) . the surface option was used ( parameters : smoothing enabled , surface grain size being equal to 0.833 m per pixel , no background elimination , the diameter of the largest sphere being 6.23 m , and thresholding being automatic ) to determine the total surface area covered by nerves and is represented as a percentage of the total cornea surface area determined by manually tracing the cornea with a closed poly - line , as in previous experiments [ 9 , 23 ] . secondly , using a 63x objective ( plan - apochromat 63x/1.4 ) a 3 3 tile scan z - stack ( 405 m 405 m 30.11 m ; 1536 pixels 1536 pixels 78 pixels ) was taken with optimum axial resolution to image the epithelial nerves of the cornea . these images were cropped to include only the epithelial nerves and subjected to volume analysis using imaris version 7.6.4 x64 software ( parameters : smooth was enabled , surface grain size equaled 0.187 m , no background elimination was used , diameter of the largest sphere was 0.701 m , and thresholding was automatic ) ; the nerve volume is represented as a percentage of total volume as used for previous experiments [ 3 , 9 , 23 ] . for presentation purposes images were adjusted using imaris and scale bars inserted with fiji . density of intraepidermal nerve fibers was determined as in previous experiments [ 3 , 23 ] . nerve profiles were imaged using a zeiss lsm710 confocal microscope with a 40x objective ( ec plan - neofluar 40x/0.75 ) , counted by two independent investigators blinded to the sample condition , and profiles were normalized to the length of the epidermis in millimeters . mouse blood was collected from the right ventricle and serum was scrutinized for triglycerides , free fatty acids , cholesterol , and resolvin d1 using commercial kits as reported in previous experiments [ 3 , 9 , 23 ] . one - way analysis of variance ( anova ) and sidak 's multiple comparisons test were performed to determine group differences ( graphpad prism 6 , graphpad , san diego , ca ) . animals were diabetic for a total of 20 weeks and for the final 12 weeks selected animals received treatment via dietary supplementation of menhaden oil , salsalate , or menhaden oil plus salsalate or daily injection of 1 ng / g resolvin d1 . as seen in table 3 , all diabetic mice failed to gain significant weight compared to control animals regardless of treatment and with only a slight nonsignificant addition of weight in the groups receiving menhaden oil supplementation ( f(5,81 ) = 14.54 , p < 0.0001 ) . throughout the experiment , diabetic animals had elevated blood glucose levels that were greater than in control mice and were not significantly impacted by treatment ( f(5 , 81 ) = 60.49 , p < 0.0001 and f(5 , 81 ) = 12.14 , p < 0.0001 , resp . ) . serum triglycerides and free fatty acids were not significantly changed in any of the groups . serum cholesterol levels were 20.0 1.2 mg / dl in control mice but trended towards an increase in all diabetic groups ( f(5,72 ) = 3.53 , p < 0.01 ; diabetic group 26.8 1.4 , diabetic plus salsalate group 28.6 4.0 , and resolvin d1 group 27.3 1.1 mg / dl ) . for mice receiving menhaden oil and menhaden oil plus salsalate serum cholesterol was increased over the control group ( 34.2 3.8 and 32.0 3.6 mg / dl , resp . as seen in figure 1 , serum resolvin d1 levels trended to be elevated in diabetic mice receiving menhaden oil and elevated to a significant level in the menhaden oil plus salsalate group compared to control ( f(5,60 ) = 8.13 , p < 0.0001 ; menhaden oil 862 130 pg / ml , menhaden oil plus salsalate 1151 117 pg / ml , and control 680 52 pg / ml ) while the untreated diabetic group ( diabetic group 613 47 pg / ml ) remained unchanged from control , salsalate , and resolvin d1 groups ( 714 127 , 612 44 and 620 63 pg / ml , resp . ) . the in vivo analysis of corneal nerves with corneal confocal microscopy is presented in figure 2 . untreated diabetic mice presented with very little observable nerves in the subepithelial layer while control animals typically have several nerve fibers easily identified and measured ( f(5,76 ) = 16 , p < 0.0001 ) . following treatment with menhaden oil , menhaden oil plus salsalate , and resolvin d1 subepithelial corneal nerve occupancy appears similar to the control mice ( 2.4 0.8 , 2.3 0.5 , 2.6 0.5 , and 2.7 0.8 mm / mm , resp . ) whereas salsalate treatment alone had marginal but significant improvement over diabetic animals ( 1.8 0.6 and 1.0 0.4 mm / mm ) . using an antibody to -iii tubulin we further examined the density of corneal nerves in the epithelial and subepithelial layers by immunohistochemistry . figure 3 shows that the surface area covered by corneal nerves in the subepithelial layer of diabetic mice is significantly reduced when compared to control ( f(5,76 ) = 4.4 , p = 0.0015 ; 49 2.7 and 61 2.2% area , resp . ) and treatment with salsalate , menhaden oil , and menhaden oil plus salsalate provides a trend towards improvement while resolvin d1 treatment significantly increases nerve surface area over diabetic animals ( 54 0.7 , 57 1.4 , 58 3.3 , and 61 2.1 , resp . ) . as observed in the representative images untreated diabetic mice show reduced subepithelial nerve bundle length and compared to control mice a greater area of the cornea is devoid of -iii tubulin staining . figure 4 provides data and representative images of the density of corneal epithelial nerves over the central whorl region of the cornea . control animals have a uniform epithelial innervation ( 1.8 0.3% volume ) and when compared to untreated diabetic animals we found the innervation volume significantly reduced ( f(5,76 ) = 14 , p < 0.0001 ) . treatment with menhaden oil , menhaden oil plus salsalate , or resolvin d1 provided significant benefits compared to untreated diabetic mice ( 1.3 0.3 , 1.4 0.3 , 1.4 0.4 , and 0.6 0.3% volume , resp . ) and salsalate alone showed a modest nonsignificant increase over untreated diabetic mice ( 1.1 0.1% volume ) . following 20 weeks of hyperglycemia untreated diabetic mice had motor and sensory nerve conduction velocities significantly reduced when compared to control animals ( mncv f(5,81 ) = 12.47 , p < 0.0001 , and control 41.1 1.5 versus diabetic group 27.7 1.0 m / sec ; sncv f(5,81 ) = 21.49 , p < 0.0001 , and control 29.9 0.7 versus diabetic group 22.5 0.6 ) and treatment with menhaden oil , menhaden oil plus salsalate , and resolvin d1 produced significantly faster conduction velocities compared to untreated diabetic mice ( mncv 37.2 1.1 , 39.2 1.0 , and 38.8 1.1 , resp . , versus 27.7 1.0 m / sec ; sncv 29.5 0.8 , 30.2 0.6 , and 29.3 0.5 , resp . treatment with salsalate alone significantly improved sensory nerve conduction velocities compared to untreated diabetic mice ( 27.0 0.5 versus 22.5 0.6 m / sec ) ; however , motor nerve conduction velocity did not reach significance over untreated diabetic mice ( 35.2 2.4 versus 27.7 1.0 m / sec ) . both motor and sensory nerve conduction velocity in diabetic mice treated with salsalate alone remained significantly reduced compared to control mice . untreated diabetic mice show a reduction in intraepidermal nerve profiles compared to control mice and treatment with salsalate , menhaden oil , menhaden oil plus salsalate , and resolvin d1 showed significantly more nerve profiles ( f(5,81 ) = 44.20 , p < 0.0001 ; 14.6 0.4 , 24.7 0.7 , 18.4 0.4 , 21.3 0.5 , 21.0 0.6 , and 20.4 0.3 profiles / mm , resp . ) . all diabetic mice showed a reduced thermal sensitivity compared to control animals , except those treated with resolvin d1 ( f(5,81 ) = 32.0 , p < 0.0001 ; control 5.7 0.2 versus diabetic group 8.6 0.2 sec ) . treatment with salsalate , menhaden oil , menhaden oil plus salsalate , and resolvin d1 significantly improved thermal sensitivity over untreated diabetic mice ( 7.3 0.2 , 6.6 0.2 , 6.7 0.1 , and 5.8 0.2 , resp . pharmaceutics used in the treatment of diabetic neuropathy range from nonsteroidal anti - inflammatory drugs such as aspirin , anticonvulsants like gabapentin and pregabalin , antidepressants such as tricyclic amitriptyline or serotonin - norepinephrine reuptake inhibitors like duloxetine and venlafaxine , and opioids like tramadol . many of the available treatments have shown promise for reducing pain yet with side effects such as sedation , fatigue , nausea , sweating , and peripheral edema , and respiratory depression patients are reluctant to comply with doctor recommendations . complicating the matter , the typical diabetic patient uses more than one medicine which may hinder the identification of side effects and may lead to discontinuation of treatment . most importantly , these drugs do not constitute a treatment for diabetic peripheral neuropathy and they merely target painful symptoms . in this experiment we have used a series of compounds that have a large therapeutic index and minimal side effects and targeted endpoints constituting a treatment to prevent or slow the progression of diabetic peripheral neuropathy . salsalate and its active metabolite salicylic acid are structurally and functionally similar to acetylsalicylic acid , but contrary to aspirin , salsalate passes through the stomach into the small intestine before being hydrolyzed into two salicylic acid molecules . salicylic acid exerts its effects by reducing the generation of prostaglandins via cyclooxygenase inactivation . in this study we extend previous findings by showing that salsalate provides benefit when used alone for the treatment of diabetes . as we show in table 3 , salsalate treated diabetic mice had improved motor and sensory nerve conduction velocities , salsalate benefited animals ' thermal sensitivity , and the foot pad of salsalate treated animals had greater number of nerve profiles . these data indicate that salsalate alone reduced the pathological consequences of diabetic neuropathy . in humans , salsalate has been used to treat the pain and inflammation associated with rheumatoid arthritis , osteoarthritis , and other rheumatic disorders [ 30 , 31 ] . there are multiple noncyclooxygenase uses for salsalate in diabetes including results from randomized placebo - controlled studies showing salsalate reduced fasting blood glucose , hba1c , and circulating triglycerides [ 18 , 19 , 32 ] . furthermore , studies have shown salsalate treatment blocks free fatty acid mediated vascular insulin resistance with improved glycemic control and with reduced inflammatory outcomes measures [ 17 , 20 ] . some beneficial metabolic effects may be mediated by increased energy expenditure from activated brown adipose tissue and reduced microvascular complications via inhibition of inhibitor of kappa b kinase ( ikk ) , the subsequent reduction in nf-b related genes , and increased nitric oxide ( no ) preventing ischemic nerve damage [ 35 , 36 ] . this study demonstrated for the first time that salsalate alone can improve diabetic neuropathic endpoints in a mouse model of type 1 diabetes even when treatment was delayed for eight weeks after the onset of hyperglycemia . fish oils , like menhaden oil , contain a high concentration of epa and dha and their consumption elevates the plasma concentration of proresolving inflammatory lipid mediators like resolvin d1 . this study extends our previous findings by demonstrating the effectiveness of menhaden oil and resolvin d1 in alleviation of diabetic neuropathy outcome measures in a type 1 diabetes mouse model . as seen in table 4 , diabetic mice receiving menhaden oil or resolvin d1 showed improved motor and sensory nerve conduction velocities and improved thermal sensitivity and more nerve profiles in the skin compared to untreated diabetic mice . further , as shown in figures 2 , 3 , and 4 diabetic mice receiving menhaden oil treatment or resolvin d1 presented with greater nerve coverage in the subepithelial layer , both by in vivo confocal microscopy and by immunohistochemistry , and had a greater volume of epithelial nerves in the corneal epithelium . we attribute these results in part to the increased circulating levels of proresolving lipid mediators found following dietary enrichment with fish oil or exogenous treatment with the derivate resolvin d1 . it is likely that other proresolving inflammatory lipid mediators such as resolvin e1 were also elevated following treatment with menhaden oil . resolvin d1 levels were not found to be elevated in serum of diabetic mice treated exogenously with resolvin d1 . this was likely due to the serum being collected at the termination of the study which was not performed until 24 h after the final resolvin d1 treatment . dha may be acted upon by 15-lipoxygenase , peroxidases , 5-lipoxygenase , and hydrolases before the derivate resolvin d1 is generated . additionally , aspirin - triggered resolvin d1 can be generated by acetylated cyclooxygenase followed by activity of a peroxidase and hydrolase . we previously showed that ingestion of menhaden oil will elevate serum levels of resolvin d1 and in this study we confirm this finding and extend the result by implicating salsalate as a possible agent for further enhancing the circulating concentration of proresolving lipid mediators , such as resolvin d1 . this finding can be observed in figure 1 where diabetic mice receiving a combination of menhaden oil and salsalate were found to have a resolvin d1 average of nearly three hundred pg / ml more than diabetic mice treated with menhaden oil alone . this indicates , for the first time , that salsalate , or its active molecule salicylic acid , triggers an elevation of resolvin d1 levels beyond that observed with menhaden oil treatment alone . in this study , when animals received the combined treatment of salsalate and menhaden oil we did not observe an improvement in diabetic neuropathy outcome measures beyond monotherapy with menhaden oil . this was not surprising since the study was not designed to examine the potential for elevating lipid mediator levels following low dose fish oil combined with salsalate treatment . in this study we used the maximum concentration of menhaden oil possible for treatment of type 1 diabetic rodents . and while the amount of fish oil tested in this study ( 25% kcal fat from fish oil ) is not an unrealistic dose , we are pushing the limits of what a human will consume daily . results from this study do provide rationale for a more comprehensive study to examine whether increased resolvin levels can be achieved with lower levels of dietary fish oil when combined with salsalate and if this combination can be as effective in treating diabetic neuropathy as the maximum dietary levels of fish oil . the principle finding of our study is that anti - inflammatory compounds such as salsalate and dietary supplements such as fish oil can provide benefit to patients suffering from diabetic peripheral neuropathy . whether the combination of salsalate and lower doses of fish oil may provide a similar benefit for treatment of diabetic neuropathy as the higher doses of fish oil will require further investigation .

aims . in this study a streptozotocin induced type 1 diabetes mouse model was used to assess the effectiveness of salsalate , menhaden oil , the combination of salsalate and menhaden oil , or resolvin d1 on neuropathic endpoints . materials and methods . changes in body weight , blood glucose , serum markers for triglycerides , free fatty acids , cholesterol , and resolvin d1 , motor and sensory nerve conduction velocities and thermal sensitivity were assessed , as well as performing in vivo confocal microscopy of subepithelial corneal nerves and immunohistochemistry of nerves in the cornea and foot pad . results . diabetic animals failed to gain weight and had elevated blood glucose levels . diabetic mice had slowed nerve conduction velocity , reduced innervation of the foot pad and cornea subepithelial and epithelial layers , and reduced thermal sensitivity . monotherapy treatment with salsalate , menhaden oil , and resolvin d1 reduced the pathological signs of diabetic neuropathy . the combination of salsalate and menhaden oil also reduced signs of pathology and generated elevated plasma levels of resolvin d1 compared to other groups . conclusions . additional studies are needed to determine whether the combination of salsalate and menhaden oil may be more efficacious than monotherapy alone for the treatment of diabetic peripheral neuropathy .

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

approved treatments to slow progression or prevent diabetic peripheral neuropathy have failed in clinical trials . while intensive insulin therapy to maintain near - normal blood glucose can delay onset of symptoms in type 1 diabetes it is not effective in preventing the development of neuropathy or slowing the progression in type 2 diabetes [ 2 , 3 ] . the ingestion of n-3 polyunsaturated fatty acids , eicosapentaenoic acid ( epa ) , and docosahexaenoic acid ( dha ) , those concentrated in fish oil , may be acted upon by lipoxygenases , acetylated cyclooxygenase , or cytochrome p450 enzymes producing serum elevations of various eicosanoid derivatives including resolvins of e and d series [ 5 , 9 ] . our current understanding of lipid derived health benefits implicates proresolving lipid mediators , such as resolvin d1 , as the major curative entity produced from the metabolism of epa and dha . further , we have shown that treatment of c57bl/6j mice with fish oil or 1 ng / g body weight of resolvin d1 ameliorates symptoms of peripheral neuropathy in a type 2 diabetic model . in clinical trials the main side effects of salsalate were tinnitus , headache , dizziness , and gastrointestinal discomfort , which occurred at higher doses and subside following withdrawal of treatment [ 1720 ] . in this study we have used a streptozotocin induced type 1 diabetes mouse model , which we previously characterized , to assess the effectiveness of salsalate , menhaden oil , a fish oil enriched with n-3 fatty acids epa and dha , the combination of salsalate and menhaden oil , or the dha derivative resolvin d1 in treating endpoints of diabetic peripheral neuropathy . following 8 weeks of untreated diabetes animals received 12 weeks of treatment by way of diet supplementation or a daily injection of resolvin d1 . the fatty acid composition of the standard and menhaden oil enriched diets is presented in table 2 and was found to be similar to what we previously reported . control and one group of diabetic mice , the untreated diabetic group , received standard diet throughout the experiment . three groups of diabetic mice received diet containing either menhaden oil ( 25% kcal fat , in diet ) , salsalate ( 250 mg / kg , in diet ) , or the combination of both menhaden oil and salsalate . salsalate was mixed into the meal form of the standard or menhaden oil enriched diets and pelleted . the final group of diabetic mice was fed a standard diet and received a daily injection of resolvin d1 ( 1 ng / g body weight , i.p . ) five recordings were made with a 5-minute delay between recordings , the initial recording was discarded , and the remaining four were averaged and presented in seconds and served as the thermal nociceptive response latency . , abbott laboratories , north chicago , il ) and motor and sensory nerve conduction velocities were assessed as in previous experiments [ 3 , 23 , 24 ] . motor nerve conduction velocity was calculated by using the stimulus artifact of the evoked potential , subtracting the latency measurement ( in milliseconds ) from the sciatic notch from the latency measurement of the achilles tendon and dividing the difference by the distance between the two stimulating electrodes ( measured in millimeters ) . sensory nerve conduction velocity equaled the distance between stimulating and recording electrodes over the latency to initial peak negative deflection . the rostock cornea module for the heidelberg retina tomograph ( heidelberg engineering , vista , ca ) was used for in vivo assessment of subepithelial nerves in the mouse cornea as described previously [ 3 , 23 ] . three more 15-minute washes in pbs containing 0.2% triton x-100 preceded the mounting of the cornea ( epithelium facing up ) to a microscope slide in prolong gold ( life technologies , carlsbad , ca ) . a cover slip was placed on the cornea and sealed with clear fingernail polish . firstly , a 20x objective ( plan - apochromat 20x/0.8 ) was used to make 8 8 tile scan z - stack ( 3400 m 3400 m 30 m ; 4096 pixels 4096 pixels 30 pixels ) images of the entire mouse cornea that were further processed to make a maximum projection intensity image for analysis . the surface option was used ( parameters : smoothing enabled , surface grain size being equal to 0.833 m per pixel , no background elimination , the diameter of the largest sphere being 6.23 m , and thresholding being automatic ) to determine the total surface area covered by nerves and is represented as a percentage of the total cornea surface area determined by manually tracing the cornea with a closed poly - line , as in previous experiments [ 9 , 23 ] . secondly , using a 63x objective ( plan - apochromat 63x/1.4 ) a 3 3 tile scan z - stack ( 405 m 405 m 30.11 m ; 1536 pixels 1536 pixels 78 pixels ) was taken with optimum axial resolution to image the epithelial nerves of the cornea . these images were cropped to include only the epithelial nerves and subjected to volume analysis using imaris version 7.6.4 x64 software ( parameters : smooth was enabled , surface grain size equaled 0.187 m , no background elimination was used , diameter of the largest sphere was 0.701 m , and thresholding was automatic ) ; the nerve volume is represented as a percentage of total volume as used for previous experiments [ 3 , 9 , 23 ] . nerve profiles were imaged using a zeiss lsm710 confocal microscope with a 40x objective ( ec plan - neofluar 40x/0.75 ) , counted by two independent investigators blinded to the sample condition , and profiles were normalized to the length of the epidermis in millimeters . mouse blood was collected from the right ventricle and serum was scrutinized for triglycerides , free fatty acids , cholesterol , and resolvin d1 using commercial kits as reported in previous experiments [ 3 , 9 , 23 ] . animals were diabetic for a total of 20 weeks and for the final 12 weeks selected animals received treatment via dietary supplementation of menhaden oil , salsalate , or menhaden oil plus salsalate or daily injection of 1 ng / g resolvin d1 . as seen in table 3 , all diabetic mice failed to gain significant weight compared to control animals regardless of treatment and with only a slight nonsignificant addition of weight in the groups receiving menhaden oil supplementation ( f(5,81 ) = 14.54 , p < 0.0001 ) . throughout the experiment , diabetic animals had elevated blood glucose levels that were greater than in control mice and were not significantly impacted by treatment ( f(5 , 81 ) = 60.49 , p < 0.0001 and f(5 , 81 ) = 12.14 , p < 0.0001 , resp . ) serum triglycerides and free fatty acids were not significantly changed in any of the groups . serum cholesterol levels were 20.0 1.2 mg / dl in control mice but trended towards an increase in all diabetic groups ( f(5,72 ) = 3.53 , p < 0.01 ; diabetic group 26.8 1.4 , diabetic plus salsalate group 28.6 4.0 , and resolvin d1 group 27.3 1.1 mg / dl ) . for mice receiving menhaden oil and menhaden oil plus salsalate serum cholesterol was increased over the control group ( 34.2 3.8 and 32.0 3.6 mg / dl , resp . as seen in figure 1 , serum resolvin d1 levels trended to be elevated in diabetic mice receiving menhaden oil and elevated to a significant level in the menhaden oil plus salsalate group compared to control ( f(5,60 ) = 8.13 , p < 0.0001 ; menhaden oil 862 130 pg / ml , menhaden oil plus salsalate 1151 117 pg / ml , and control 680 52 pg / ml ) while the untreated diabetic group ( diabetic group 613 47 pg / ml ) remained unchanged from control , salsalate , and resolvin d1 groups ( 714 127 , 612 44 and 620 63 pg / ml , resp . ) the in vivo analysis of corneal nerves with corneal confocal microscopy is presented in figure 2 . untreated diabetic mice presented with very little observable nerves in the subepithelial layer while control animals typically have several nerve fibers easily identified and measured ( f(5,76 ) = 16 , p < 0.0001 ) . following treatment with menhaden oil , menhaden oil plus salsalate , and resolvin d1 subepithelial corneal nerve occupancy appears similar to the control mice ( 2.4 0.8 , 2.3 0.5 , 2.6 0.5 , and 2.7 0.8 mm / mm , resp . ) using an antibody to -iii tubulin we further examined the density of corneal nerves in the epithelial and subepithelial layers by immunohistochemistry . figure 3 shows that the surface area covered by corneal nerves in the subepithelial layer of diabetic mice is significantly reduced when compared to control ( f(5,76 ) = 4.4 , p = 0.0015 ; 49 2.7 and 61 2.2% area , resp . ) and treatment with salsalate , menhaden oil , and menhaden oil plus salsalate provides a trend towards improvement while resolvin d1 treatment significantly increases nerve surface area over diabetic animals ( 54 0.7 , 57 1.4 , 58 3.3 , and 61 2.1 , resp . ) as observed in the representative images untreated diabetic mice show reduced subepithelial nerve bundle length and compared to control mice a greater area of the cornea is devoid of -iii tubulin staining . figure 4 provides data and representative images of the density of corneal epithelial nerves over the central whorl region of the cornea . control animals have a uniform epithelial innervation ( 1.8 0.3% volume ) and when compared to untreated diabetic animals we found the innervation volume significantly reduced ( f(5,76 ) = 14 , p < 0.0001 ) . treatment with menhaden oil , menhaden oil plus salsalate , or resolvin d1 provided significant benefits compared to untreated diabetic mice ( 1.3 0.3 , 1.4 0.3 , 1.4 0.4 , and 0.6 0.3% volume , resp . ) following 20 weeks of hyperglycemia untreated diabetic mice had motor and sensory nerve conduction velocities significantly reduced when compared to control animals ( mncv f(5,81 ) = 12.47 , p < 0.0001 , and control 41.1 1.5 versus diabetic group 27.7 1.0 m / sec ; sncv f(5,81 ) = 21.49 , p < 0.0001 , and control 29.9 0.7 versus diabetic group 22.5 0.6 ) and treatment with menhaden oil , menhaden oil plus salsalate , and resolvin d1 produced significantly faster conduction velocities compared to untreated diabetic mice ( mncv 37.2 1.1 , 39.2 1.0 , and 38.8 1.1 , resp . treatment with salsalate alone significantly improved sensory nerve conduction velocities compared to untreated diabetic mice ( 27.0 0.5 versus 22.5 0.6 m / sec ) ; however , motor nerve conduction velocity did not reach significance over untreated diabetic mice ( 35.2 2.4 versus 27.7 1.0 m / sec ) . both motor and sensory nerve conduction velocity in diabetic mice treated with salsalate alone remained significantly reduced compared to control mice . untreated diabetic mice show a reduction in intraepidermal nerve profiles compared to control mice and treatment with salsalate , menhaden oil , menhaden oil plus salsalate , and resolvin d1 showed significantly more nerve profiles ( f(5,81 ) = 44.20 , p < 0.0001 ; 14.6 0.4 , 24.7 0.7 , 18.4 0.4 , 21.3 0.5 , 21.0 0.6 , and 20.4 0.3 profiles / mm , resp . ) all diabetic mice showed a reduced thermal sensitivity compared to control animals , except those treated with resolvin d1 ( f(5,81 ) = 32.0 , p < 0.0001 ; control 5.7 0.2 versus diabetic group 8.6 0.2 sec ) . treatment with salsalate , menhaden oil , menhaden oil plus salsalate , and resolvin d1 significantly improved thermal sensitivity over untreated diabetic mice ( 7.3 0.2 , 6.6 0.2 , 6.7 0.1 , and 5.8 0.2 , resp . pharmaceutics used in the treatment of diabetic neuropathy range from nonsteroidal anti - inflammatory drugs such as aspirin , anticonvulsants like gabapentin and pregabalin , antidepressants such as tricyclic amitriptyline or serotonin - norepinephrine reuptake inhibitors like duloxetine and venlafaxine , and opioids like tramadol . many of the available treatments have shown promise for reducing pain yet with side effects such as sedation , fatigue , nausea , sweating , and peripheral edema , and respiratory depression patients are reluctant to comply with doctor recommendations . most importantly , these drugs do not constitute a treatment for diabetic peripheral neuropathy and they merely target painful symptoms . in this experiment we have used a series of compounds that have a large therapeutic index and minimal side effects and targeted endpoints constituting a treatment to prevent or slow the progression of diabetic peripheral neuropathy . in this study we extend previous findings by showing that salsalate provides benefit when used alone for the treatment of diabetes . as we show in table 3 , salsalate treated diabetic mice had improved motor and sensory nerve conduction velocities , salsalate benefited animals ' thermal sensitivity , and the foot pad of salsalate treated animals had greater number of nerve profiles . these data indicate that salsalate alone reduced the pathological consequences of diabetic neuropathy . in humans , salsalate has been used to treat the pain and inflammation associated with rheumatoid arthritis , osteoarthritis , and other rheumatic disorders [ 30 , 31 ] . there are multiple noncyclooxygenase uses for salsalate in diabetes including results from randomized placebo - controlled studies showing salsalate reduced fasting blood glucose , hba1c , and circulating triglycerides [ 18 , 19 , 32 ] . some beneficial metabolic effects may be mediated by increased energy expenditure from activated brown adipose tissue and reduced microvascular complications via inhibition of inhibitor of kappa b kinase ( ikk ) , the subsequent reduction in nf-b related genes , and increased nitric oxide ( no ) preventing ischemic nerve damage [ 35 , 36 ] . this study demonstrated for the first time that salsalate alone can improve diabetic neuropathic endpoints in a mouse model of type 1 diabetes even when treatment was delayed for eight weeks after the onset of hyperglycemia . fish oils , like menhaden oil , contain a high concentration of epa and dha and their consumption elevates the plasma concentration of proresolving inflammatory lipid mediators like resolvin d1 . this study extends our previous findings by demonstrating the effectiveness of menhaden oil and resolvin d1 in alleviation of diabetic neuropathy outcome measures in a type 1 diabetes mouse model . as seen in table 4 , diabetic mice receiving menhaden oil or resolvin d1 showed improved motor and sensory nerve conduction velocities and improved thermal sensitivity and more nerve profiles in the skin compared to untreated diabetic mice . further , as shown in figures 2 , 3 , and 4 diabetic mice receiving menhaden oil treatment or resolvin d1 presented with greater nerve coverage in the subepithelial layer , both by in vivo confocal microscopy and by immunohistochemistry , and had a greater volume of epithelial nerves in the corneal epithelium . we attribute these results in part to the increased circulating levels of proresolving lipid mediators found following dietary enrichment with fish oil or exogenous treatment with the derivate resolvin d1 . resolvin d1 levels were not found to be elevated in serum of diabetic mice treated exogenously with resolvin d1 . dha may be acted upon by 15-lipoxygenase , peroxidases , 5-lipoxygenase , and hydrolases before the derivate resolvin d1 is generated . we previously showed that ingestion of menhaden oil will elevate serum levels of resolvin d1 and in this study we confirm this finding and extend the result by implicating salsalate as a possible agent for further enhancing the circulating concentration of proresolving lipid mediators , such as resolvin d1 . this finding can be observed in figure 1 where diabetic mice receiving a combination of menhaden oil and salsalate were found to have a resolvin d1 average of nearly three hundred pg / ml more than diabetic mice treated with menhaden oil alone . this indicates , for the first time , that salsalate , or its active molecule salicylic acid , triggers an elevation of resolvin d1 levels beyond that observed with menhaden oil treatment alone . in this study , when animals received the combined treatment of salsalate and menhaden oil we did not observe an improvement in diabetic neuropathy outcome measures beyond monotherapy with menhaden oil . in this study we used the maximum concentration of menhaden oil possible for treatment of type 1 diabetic rodents . and while the amount of fish oil tested in this study ( 25% kcal fat from fish oil ) is not an unrealistic dose , we are pushing the limits of what a human will consume daily . results from this study do provide rationale for a more comprehensive study to examine whether increased resolvin levels can be achieved with lower levels of dietary fish oil when combined with salsalate and if this combination can be as effective in treating diabetic neuropathy as the maximum dietary levels of fish oil . the principle finding of our study is that anti - inflammatory compounds such as salsalate and dietary supplements such as fish oil can provide benefit to patients suffering from diabetic peripheral neuropathy . whether the combination of salsalate and lower doses of fish oil may provide a similar benefit for treatment of diabetic neuropathy as the higher doses of fish oil will require further investigation .

telepathology is defined as the practice of pathology at a distance , transmitting macroscopic and/or microscopic images via telecommunication links for ( 1 ) remote interpretations ( telediagnosis ) , second opinions or consultations ( teleconsultation ) , and educational purposes ( teleconferencing ) . with the widespread availability of imaging technology and telecommunication , access to global expert pathologists telepathology has been shown to be applicable for : ( i ) anatomical pathology including intra - operative consultation ( frozen sections ) , surgical pathology ( second opinions , immunostains ) , telecytology ( e.g. , on - site evaluation ) , and ultrastructural pathology , as well as ( ii ) clinical pathology including telehematology , microbiology ( e.g. , parasitology ) , and chemistry ( e.g. , interpretation of gels ) . the first is static ( store - and - forward ) telepathology that involves the examination of pre - captured still images transmitted via e - mail or stored on a shared server . the second mode of telepathology involves dynamic ( live ) examination of images in real - time , employing video or robotic microscopy . finally , hybrid technology involving whole slide imaging ( wsi ) has emerged that utilizes both dynamic viewing of a digitized ( scanned ) slide as well as viewing of selected areas of the saved image at higher magnification . the university of pittsburgh medical center ( upmc ) health system operates 20 geographically diverse hospitals within and around the city of pittsburgh , and also partners with hospitals located in distant states ( e.g. , indianapolis , indiana ) and other countries ( e.g. , italy , china ) . the anatomical pathology department employs an academic centers of excellence ( coe ) subspecialty model , where pathologists in their respective specialty tend to all be located in the same hospital . with this infrastructure , telepathology has been employed at upmc for over a decade to provide remote subspecialty expertise at local , national , and international sites . the aim of this article is to review the different modes of telepathology and share the experience garnered at upmc with respect to each teleconsultation method as well as describe future aspects of practicing telepathology . static image telepathology involves capturing , storing , and forwarding individual digital images , or galleries of static images , for remote diagnosis . advantages of this form of telepathology are the low cost involved , vendor independence , technical simplicity , the fact that the recipient does not require special software to view images , small manageable files are involved ( easy to retrieve , send , review , store , and share ) , and that these systems are easy to maintain . however , disadvantages of static telepathology relate to the fact that the consulting telepathologist has no remote control of the microscope or imaging device / camera , their interpretation is limited to only captured field of views , the host capturing images needs to have some expertise , acquiring images is labor intensive and may cause sampling error if the incorrect images are captured , there is frequent lack of clarity with low power magnification images , and not all still images are in focus . at upmc , , consultations were received from ismett ( mediterranean institute for transplantation and high specialization therapies ) located in palermo , italy . transplant - related biopsies are usually challenging and hence best interpreted by experts in the field . access to transplant pathologists is highly desirable for second opinions when dealing with difficult cases . teleconsultation in this setting also needs to be performed in a timely manner , since rapid and accurate interpretation of allograft biopsies influences outcome after organ transplantation . moreover , histopathologic interpretation determines whether a donor organ should be used for transplantation or disposed . the transplant telepathology system was , therefore , developed to support coverage 24 hours a day , 7 days a week . initially , this employed static images run in a store - and - forward mode . communication was limited to a private network using a thick client - server architecture between the host ( e.g. , italy ) and upmc consulting pathologists . analysis of early ( 14-month period ) accrued data for 102 transmitted cases showed full agreement with the original diagnosis in 86% of cases . for cases with disagreement ( 14% ) , 8 resulted in minor and 3 clinically significant differences in opinion . subsequently , during the 12-year partnership between upmc and ismett , approximately 3000 cases have been reviewed by telepathology . teleconsultation using static images improved with respect to workflow with only infrequent discrepancies being noted . this first generation home - grown static telepathology system has since been replaced by second generation dynamic robotic microscopy ( nikon coolscope streaming ) , third generation hybrid rapid virtual microscopy ( trestle live viewing ) , and most recently , in 2009 , with fourth generation wsi ultra - rapid virtual microscopy ( mirax midi ) [ figure 1 ] . with better technology allowing pathologists to view entire slides at high resolution , the performance of telepathology has improved . however , we did not change to these newer technologies until these approaches were proven to be technically feasible . although static images were quicker to read than robotic microscopy , being limited to specific field of views forced evolution . the evolution from static imaging to a wsi scanning system was necessitated due to the lag time required for robotic objective magnification changes and positional adjustments of the slide . with wsi scanning , these magnifications are digitally incorporated into the resultant image , as such the time required for biopsy interpretation was greatly reduced . secondly , configuring the wsi system with a high numerical aperture ( na ) objective ( e.g. , 40x , 95na ) enabled an image resolution that maximized image clarity ( detail ) , therefore , reducing eye fatigue of the reviewing pathologist , enabling longer review sessions . lower resolution imagery ( e.g. , 25 micron ) forces the human eye to continually focus trying to pull out acute pathology details , which leads to fatigue . lastly , high resolution scanning enabled subtle details pertaining to tissue rejection to be clearly identified , such as visual confirmation of splitting of the glomerular basement membranes . combined , these advancements improved the performance of the system such that routine consultation was practical and efficient . early and continued adoption of telepathology has provided invaluable experience with digital pathology , improved workflow , and accumulated resources ( facilitated funding for equipment , it infrastructure , and staffing ) within the transplant pathology division . today , digital pathology has evolved whereby multiplex - stained wsi is being used for microscopy and image analytics . multiplexing is defined as the application and analysis of multiple fluorescence markers ( typically greater than 3 ) to a single histologically prepared section . these markers can then be analyzed in the tissue context , quantifying spatial relationships between multiple markers as related to surrounding morphology expression patterns . diagram showing the information technology and server components for the upmc / ismett telepathology platform . cases are accessioned in the mirax digital slide desktop ( mdsd ) image repository ( blue box ) , individual slides are then scanned on the mirax midi wsi scanner and transferred via network file share to the mdsd repository . access to the images is direct via secure ( username / password ) connection to the mdsd system from client pc / mac using a java applet for viewing , or via affiliated / specific workflow applications for the purpose of transplant immunology assessment . the java applet viewer can be embedded in any website or client / server application for flexibility of workflow integration the division of hematopathology at our institution is another area that employed static telepathology . in several of the academic hospitals , without a hematopathologist on site , telehematology has ensured that interpretation of peripheral blood smears and differentials continue to be performed rapidly and accurately . expert hematopathologist interpretation is often required for difficult cases ( e.g. , blasts in leukemia ) . to accomplish this , the hematology laboratory relied on the cellavision automated digital system to digitize glass slides containing peripheral blood smears . the instrument was used to automatically locate and pre - classify digital images of white blood cells , red blood cells , and platelets . using this system , a technologist in the laboratory was able to email static images of concerning blood cells to a hematopathologist for interpretation [ figure 2 ] , while , in turn , the hematopathologist could access the system and the entire stored differential of a patient remotely from their office computer . analysis of our experience has shown that this device correctly classified up to 94% of cells , with some reduced accuracy for immature granulocytes . . this will also allow clinicians to view digitized blood smears in real time , instead of having to come to the laboratory to physically review the blood smear . ( a ) cellavision dm96 instrument ; ( b ) review monitor displaying captured images of different blood cells ; ( c ) e - mail with embedded selected static images generated using the systems remote review software the host driving ( navigating and focusing ) the slide on the microscope , to be viewed by a remote pathologist on a monitor , requires some expertise to perform this task . the remote consulting pathologist is usually in communication ( e.g. , via telephone or teleconferencing ) with the host . however , in order to transmit the image over the internet , the analog video signal needs to be converted ( using an ad converter ) to a digital signal . analog video signals unfortunately are subject to noise and degradation , which may affect the image quality . while many prior publications regarding telecytology have predominantly utilized static images , in practice , today , most cytology laboratories that use telecytology employ video streaming . at upmc , live video streaming has remained the mode of choice for providing immediate adequacy assessment of cytology specimens by telecytology . other methods , such as robotic microscopy , have nevertheless been investigated and shown to be effective . validation studies conducted in our cytopathology division using web - based streaming have shown that adequacy assessment and diagnostic accuracy with telecytology are acceptable . nevertheless , cytopathologists found that the quality of these digital images were inferior to viewing these cases on a conventional light microscope [ figure 3 ] . moreover , cytopathologists documented spending more time reviewing cases examined by telecytology [ figure 4 ] . these factors may explain , in part , why our cytopathologists only infrequently employ telecytology , despite the fact the necessary equipment to perform telepathology for immediate on - site evaluation of fine needle aspirations is available . for remote evaluations of fine needle aspirations , cytopathologists demonstrated similar performance using a conventional microscope ( blue bars ) and the telecytology system ( yellow bars ) for providing tissue adequacy ( far left bars ) and the correct diagnosis ( far right bars ) . however , the middle bars in this graph indicate that pathologists found reviewing cases remotely to appear more complex , with inferior image quality and more obscured features . consequently , they were less confident with their telecytology diagnoses average time ( seconds ) taken to review glass and telecytology cases . cytopathologists took longer to review a cytology slide using telecytology ( 94 seconds ) than they did when examining the same slide with a conventional light microscope ( 70 seconds ) robotic ( real - time , dynamic ) telepathology differs from video microscopy because the telepathologist ( consultant ) is now actively involved in selecting fields on the glass slide to be viewed . with a robotic telepathology system , the operator has remote control of critical motorized microscope functions , including glass slide movements in the x- and y - axes on the motorized microscope stage , focus of the glass slide , and selection of various microscope objective lenses by a robotic turret . this provides the pathologist with access to the entire slide with good image quality . a trained host ( e.g. , pathology assistant ) these devices are expensive and because of their point - to - point network connectivity requirements ( i.e. not a web - based protocol ) are often not desirable to implement within a hospital it infrastructure . additional disadvantages are the relatively slow scanning of slides ( approximately 10 min / slide ) , the fact that both the host and recipient require integrated software , and a required high level of system maintenance for routine optimization , cleaning , and adjustment . robotic telepathology has been extensively used at upmc to remotely interpret intra - operative neuropathology consultations ( frozen sections ) . a considerable volume of neurosurgery is performed within our organization . however , in keeping with our coe model , neuropathologists are all housed at one hospital location . the steady demand for neuropathology frozen sections in different hospitals has been met by utilizing robotic telepathology [ figure 5 ] . several zeiss ( previously trestle ) robotic microscopes have been strategically placed within frozen section rooms at the different facilities [ figure 6 ] . pathology staff , including trainees , have received instruction on how to handle tissue received for frozen section and load prepared slides onto these microscopes . the importance of such training is a key element to the success of our teleneuropathology service and should not be overlooked . since only small portions of brain / neural tissue are usually procured and entirely submitted for frozen section , in our experience , we found that a brief gross description of the tissues received to the remote neuropathologist over the telephone by a pathology resident or assistant was sufficient . at one site , a surgical pathologist does the sampling with guidance from a consulting neuropathologist . nevertheless , a secure webcam in the frozen section room is available to facilitate gross telepathology if required . also , investigations of newer technologies ( e.g. , high definition digital cameras and teleconferencing software ) are underway in an attempt to improve our macroscopic teleneuropathology capabilities [ figure 7 ] . software ( medmicroscopy and mirax navigator ) to access scanned images and remotely control robotic microscopes has been made readily available on the internet accessible from any workstation . the diagnostic outcome from 2002 to 2006 ( comparing 1227 conventional frozen sections to 402 performed by telepathology ) demonstrated that the telepathology deferral rate ( 19.7% ) varied , and was higher than the deferral rate for conventional frozen section cases ( 10% ) . however , with the adoption of newer technology ( trestle scanner replaced the nikon coolscope ) and increased pathologist experience , in 2007 to 2008 ( comparing 547 conventional frozen sections to 262 by telepathology ) , there was acceptable concordance ( 78.2% ) between telepathology and conventional frozen sections . in addition , discrepancy and deferral rate differences during this later time period were statistically insignificant . intra - operative final discrepancies were uncommon , occurring in only 2.7% of these neuropathology cases . discrepant events were more common in non - neoplastic ( reactive ) cases , lymphomas , and with rare tumors . on account of our neuropathology division 's unique intra - institutional experience , they were able to successfully expand their teleconsultation practice across state lines ( inter - institutional ) with a financially separate medical center in indianapolis , indiana . implementation of technology during this venture proved to be easier than the accompanying administrative ( e.g. , contracts ) and legal ( e.g. , licensing ) issues involved . 2007 ) information technology infrastructure used to support intra - institutional neuropathology frozen section telepathology gross teleneuropathology showing close - up views of two portions of tissue submitted for frozen section from a glioblastoma multiforme tumor wsi telepathology systems are being increasingly employed for clinical , education , and research applications . wsi systems provide users with access to the entire case ( including sets of slides ) , offers automated scanning , and rapidly produces high resolution images . wsi scanners often have added software available for teleconferencing , image management , and image analysis . however , current impediments to their widespread adoption are related to their high expense and limited vendor interoperability . newer wsi scanners designed to hold one to four slides are smaller and less expensive . with wsi , there is a minor scan failure rate ( 2 - 5% ) . our scan failure rate is particularly low ( below 1% ) , probably because we train our staff to recognize problems before presenting images to pathologists . furthermore , pathologists maintain a close working relationship with histology to optimize slides for scanning ( e.g. , care taken to avoid folds , bubbles , etc . ) . our it staff also adjusts scanning technique with the vendor to optimize scanner device settings . such optimizations included vendor provided focus point algorithm changes to account for tissue artifact ( edge effects , folds ) . nevertheless , occasional difficulties can be experienced when scanning slides ( e.g. , misplaced cover slips and sticky wet slides ) . small tissue fragments , faint tissue or material at the slide edge or even outside the coverslip may not be recognized . users may also have to contend with long scan times , especially for thick tissue sections and if high - resolution images are desired ( e.g. , native high numerical aperture 40 scanning ) . this web - based tool was designed to facilitate digital pathology second opinion consults , especially for wsi . a general portal was developed to be used by all clients , and another that required customization for individual clients [ table 1 ] . with these portals , wsi can either be uploaded for transmission to upmc consultants ( general portal ) or accessed on a specific client 's server ( client - specific portal ) [ figure 8 ] . with the client - specific portal , we are able to avoid lengthy transfers of large wsi files over the internet as well as automate the process for image association to cases . despite distance , however , with this client - specific model image files are largely outside our control , we can not always quickly resolve problems with the client 's image server , and any network / hardware changes on the client 's side may cause unexpected instability . the general portal allows supported image file types ( static and wsi formats of major vendors ) , as well as pertinent accompanying clinical information , to be uploaded . all clients are offered secure login to submit their patient 's data and upload images , as well as to check on the status of their case and view or print second opinion reports . using this tool , upmc consultants are able to view digital images using a java applet . they have the ability to annotate and capture static image snapshots to be embedded into their reports . workflow is handled by managers who triage requests , monitor cases , and maintain personnel data . streamlined workflow , as well as training of users , has ensured prompt turn - around time and buy - in by institute pathologists . post - launch feedback from pathologists has resulted in customization to incorporate transcription services , peer - to - peer review for consultants , provision for issuing addenda and amendments , and improved wsi viewing experience . all cases received via the portal thus far have been surgical pathology cases . on average , 11 wsi per case have been received including h and e , histochemical and immunohistochemistry slides . the mean turnaround time for 22 consulting pathologists was 40 hours ( range 2 - 152 hours ) . different upmc telepathology portals data workflow with the upmc portal and one of their clients ( kingmed in china ) . as scans are transferred from the scanner to the ndp . serve database , this system allows for automated notification of the upmc assigned pathologist that the case is ready for consult . perhaps more importantly , this automated system eliminates a person having to email or notify , which scan filenames are associated with a case , thereby removing the possibility of human error of assigning the wrong slide image to a case digital pathology consultation at upmc has spanned more than a decade . during this time , all modes of telepathology have been successfully utilized to exploit our subspecialty expertise and to compete for pathology services . the practice of telepathology at our institution has evolved in concert with advances in technology , which has become more cost - effective . although several of the aforementioned modes of practicing telepathology may be outdated , we believe it is nevertheless important to share our experiences and point out that very often technology is not the limiting issue , but rather the people and processes involved . of note , diagnostic accuracy and turnaround time telecytology has been accomplished largely using real - time video streaming , and for wsi of cytology slides , the low volume of cases received for teleconsultation have been interpretable without the need for scanning slides with z - stacking . early and continued adoption has promoted several digital pathology resources ( e.g. , facilitated funding for equipment , it infrastructure , and staff ) that are now being leveraged for other clinical , educational , and research purposes . table 2 lists many of the other factors that may need to be considered when practicing telepathology . a key aspect of a successful digital teleconsultation program is integration of digital images ( including wsi ) into the laboratory information system ( lis ) and electronic health record . such integration , for example , was one of the main objectives of the cost action efforts towards integrating the lis with wsi are underway at our institution in partnership with omnyx . telepathology requirements are different for developed and developing countries . based on our experience with china , some bottlenecks to telepathology included the high price of wsi devices and regulatory issues . technological issues , however , are often easier to overcome than administrative , contractual , and legal challenges our institution is currently evaluating newer approaches such as grid technology , open access forums , and mobile solutions to determine how they can be leveraged to enhance telepathology at our institution . grid technology , which uses open standards to access distributed information , has been suggested by some authors as a way to improve quality in image - based diagnosis . we have provided our pathologists with access to a suite of cloud - based applications through a web browser [ figure 9 ] . since the computer programs that support these various telepathology devices are stored on servers at a remote location , the hardware and software demands placed on the end users are greatly diminished . while the use of commercial web conferencing systems ( e.g. , skype ) has been shown to be feasible for telepathology , in our institutional setting , reliance on public servers raises potential security issues , which has proven to be a barrier for telepathology . more recently , the field of telepathology is witnessing the emergence of specific open access forums ( e.g. , medical electronic expert communication system [ meces ] with embedded virtual slide technology ) . these new generation forums , which employ browser - friendly w3c conform standards , offer additional benefits such as acoustic information transfer and assistance in image screening . finally , taking advantage of mobile devices ( e.g. , cellular phones ) , which are almost ubiquitous today , provides another opportunity to perform telepathology almost anywhere and anytime .

several modes of telepathology exist including static ( store - and - forward ) , dynamic ( live video streaming or robotic microscopy ) , and hybrid technology involving whole slide imaging ( wsi ) . telepathology has been employed at the university of pittsburgh medical center ( upmc ) for over a decade at local , national , and international sites . all modes of telepathology have been successfully utilized to exploit our institutions subspecialty expertise and to compete for pathology services . this article discusses the experience garnered at upmc with each of these teleconsultation methods . static and wsi telepathology systems have been utilized for many years in transplant pathology using a private network and client - server architecture . only minor clinically significant differences of opinion were documented . in hematopathology , the cellavision system is used to transmit , via email , static images of blood cells in peripheral blood smears for remote interpretation . while live video streaming has remained the mode of choice for providing immediate adequacy assessment of cytology specimens by telecytology , other methods such as robotic microscopy have been validated and shown to be effective . robotic telepathology has been extensively used to remotely interpret intra - operative neuropathology consultations ( frozen sections ) . adoption of newer technology and increased pathologist experience has improved accuracy and deferral rates in teleneuropathology . a digital pathology consultation portal ( https://pathconsult.upmc.com/ ) was recently created at our institution to facilitate digital pathology second opinion consults , especially for wsi . the success of this web - based tool is the ability to handle vendor agnostic , large image files of digitized slides , and ongoing user - friendly customization for clients and teleconsultants . it is evident that the practice of telepathology at our institution has evolved in concert with advances in technology and user experience . early and continued adoption of telepathology has promoted additional digital pathology resources that are now being leveraged for other clinical , educational , and research purposes .

INTRODUCTION STATIC TELEPATHOLOGY DYNAMIC VIDEO MICROSCOPY ROBOTIC TELEPATHOLOGY WSI TELEPATHOLOGY CONCLUSIONS POTENTIAL FUTURE SOLUTIONS

telepathology is defined as the practice of pathology at a distance , transmitting macroscopic and/or microscopic images via telecommunication links for ( 1 ) remote interpretations ( telediagnosis ) , second opinions or consultations ( teleconsultation ) , and educational purposes ( teleconferencing ) . with the widespread availability of imaging technology and telecommunication , access to global expert pathologists telepathology has been shown to be applicable for : ( i ) anatomical pathology including intra - operative consultation ( frozen sections ) , surgical pathology ( second opinions , immunostains ) , telecytology ( e.g. , on - site evaluation ) , and ultrastructural pathology , as well as ( ii ) clinical pathology including telehematology , microbiology ( e.g. , parasitology ) , and chemistry ( e.g. the first is static ( store - and - forward ) telepathology that involves the examination of pre - captured still images transmitted via e - mail or stored on a shared server . the second mode of telepathology involves dynamic ( live ) examination of images in real - time , employing video or robotic microscopy . finally , hybrid technology involving whole slide imaging ( wsi ) has emerged that utilizes both dynamic viewing of a digitized ( scanned ) slide as well as viewing of selected areas of the saved image at higher magnification . the university of pittsburgh medical center ( upmc ) health system operates 20 geographically diverse hospitals within and around the city of pittsburgh , and also partners with hospitals located in distant states ( e.g. , italy , china ) . with this infrastructure , telepathology has been employed at upmc for over a decade to provide remote subspecialty expertise at local , national , and international sites . the aim of this article is to review the different modes of telepathology and share the experience garnered at upmc with respect to each teleconsultation method as well as describe future aspects of practicing telepathology . static image telepathology involves capturing , storing , and forwarding individual digital images , or galleries of static images , for remote diagnosis . advantages of this form of telepathology are the low cost involved , vendor independence , technical simplicity , the fact that the recipient does not require special software to view images , small manageable files are involved ( easy to retrieve , send , review , store , and share ) , and that these systems are easy to maintain . however , disadvantages of static telepathology relate to the fact that the consulting telepathologist has no remote control of the microscope or imaging device / camera , their interpretation is limited to only captured field of views , the host capturing images needs to have some expertise , acquiring images is labor intensive and may cause sampling error if the incorrect images are captured , there is frequent lack of clarity with low power magnification images , and not all still images are in focus . at upmc , , consultations were received from ismett ( mediterranean institute for transplantation and high specialization therapies ) located in palermo , italy . teleconsultation in this setting also needs to be performed in a timely manner , since rapid and accurate interpretation of allograft biopsies influences outcome after organ transplantation . initially , this employed static images run in a store - and - forward mode . communication was limited to a private network using a thick client - server architecture between the host ( e.g. analysis of early ( 14-month period ) accrued data for 102 transmitted cases showed full agreement with the original diagnosis in 86% of cases . for cases with disagreement ( 14% ) , 8 resulted in minor and 3 clinically significant differences in opinion . subsequently , during the 12-year partnership between upmc and ismett , approximately 3000 cases have been reviewed by telepathology . teleconsultation using static images improved with respect to workflow with only infrequent discrepancies being noted . this first generation home - grown static telepathology system has since been replaced by second generation dynamic robotic microscopy ( nikon coolscope streaming ) , third generation hybrid rapid virtual microscopy ( trestle live viewing ) , and most recently , in 2009 , with fourth generation wsi ultra - rapid virtual microscopy ( mirax midi ) [ figure 1 ] . with better technology allowing pathologists to view entire slides at high resolution , the performance of telepathology has improved . however , we did not change to these newer technologies until these approaches were proven to be technically feasible . although static images were quicker to read than robotic microscopy , being limited to specific field of views forced evolution . with wsi scanning , these magnifications are digitally incorporated into the resultant image , as such the time required for biopsy interpretation was greatly reduced . , 40x , 95na ) enabled an image resolution that maximized image clarity ( detail ) , therefore , reducing eye fatigue of the reviewing pathologist , enabling longer review sessions . , 25 micron ) forces the human eye to continually focus trying to pull out acute pathology details , which leads to fatigue . lastly , high resolution scanning enabled subtle details pertaining to tissue rejection to be clearly identified , such as visual confirmation of splitting of the glomerular basement membranes . combined , these advancements improved the performance of the system such that routine consultation was practical and efficient . early and continued adoption of telepathology has provided invaluable experience with digital pathology , improved workflow , and accumulated resources ( facilitated funding for equipment , it infrastructure , and staffing ) within the transplant pathology division . today , digital pathology has evolved whereby multiplex - stained wsi is being used for microscopy and image analytics . these markers can then be analyzed in the tissue context , quantifying spatial relationships between multiple markers as related to surrounding morphology expression patterns . diagram showing the information technology and server components for the upmc / ismett telepathology platform . cases are accessioned in the mirax digital slide desktop ( mdsd ) image repository ( blue box ) , individual slides are then scanned on the mirax midi wsi scanner and transferred via network file share to the mdsd repository . access to the images is direct via secure ( username / password ) connection to the mdsd system from client pc / mac using a java applet for viewing , or via affiliated / specific workflow applications for the purpose of transplant immunology assessment . the java applet viewer can be embedded in any website or client / server application for flexibility of workflow integration the division of hematopathology at our institution is another area that employed static telepathology . in several of the academic hospitals , without a hematopathologist on site , telehematology has ensured that interpretation of peripheral blood smears and differentials continue to be performed rapidly and accurately . to accomplish this , the hematology laboratory relied on the cellavision automated digital system to digitize glass slides containing peripheral blood smears . the instrument was used to automatically locate and pre - classify digital images of white blood cells , red blood cells , and platelets . using this system , a technologist in the laboratory was able to email static images of concerning blood cells to a hematopathologist for interpretation [ figure 2 ] , while , in turn , the hematopathologist could access the system and the entire stored differential of a patient remotely from their office computer . analysis of our experience has shown that this device correctly classified up to 94% of cells , with some reduced accuracy for immature granulocytes . . this will also allow clinicians to view digitized blood smears in real time , instead of having to come to the laboratory to physically review the blood smear . ( a ) cellavision dm96 instrument ; ( b ) review monitor displaying captured images of different blood cells ; ( c ) e - mail with embedded selected static images generated using the systems remote review software the host driving ( navigating and focusing ) the slide on the microscope , to be viewed by a remote pathologist on a monitor , requires some expertise to perform this task . , via telephone or teleconferencing ) with the host . however , in order to transmit the image over the internet , the analog video signal needs to be converted ( using an ad converter ) to a digital signal . analog video signals unfortunately are subject to noise and degradation , which may affect the image quality . while many prior publications regarding telecytology have predominantly utilized static images , in practice , today , most cytology laboratories that use telecytology employ video streaming . at upmc , live video streaming has remained the mode of choice for providing immediate adequacy assessment of cytology specimens by telecytology . other methods , such as robotic microscopy , have nevertheless been investigated and shown to be effective . validation studies conducted in our cytopathology division using web - based streaming have shown that adequacy assessment and diagnostic accuracy with telecytology are acceptable . nevertheless , cytopathologists found that the quality of these digital images were inferior to viewing these cases on a conventional light microscope [ figure 3 ] . moreover , cytopathologists documented spending more time reviewing cases examined by telecytology [ figure 4 ] . these factors may explain , in part , why our cytopathologists only infrequently employ telecytology , despite the fact the necessary equipment to perform telepathology for immediate on - site evaluation of fine needle aspirations is available . for remote evaluations of fine needle aspirations , cytopathologists demonstrated similar performance using a conventional microscope ( blue bars ) and the telecytology system ( yellow bars ) for providing tissue adequacy ( far left bars ) and the correct diagnosis ( far right bars ) . however , the middle bars in this graph indicate that pathologists found reviewing cases remotely to appear more complex , with inferior image quality and more obscured features . consequently , they were less confident with their telecytology diagnoses average time ( seconds ) taken to review glass and telecytology cases . cytopathologists took longer to review a cytology slide using telecytology ( 94 seconds ) than they did when examining the same slide with a conventional light microscope ( 70 seconds ) robotic ( real - time , dynamic ) telepathology differs from video microscopy because the telepathologist ( consultant ) is now actively involved in selecting fields on the glass slide to be viewed . with a robotic telepathology system , the operator has remote control of critical motorized microscope functions , including glass slide movements in the x- and y - axes on the motorized microscope stage , focus of the glass slide , and selection of various microscope objective lenses by a robotic turret . this provides the pathologist with access to the entire slide with good image quality . not a web - based protocol ) are often not desirable to implement within a hospital it infrastructure . additional disadvantages are the relatively slow scanning of slides ( approximately 10 min / slide ) , the fact that both the host and recipient require integrated software , and a required high level of system maintenance for routine optimization , cleaning , and adjustment . robotic telepathology has been extensively used at upmc to remotely interpret intra - operative neuropathology consultations ( frozen sections ) . however , in keeping with our coe model , neuropathologists are all housed at one hospital location . the steady demand for neuropathology frozen sections in different hospitals has been met by utilizing robotic telepathology [ figure 5 ] . several zeiss ( previously trestle ) robotic microscopes have been strategically placed within frozen section rooms at the different facilities [ figure 6 ] . pathology staff , including trainees , have received instruction on how to handle tissue received for frozen section and load prepared slides onto these microscopes . the importance of such training is a key element to the success of our teleneuropathology service and should not be overlooked . since only small portions of brain / neural tissue are usually procured and entirely submitted for frozen section , in our experience , we found that a brief gross description of the tissues received to the remote neuropathologist over the telephone by a pathology resident or assistant was sufficient . nevertheless , a secure webcam in the frozen section room is available to facilitate gross telepathology if required . also , investigations of newer technologies ( e.g. software ( medmicroscopy and mirax navigator ) to access scanned images and remotely control robotic microscopes has been made readily available on the internet accessible from any workstation . the diagnostic outcome from 2002 to 2006 ( comparing 1227 conventional frozen sections to 402 performed by telepathology ) demonstrated that the telepathology deferral rate ( 19.7% ) varied , and was higher than the deferral rate for conventional frozen section cases ( 10% ) . however , with the adoption of newer technology ( trestle scanner replaced the nikon coolscope ) and increased pathologist experience , in 2007 to 2008 ( comparing 547 conventional frozen sections to 262 by telepathology ) , there was acceptable concordance ( 78.2% ) between telepathology and conventional frozen sections . in addition , discrepancy and deferral rate differences during this later time period were statistically insignificant . intra - operative final discrepancies were uncommon , occurring in only 2.7% of these neuropathology cases . discrepant events were more common in non - neoplastic ( reactive ) cases , lymphomas , and with rare tumors . on account of our neuropathology division 's unique intra - institutional experience , they were able to successfully expand their teleconsultation practice across state lines ( inter - institutional ) with a financially separate medical center in indianapolis , indiana . implementation of technology during this venture proved to be easier than the accompanying administrative ( e.g. , licensing ) issues involved . 2007 ) information technology infrastructure used to support intra - institutional neuropathology frozen section telepathology gross teleneuropathology showing close - up views of two portions of tissue submitted for frozen section from a glioblastoma multiforme tumor wsi telepathology systems are being increasingly employed for clinical , education , and research applications . wsi systems provide users with access to the entire case ( including sets of slides ) , offers automated scanning , and rapidly produces high resolution images . wsi scanners often have added software available for teleconferencing , image management , and image analysis . newer wsi scanners designed to hold one to four slides are smaller and less expensive . our scan failure rate is particularly low ( below 1% ) , probably because we train our staff to recognize problems before presenting images to pathologists . small tissue fragments , faint tissue or material at the slide edge or even outside the coverslip may not be recognized . users may also have to contend with long scan times , especially for thick tissue sections and if high - resolution images are desired ( e.g. this web - based tool was designed to facilitate digital pathology second opinion consults , especially for wsi . a general portal was developed to be used by all clients , and another that required customization for individual clients [ table 1 ] . with these portals , wsi can either be uploaded for transmission to upmc consultants ( general portal ) or accessed on a specific client 's server ( client - specific portal ) [ figure 8 ] . with the client - specific portal , we are able to avoid lengthy transfers of large wsi files over the internet as well as automate the process for image association to cases . despite distance , however , with this client - specific model image files are largely outside our control , we can not always quickly resolve problems with the client 's image server , and any network / hardware changes on the client 's side may cause unexpected instability . the general portal allows supported image file types ( static and wsi formats of major vendors ) , as well as pertinent accompanying clinical information , to be uploaded . all clients are offered secure login to submit their patient 's data and upload images , as well as to check on the status of their case and view or print second opinion reports . using this tool , upmc consultants are able to view digital images using a java applet . they have the ability to annotate and capture static image snapshots to be embedded into their reports . workflow is handled by managers who triage requests , monitor cases , and maintain personnel data . streamlined workflow , as well as training of users , has ensured prompt turn - around time and buy - in by institute pathologists . post - launch feedback from pathologists has resulted in customization to incorporate transcription services , peer - to - peer review for consultants , provision for issuing addenda and amendments , and improved wsi viewing experience . all cases received via the portal thus far have been surgical pathology cases . on average , 11 wsi per case have been received including h and e , histochemical and immunohistochemistry slides . different upmc telepathology portals data workflow with the upmc portal and one of their clients ( kingmed in china ) . serve database , this system allows for automated notification of the upmc assigned pathologist that the case is ready for consult . perhaps more importantly , this automated system eliminates a person having to email or notify , which scan filenames are associated with a case , thereby removing the possibility of human error of assigning the wrong slide image to a case digital pathology consultation at upmc has spanned more than a decade . during this time , all modes of telepathology have been successfully utilized to exploit our subspecialty expertise and to compete for pathology services . the practice of telepathology at our institution has evolved in concert with advances in technology , which has become more cost - effective . although several of the aforementioned modes of practicing telepathology may be outdated , we believe it is nevertheless important to share our experiences and point out that very often technology is not the limiting issue , but rather the people and processes involved . of note , diagnostic accuracy and turnaround time telecytology has been accomplished largely using real - time video streaming , and for wsi of cytology slides , the low volume of cases received for teleconsultation have been interpretable without the need for scanning slides with z - stacking . early and continued adoption has promoted several digital pathology resources ( e.g. , facilitated funding for equipment , it infrastructure , and staff ) that are now being leveraged for other clinical , educational , and research purposes . table 2 lists many of the other factors that may need to be considered when practicing telepathology . a key aspect of a successful digital teleconsultation program is integration of digital images ( including wsi ) into the laboratory information system ( lis ) and electronic health record . such integration , for example , was one of the main objectives of the cost action efforts towards integrating the lis with wsi are underway at our institution in partnership with omnyx . technological issues , however , are often easier to overcome than administrative , contractual , and legal challenges our institution is currently evaluating newer approaches such as grid technology , open access forums , and mobile solutions to determine how they can be leveraged to enhance telepathology at our institution . grid technology , which uses open standards to access distributed information , has been suggested by some authors as a way to improve quality in image - based diagnosis . we have provided our pathologists with access to a suite of cloud - based applications through a web browser [ figure 9 ] . since the computer programs that support these various telepathology devices are stored on servers at a remote location , the hardware and software demands placed on the end users are greatly diminished . while the use of commercial web conferencing systems ( e.g. , skype ) has been shown to be feasible for telepathology , in our institutional setting , reliance on public servers raises potential security issues , which has proven to be a barrier for telepathology . more recently , the field of telepathology is witnessing the emergence of specific open access forums ( e.g. these new generation forums , which employ browser - friendly w3c conform standards , offer additional benefits such as acoustic information transfer and assistance in image screening . , cellular phones ) , which are almost ubiquitous today , provides another opportunity to perform telepathology almost anywhere and anytime .

the study design and baseline data of the alert trial have been described previously 12 . in brief , alert was a randomized , double - blind , placebo - controlled study of the effect of fluvastatin ( 4080 mg / d vs. placebo ) on cardiac and renal outcomes in 2102 male and female ktr aged 3075 yr , included from june 1996 to october 1997 . patients had received a renal transplant more than six months previously , had a stable graft function and a total serum cholesterol between 4.0 and 9.0 mm ( 155348 mg / dl ) . exclusion criteria were familial hypercholesterolemia , recent acute rejection episodes , predicted life expectancy of less than one yr or ongoing statin therapy . follow - up was 56 yr in the core study , after which trial participants were offered open - label fluvastatin 80 mg / d in a two - yr extension trial . mean total follow - up time for the extension study was 6.7 yr . prior to unblinding the alert study , neopterin was chosen as one of the pre - specified cardiovascular risk factors to be analyzed . serum neopterin concentration was measured in 30% of patients ( randomly chosen ) by radioimmunoassay ( ibl diagnostics , hamburg , germany ) in samples taken at the time of study entry ( baseline ) , a mean of 5.4 yr after transplantation . the study adhered to the international conference on harmonization guidelines for good clinical practice and was conducted in accordance with the declaration of helsinki principles . all participants provided written informed consent , and the ethics committee at each participating center approved the trial . renal endpoint was the time to graft loss ( rgl ) , defined as return to dialysis or retransplantation . cardiac endpoint was the occurrence of a mace , defined as cardiac death , non - fatal myocardial infarction verified by hospital records , or coronary revascularization procedure , including coronary artery bypass graft or percutaneous coronary intervention . all endpoints were validated by an independent clinical end point committee blinded to study randomization . since treatment and placebo arms of the original study showed no significant heterogeneity in relation to demographics , known cardiovascular or renal risk factors or levels of inflammatory markers 12 , the current analysis was based on the pooled patient population . in comparing baseline characteristics between groups , independent samples t - test and mann whitney u - test for continuous variables and chi - square test for associations between categorical variables were used . spearman s rank correlation was used in checking for statistical associations between neopterin and creatinine , as well as the inflammatory markers il-6 and crp . univariable and multivariable cox proportional hazard models were used to evaluate the influence of possible prognostic variables , including conventional cardiovascular risk factors , other inflammatory markers and factors associated with graft survival . these models were used to examine the association between neopterin and mace , all - cause mortality and graft loss . since high sensitivity crp ( hscrp ) and il-6 are closely linked etiologically and considered to be mutual confounders , they were included as covariates in separate multivariable regression models , the other variables remaining the same . as neopterin is chemically inert and its elimination is solely through the kidneys , compromised renal function leads to a rise in serum neopterin that is not caused by increased inflammatory activity 13 . therefore , as done in the majority of previous studies on neopterin in populations with compromised renal function , neopterin levels were calculated relative to the serum concentration of creatinine , thus adjusting for kidney function . spss version 18 ( ibm corp . , armonk , ny , usa ) was used for all statistical analyses except for generation of figs . 1 and 2 where we used stata version 11 ( statacorp , college station , tx , usa ) . cumulative all - cause mortality according to quartiles of neopterin - to - creatinine ratio . cumulative major cardiovascular events ( mace ) according to quartiles of neopterin - to - creatinine ratio . the study design and baseline data of the alert trial have been described previously 12 . in brief , alert was a randomized , double - blind , placebo - controlled study of the effect of fluvastatin ( 4080 mg / d vs. placebo ) on cardiac and renal outcomes in 2102 male and female ktr aged 3075 yr , included from june 1996 to october 1997 . patients had received a renal transplant more than six months previously , had a stable graft function and a total serum cholesterol between 4.0 and 9.0 mm ( 155348 mg / dl ) . exclusion criteria were familial hypercholesterolemia , recent acute rejection episodes , predicted life expectancy of less than one yr or ongoing statin therapy . follow - up was 56 yr in the core study , after which trial participants were offered open - label fluvastatin 80 mg / d in a two - yr extension trial . mean total follow - up time for the extension study was 6.7 yr . prior to unblinding the alert study , neopterin was chosen as one of the pre - specified cardiovascular risk factors to be analyzed . serum neopterin concentration was measured in 30% of patients ( randomly chosen ) by radioimmunoassay ( ibl diagnostics , hamburg , germany ) in samples taken at the time of study entry ( baseline ) , a mean of 5.4 yr after transplantation . the study adhered to the international conference on harmonization guidelines for good clinical practice and was conducted in accordance with the declaration of helsinki principles . all participants provided written informed consent , and the ethics committee at each participating center approved the trial . renal endpoint was the time to graft loss ( rgl ) , defined as return to dialysis or retransplantation . cardiac endpoint was the occurrence of a mace , defined as cardiac death , non - fatal myocardial infarction verified by hospital records , or coronary revascularization procedure , including coronary artery bypass graft or percutaneous coronary intervention . all endpoints were validated by an independent clinical end point committee blinded to study randomization . since treatment and placebo arms of the original study showed no significant heterogeneity in relation to demographics , known cardiovascular or renal risk factors or levels of inflammatory markers 12 , the current analysis was based on the pooled patient population . in comparing baseline characteristics between groups , independent samples t - test and mann whitney u - test for continuous variables and chi - square test for associations between categorical variables were used . spearman s rank correlation was used in checking for statistical associations between neopterin and creatinine , as well as the inflammatory markers il-6 and crp . univariable and multivariable cox proportional hazard models were used to evaluate the influence of possible prognostic variables , including conventional cardiovascular risk factors , other inflammatory markers and factors associated with graft survival . these models were used to examine the association between neopterin and mace , all - cause mortality and graft loss . since high sensitivity crp ( hscrp ) and il-6 are closely linked etiologically and considered to be mutual confounders , they were included as covariates in separate multivariable regression models , the other variables remaining the same . as neopterin is chemically inert and its elimination is solely through the kidneys , compromised renal function leads to a rise in serum neopterin that is not caused by increased inflammatory activity 13 . therefore , as done in the majority of previous studies on neopterin in populations with compromised renal function , neopterin levels were calculated relative to the serum concentration of creatinine , thus adjusting for kidney function . spss version 18 ( ibm corp . , armonk , ny , usa ) was used for all statistical analyses except for generation of figs . 1 and 2 where we used stata version 11 ( statacorp , college station , tx , usa ) . cumulative all - cause mortality according to quartiles of neopterin - to - creatinine ratio . cumulative major cardiovascular events ( mace ) according to quartiles of neopterin - to - creatinine ratio . table 1 lists baseline characteristics for the alert participants , comparing those with and without available baseline neopterin data . demographics , risk factors , and neopterin levels were similar in the fluvastatin and placebo arms ( not shown ) . since there were no clinically important differences , neither between the two treatment arms nor between the overall study population and those for whom neopterin levels were available demographic and baseline data for patients with or without measurement of neopterin continuous variables are shown as mean ( sd ) ; categorical variables as n ( % ) . hdl , high - density lipoprotein ; ldl , low - density lipoprotein ; hscrp , high sensitivity crp ; il-6 , interleukin-6 . the correlation coefficient between neopterin and creatinine was 0.61 . for purposes of survival analysis , we categorized patients into quartiles according to their neopterin - to - creatinine ratios at baseline . levels ranged from 33 to 325 mol / mol . in table 2 , demographic data and background risk factors are presented for each quartile of neopterin / creatinine . there was a tendency towards higher proportions of patients with hypertension , chronic heart disease , panel reactive antibodies , delayed graft function ( dgf ) , longer time on dialysis prior to transplantation and treatment for cmv - infection / reactivation in the highest or the two highest quartiles . demographic and baseline data according to quartiles of neopterin - to - creatinine ratio continuous variables are shown as mean ( sd ) ; categorical variables as n ( % ) . hdl , high - density lipoprotein ; ldl , low - density lipoprotein ; hscrp , high sensitivity crp ; il-6 , interleukin-6 . the proportions of patients reaching the renal , cardiac and mortality endpoints in each quartile for neopterin - to - creatinine ratio are listed in table 3 . log - rank tests were used to check the significance of differences between each of the three upper quartiles compared with the lowest . event occurrence in stable renal transplant patients according to neopterin / creatinine quartiles data expressed as number of patients with the event in each quartile ( % ) . the rate of death from all causes increased in higher neopterin / creatinine quartiles , and the differences were statistically significant between all three upper quartiles and the first quartile . the number of events more than trebled from the first to the fourth quartile ( 9.533.6% ) . for mace , the incidence rate was more than twice as high ( 11.125.0% ) in the fourth neopterin / creatinine quartile compared with the first one , and the difference was statistically significant . for renal graft loss the incidence rates was slightly higher in the fourth quartile , but no statistical significance was reached . figs . meier failure estimates plots for all - cause mortality and mace showing time to event , or end of follow - up , by neopterin / creatinine quartile . table 4 shows risk factor evaluation using univariable and multivariable models of cox regression analyses . for all study outcomes we present hr with 95% confidence intervals ( 95% ci ) and their respective p - values . in the univariable model , baseline neopterin expressed as neopterin - to - creatinine ratio , as well as potential risk factors and baseline demographics , the multivariable model is adjusted for the following baseline covariates : age , gender , smoking habit , diagnosis of coronary heart disease , ldl - cholesterol , systolic blood pressure , diabetes mellitus , serum creatinine , and proteinuria . il-6 and hscrp were included in separate models , as they are part of the same etiological inflammatory pathway and thus have a high colinearity . results are shown only for the analysis including hscrp , as the hazard ratio for neopterin was virtually identical in the two models . hazard ratios for neopterin - to - creatinine ratio ( per 10 units in mol / mol ) with covariates in relation to outcomes in 628 stable renal transplant patients by univariable and multivariable cox regression analysis the multivariable analysis adjusts for relevant demographic covariates ( age , gender ) , known renal / cardiovascular risk factors ( smoking , coronary heart disease , ldl - cholesterol , systolic blood pressure , diabetes mellitus , creatinine , level of proteinuria ) , and the inflammation marker hscrp . ci , confidence interval ; hdl , high - density lipoprotein ; hr , hazard ration ; hscrp , high sensitivity crp ; ldl , low - density lipoprotein ; mace , major adverse cardiac event . in the univariable model , after adjustment for other established and potentially important risk factors , we found neopterin ( in mol / mol creatinine ) to have a significant positive association with all - cause of death ( hr 1.06 per 10 units , p = 0.002 , 95% ci 1.021.09 ) and mace ( hr 1.06 per 10 units , p = 0.009 , 95% ci 1.011.10 ) but no independent predictive value for graft loss . hr and p - values for neopterin remained the same for all endpoints when randomization group was included in the multivariate model ( not shown ) . though reaching significance for mace in the univariable analyses , hscrp was not independently associated with any of the study endpoints in the multivariable analyses , nor was il-6 . using spearman s rank correlation , we found the correlation coefficient between neopterin and il-6 to be 0.26 , p < 0.001 , while for neopterin and crp it was 0.14 , p < 0.001 . among the remaining risk factors entered into the model , diabetes mellitus was strongly associated with all outcomes , as was current smoking for all outcomes but mace . as might have been expected , coronary heart disease was predictive of future mace and all - cause mortality , while serum creatinine and level of proteinuria was significantly associated with the renal endpoint . neopterin was also included in a multivariate model with osteoprotegrin , asymmetric dimethylarginine ( adma ) , and symmetric dimethylarginine ( sdma ) to assess its independency of other markers related to inflammation and endothelial function . no relevant change in hr was seen , and the association with mace and all - cause death remained highly significant ( data not shown ) . table 1 lists baseline characteristics for the alert participants , comparing those with and without available baseline neopterin data . demographics , risk factors , and neopterin levels were similar in the fluvastatin and placebo arms ( not shown ) . since there were no clinically important differences , neither between the two treatment arms nor between the overall study population and those for whom neopterin levels were available demographic and baseline data for patients with or without measurement of neopterin continuous variables are shown as mean ( sd ) ; categorical variables as n ( % ) . hdl , high - density lipoprotein ; ldl , low - density lipoprotein ; hscrp , high sensitivity crp ; il-6 , interleukin-6 . the correlation coefficient between neopterin and creatinine was 0.61 . for purposes of survival analysis , we categorized patients into quartiles according to their neopterin - to - creatinine ratios at baseline . levels ranged from 33 to 325 mol / mol . in table 2 , demographic data and background risk factors are presented for each quartile of neopterin / creatinine . there was a tendency towards higher proportions of patients with hypertension , chronic heart disease , panel reactive antibodies , delayed graft function ( dgf ) , longer time on dialysis prior to transplantation and treatment for cmv - infection / reactivation in the highest or the two highest quartiles . demographic and baseline data according to quartiles of neopterin - to - creatinine ratio continuous variables are shown as mean ( sd ) ; categorical variables as n ( % ) . hdl , high - density lipoprotein ; ldl , low - density lipoprotein ; hscrp , high sensitivity crp ; il-6 , interleukin-6 . the proportions of patients reaching the renal , cardiac and mortality endpoints in each quartile for neopterin - to - creatinine ratio are listed in table 3 . log - rank tests were used to check the significance of differences between each of the three upper quartiles compared with the lowest . event occurrence in stable renal transplant patients according to neopterin / creatinine quartiles data expressed as number of patients with the event in each quartile ( % ) . the rate of death from all causes increased in higher neopterin / creatinine quartiles , and the differences were statistically significant between all three upper quartiles and the first quartile . the number of events more than trebled from the first to the fourth quartile ( 9.533.6% ) . for mace , the incidence rate was more than twice as high ( 11.125.0% ) in the fourth neopterin / creatinine quartile compared with the first one , and the difference was statistically significant . for renal graft loss the incidence rates was slightly higher in the fourth quartile , but no statistical significance was reached . meier failure estimates plots for all - cause mortality and mace showing time to event , or end of follow - up , by neopterin / creatinine quartile . table 4 shows risk factor evaluation using univariable and multivariable models of cox regression analyses . for all study outcomes we present hr with 95% confidence intervals ( 95% ci ) and their respective p - values . in the univariable model , baseline neopterin expressed as neopterin - to - creatinine ratio , as well as potential risk factors and baseline demographics , the multivariable model is adjusted for the following baseline covariates : age , gender , smoking habit , diagnosis of coronary heart disease , ldl - cholesterol , systolic blood pressure , diabetes mellitus , serum creatinine , and proteinuria . il-6 and hscrp were included in separate models , as they are part of the same etiological inflammatory pathway and thus have a high colinearity . results are shown only for the analysis including hscrp , as the hazard ratio for neopterin was virtually identical in the two models . hazard ratios for neopterin - to - creatinine ratio ( per 10 units in mol / mol ) with covariates in relation to outcomes in 628 stable renal transplant patients by univariable and multivariable cox regression analysis the multivariable analysis adjusts for relevant demographic covariates ( age , gender ) , known renal / cardiovascular risk factors ( smoking , coronary heart disease , ldl - cholesterol , systolic blood pressure , diabetes mellitus , creatinine , level of proteinuria ) , and the inflammation marker hscrp . ci , confidence interval ; hdl , high - density lipoprotein ; hr , hazard ration ; hscrp , high sensitivity crp ; ldl , low - density lipoprotein ; mace , major adverse cardiac event . in the univariable model , after adjustment for other established and potentially important risk factors , we found neopterin ( in mol / mol creatinine ) to have a significant positive association with all - cause of death ( hr 1.06 per 10 units , p = 0.002 , 95% ci 1.021.09 ) and mace ( hr 1.06 per 10 units , p = 0.009 , 95% ci 1.011.10 ) but no independent predictive value for graft loss . hr and p - values for neopterin remained the same for all endpoints when randomization group was included in the multivariate model ( not shown ) . though reaching significance for mace in the univariable analyses , hscrp was not independently associated with any of the study endpoints in the multivariable analyses , nor was il-6 . using spearman s rank correlation , we found the correlation coefficient between neopterin and il-6 to be 0.26 , p < 0.001 , while for neopterin and crp it was 0.14 , p < 0.001 . among the remaining risk factors entered into the model , diabetes mellitus was strongly associated with all outcomes , as was current smoking for all outcomes but mace . as might have been expected , coronary heart disease was predictive of future mace and all - cause mortality , while serum creatinine and level of proteinuria was significantly associated with the renal endpoint . neopterin was also included in a multivariate model with osteoprotegrin , asymmetric dimethylarginine ( adma ) , and symmetric dimethylarginine ( sdma ) to assess its independency of other markers related to inflammation and endothelial function . no relevant change in hr was seen , and the association with mace and all - cause death remained highly significant ( data not shown ) . in this analysis of a large cohort of ktr from the alert study , we have shown that the inflammatory marker neopterin is significantly associated with cardiovascular events and all cause mortality in ktr , even after adjustment for conventional and new risk factors . in patients with pre - dialysis chronic kidney disease ( ckd ) , serum neopterin is elevated and significantly correlated with markers of inflammation including hscrp , il-6 , and ifn-. 15 . we have shown previously that levels of il-6 and hscrp are associated with cardiovascular endpoints and all - cause mortality following kidney transplantation 4,16 . however , and of central importance , this study shows that in ktr the predictive power of neopterin was maintained after adjustment for hscrp and il-6 . in the multivariable analyses including neopterin , crp and in addition , though statistically significant , the correlations between neopterin and these two inflammation markers were weak ones . our epidemiological data on the predictive value of neopterin are in line with previous literature showing that neopterin rises quickly after macrophage activation 17 , is an excellent marker for longer term activation of cellular immunity during the maintenance phase , and appear to remain relatively stable over time 18 . inflammation is a key element of the development of atherosclerosis , with t - lymphocytes and monocyte - derived macrophages being detected in atherosclerotic lesions . in accordance with our results , studies have highlighted neopterin as a useful marker for long - term risk of all - cause and cardiovascular death in patients from diverse populations , including individuals undergoing coronary angiography 19 , patients with stable coronary artery disease 20 , newly diagnosed diabetics 21 , and dialysis - dependent ckd patients 22 . furthermore , it has recently been shown that elevated neopterin , but not crp level , predicts left ventricular dysfunction in patients with chronic stable angina pectoris 23 . a recent report from the hordaland study demonstrated that in elderly patients , without pre - existing coronary heart disease , higher levels of neopterin are associated with an increased risk of subsequent coronary events 24 . chronic low - grade inflammation is one of the main conditions associated with increased cardiovascular morbidity and mortality in patients with ckd , especially those on dialysis 25 . thus , it is not surprising that persistent inflammation , endothelial dysfunction , and associated oxidative stress in ktr 26 is reflected in progression of atherosclerosis 27 and adversely affects cardiovascular outcomes 4 . the significant association between neopterin and outcomes was maintained even after adjustment for markers of endothelial function ( sdma , adma ) . immunologic responses to allografts involve humoral and cellular components of both the adaptive and innate immune system , the t - cell playing a pivotal role in the initial recognition of anti - self 28 . the stronger association that we found between neopterin and the clinical endpoints than for other inflammatory markers may possibly reflect the dominance of t - cell and macrophage activation in the ongoing inflammatory status of allograft recipients . consistent with this , baseline neopterin values were substantially higher in our cohort compared with the general population 29 and patients with known cv disease 18 . this is in harmony with earlier findings on clinically stable ktr 30,31 . in one of the earliest publications to assess levels of neopterin in ktr 32 , margreiter et al . measured urinary neopterin daily during the early post - operative period and found that acute rejection and early viral infection were preceded by a rising or high level of urinary neopterin in 95% and 100% of cases , respectively . they later extended their data to include urinary neopterin measurements in 294 kidney allograft recipients 33 . subsequent studies by others have confirmed that elevation of serum or urinary neopterin precedes the rise in creatinine by up to several days in patients with acute early complications 34,35 , and routine daily post - operative neopterin measurements have been suggested for the early detection of immunologic complications in kidney allograft recipients 36 . in the trial 33 conducted by reibnegger and colleagues , a high neopterin level in the early post - transplant period was associated with a higher risk of graft failure in the long term . in a smaller cohort of patients , 37 observed that elevated neopterin levels following transplantation were associated with inferior graft and patient survival , while a recent prospective study in 216 ktr showed an association between higher levels of neopterin and acute rejection in the first year of follow - up 38 . our population was recruited five yr after transplantation and was clinically stable with a reasonably good renal function . elevated neopterin was not significantly associated with renal graft loss after adjusting for level of proteinuria . while grebe et al . 37 were primarily interested in early post - transplant macrophage activation associated with elevated neopterin , our study suggests that in clinically stable allograft recipients , serum neopterin is not independently associated with renal graft failure and does not add significantly to the prognostic information given by the degree of proteinuria . 39,40 reported significant associations between neopterin concentration at one yr post - transplant and the development of chronic rejection and chronic allograft dysfunction within two yr , but proteinuria was not included in their multivariable analysis , possibly explaining this discrepancy . a small study on children with primary nephrotic syndrome 41 showed a positive correlation between serum neopterin levels and proteinuria in patients with active disease . a link between neopterin levels and the progression rate in proteinuric diabetic nephropathy has been demonstrated 42 , and one group 43 observed marked differences in serum and urine neopterin levels among diabetic patients with and without microalbuminuria . we are not proposing that enhanced macrophage activity is not an important factor in the development of a dysfunctional renal allograft , but the potential clinical value of neopterin with respect to graft function may be limited as long as proteinuria is considered a well established risk factor for poorer long - term renal outcomes in kidney allograft recipients 44,45 . reference ranges for neopterin are higher in the healthy elderly population ( > 75 yr ) . several studies report rising neopterin levels from the age of 6070 46 or even earlier 47 , probably reflecting the involvement of cellular immunity in the aging process , as well as the presence of low - grade inflammatory processes such as atherosclerosis , neurodegeneration , or unrecognized evolving disease ( autoimmunity or malignancy ) 29 . however , we did not find significant differences in baseline neopterin between different age groups , perhaps because kidney transplants are not performed for the oldest ckd patients ( recipients in our cohort were aged 23.674.8 yr ) , and because comorbidity , the immunological consequences of long - standing uremia and the anti - allograft immune response may overshadow the component of neopterin production related to aging . the strengths of this analysis are the prospective controlled design , the long follow - up time , the large patient cohort and the independent adjudication of all clinical endpoints . although the results show a strong association between neopterin and clinical outcomes , the data do not prove a causal relationship . residual confounding can not be ruled out , though we have carefully adjusted for a wide range of covariates in our statistical models . although neopterin may , at present , not be a clinically useful single parameter for risk prediction in ktr , it is conceivable that neopterin could be valuable for multi - marker risk prediction or for the evaluation of the clinical efficacy of established treatments in this patient group . as in most large prospective trials , a cohort selected for entry into a clinical trial , is not necessarily fully representative of the general renal transplant population , although the qualitative relationships between neopterin and the specified clinical outcomes are likely to apply , at least for caucasians . mean neopterin values were not different for the two randomization groups , and there was no significant skewing in the proportion of patients receiving fluvastatin in each quartile of neopterin - to - creatinine ratio . entering treatment group as a covariate in the multiple regression analysis did not change the hazard ratio for neopterin . in conclusion , in clinically stable renal transplant recipients there appears to be an independent association between serum neopterin concentration and long - term clinical outcomes of mace and all - cause mortality . hege pihlstrm : data collection , data analysis / interpretation , statistics , drafting the article . hallvard holdaas : concept / design , study protocol , data collection , drafting and critical revision of the article . bengt fellstrm and alan jardine : concept / design , study protocol , data collection , critical revision of the article . geir mjen , ingar holme , sadollah abedini , and dag olav dahle : data analysis / interpretation , statistics , critical revision of the article . winfried mrz and stephan pilz : data analysis / interpretation , drafting and critical revision of the article .

backgroundinflammatory markers show significant associations with cardiovascular events and all - cause mortality after kidney transplantation . neopterin , reflecting interferon--release , may better reflect the proinflammatory state of recipients than less specific markers.methodskidney transplant recipients in the assessment of lescol in renal transplant ( alert ) trial were examined and investigated for an association between serum neopterin and subsequent clinical events : graft loss , major cardiovascular events ( mace ) and all - cause mortality.resultsafter adjustment for established and emerging risk factors neopterin expressed as neopterin - to - creatinine ratio was significantly associated with mace ( p = 0.009 ) and all - cause mortality ( p = 0.002 ) . endpoints were more frequent with increasing quartiles of neopterin - to - creatinine ratio . the incidence rates of mace and all - cause mortality were significantly increased in the upper quartiles compared with the first.conclusionsthis long - term prospective analysis in stable kidney allograft recipients suggests that neopterin is associated with long - term risk of cardiovascular events and all - cause mortality , but not renal outcomes .

Patients and methods Study design Outcome definitions Statistical analysis Neopterin estimates Results Baseline characteristics Outcomes Multiple risk factor analysis Discussion Authors contributions

in brief , alert was a randomized , double - blind , placebo - controlled study of the effect of fluvastatin ( 4080 mg / d vs. placebo ) on cardiac and renal outcomes in 2102 male and female ktr aged 3075 yr , included from june 1996 to october 1997 . patients had received a renal transplant more than six months previously , had a stable graft function and a total serum cholesterol between 4.0 and 9.0 mm ( 155348 mg / dl ) . prior to unblinding the alert study , neopterin was chosen as one of the pre - specified cardiovascular risk factors to be analyzed . the study adhered to the international conference on harmonization guidelines for good clinical practice and was conducted in accordance with the declaration of helsinki principles . renal endpoint was the time to graft loss ( rgl ) , defined as return to dialysis or retransplantation . all endpoints were validated by an independent clinical end point committee blinded to study randomization . univariable and multivariable cox proportional hazard models were used to evaluate the influence of possible prognostic variables , including conventional cardiovascular risk factors , other inflammatory markers and factors associated with graft survival . these models were used to examine the association between neopterin and mace , all - cause mortality and graft loss . since high sensitivity crp ( hscrp ) and il-6 are closely linked etiologically and considered to be mutual confounders , they were included as covariates in separate multivariable regression models , the other variables remaining the same . as neopterin is chemically inert and its elimination is solely through the kidneys , compromised renal function leads to a rise in serum neopterin that is not caused by increased inflammatory activity 13 . therefore , as done in the majority of previous studies on neopterin in populations with compromised renal function , neopterin levels were calculated relative to the serum concentration of creatinine , thus adjusting for kidney function . cumulative all - cause mortality according to quartiles of neopterin - to - creatinine ratio . cumulative major cardiovascular events ( mace ) according to quartiles of neopterin - to - creatinine ratio . in brief , alert was a randomized , double - blind , placebo - controlled study of the effect of fluvastatin ( 4080 mg / d vs. placebo ) on cardiac and renal outcomes in 2102 male and female ktr aged 3075 yr , included from june 1996 to october 1997 . serum neopterin concentration was measured in 30% of patients ( randomly chosen ) by radioimmunoassay ( ibl diagnostics , hamburg , germany ) in samples taken at the time of study entry ( baseline ) , a mean of 5.4 yr after transplantation . the study adhered to the international conference on harmonization guidelines for good clinical practice and was conducted in accordance with the declaration of helsinki principles . all endpoints were validated by an independent clinical end point committee blinded to study randomization . univariable and multivariable cox proportional hazard models were used to evaluate the influence of possible prognostic variables , including conventional cardiovascular risk factors , other inflammatory markers and factors associated with graft survival . these models were used to examine the association between neopterin and mace , all - cause mortality and graft loss . since high sensitivity crp ( hscrp ) and il-6 are closely linked etiologically and considered to be mutual confounders , they were included as covariates in separate multivariable regression models , the other variables remaining the same . as neopterin is chemically inert and its elimination is solely through the kidneys , compromised renal function leads to a rise in serum neopterin that is not caused by increased inflammatory activity 13 . therefore , as done in the majority of previous studies on neopterin in populations with compromised renal function , neopterin levels were calculated relative to the serum concentration of creatinine , thus adjusting for kidney function . cumulative all - cause mortality according to quartiles of neopterin - to - creatinine ratio . cumulative major cardiovascular events ( mace ) according to quartiles of neopterin - to - creatinine ratio . demographics , risk factors , and neopterin levels were similar in the fluvastatin and placebo arms ( not shown ) . for purposes of survival analysis , we categorized patients into quartiles according to their neopterin - to - creatinine ratios at baseline . in table 2 , demographic data and background risk factors are presented for each quartile of neopterin / creatinine . demographic and baseline data according to quartiles of neopterin - to - creatinine ratio continuous variables are shown as mean ( sd ) ; categorical variables as n ( % ) . the proportions of patients reaching the renal , cardiac and mortality endpoints in each quartile for neopterin - to - creatinine ratio are listed in table 3 . log - rank tests were used to check the significance of differences between each of the three upper quartiles compared with the lowest . event occurrence in stable renal transplant patients according to neopterin / creatinine quartiles data expressed as number of patients with the event in each quartile ( % ) . the rate of death from all causes increased in higher neopterin / creatinine quartiles , and the differences were statistically significant between all three upper quartiles and the first quartile . the number of events more than trebled from the first to the fourth quartile ( 9.533.6% ) . for mace , the incidence rate was more than twice as high ( 11.125.0% ) in the fourth neopterin / creatinine quartile compared with the first one , and the difference was statistically significant . for renal graft loss the incidence rates was slightly higher in the fourth quartile , but no statistical significance was reached . meier failure estimates plots for all - cause mortality and mace showing time to event , or end of follow - up , by neopterin / creatinine quartile . in the univariable model , baseline neopterin expressed as neopterin - to - creatinine ratio , as well as potential risk factors and baseline demographics , the multivariable model is adjusted for the following baseline covariates : age , gender , smoking habit , diagnosis of coronary heart disease , ldl - cholesterol , systolic blood pressure , diabetes mellitus , serum creatinine , and proteinuria . hazard ratios for neopterin - to - creatinine ratio ( per 10 units in mol / mol ) with covariates in relation to outcomes in 628 stable renal transplant patients by univariable and multivariable cox regression analysis the multivariable analysis adjusts for relevant demographic covariates ( age , gender ) , known renal / cardiovascular risk factors ( smoking , coronary heart disease , ldl - cholesterol , systolic blood pressure , diabetes mellitus , creatinine , level of proteinuria ) , and the inflammation marker hscrp . ci , confidence interval ; hdl , high - density lipoprotein ; hr , hazard ration ; hscrp , high sensitivity crp ; ldl , low - density lipoprotein ; mace , major adverse cardiac event . in the univariable model , after adjustment for other established and potentially important risk factors , we found neopterin ( in mol / mol creatinine ) to have a significant positive association with all - cause of death ( hr 1.06 per 10 units , p = 0.002 , 95% ci 1.021.09 ) and mace ( hr 1.06 per 10 units , p = 0.009 , 95% ci 1.011.10 ) but no independent predictive value for graft loss . though reaching significance for mace in the univariable analyses , hscrp was not independently associated with any of the study endpoints in the multivariable analyses , nor was il-6 . using spearman s rank correlation , we found the correlation coefficient between neopterin and il-6 to be 0.26 , p < 0.001 , while for neopterin and crp it was 0.14 , p < 0.001 . among the remaining risk factors entered into the model , diabetes mellitus was strongly associated with all outcomes , as was current smoking for all outcomes but mace . as might have been expected , coronary heart disease was predictive of future mace and all - cause mortality , while serum creatinine and level of proteinuria was significantly associated with the renal endpoint . no relevant change in hr was seen , and the association with mace and all - cause death remained highly significant ( data not shown ) . demographics , risk factors , and neopterin levels were similar in the fluvastatin and placebo arms ( not shown ) . since there were no clinically important differences , neither between the two treatment arms nor between the overall study population and those for whom neopterin levels were available demographic and baseline data for patients with or without measurement of neopterin continuous variables are shown as mean ( sd ) ; categorical variables as n ( % ) . for purposes of survival analysis , we categorized patients into quartiles according to their neopterin - to - creatinine ratios at baseline . in table 2 , demographic data and background risk factors are presented for each quartile of neopterin / creatinine . there was a tendency towards higher proportions of patients with hypertension , chronic heart disease , panel reactive antibodies , delayed graft function ( dgf ) , longer time on dialysis prior to transplantation and treatment for cmv - infection / reactivation in the highest or the two highest quartiles . demographic and baseline data according to quartiles of neopterin - to - creatinine ratio continuous variables are shown as mean ( sd ) ; categorical variables as n ( % ) . the proportions of patients reaching the renal , cardiac and mortality endpoints in each quartile for neopterin - to - creatinine ratio are listed in table 3 . log - rank tests were used to check the significance of differences between each of the three upper quartiles compared with the lowest . event occurrence in stable renal transplant patients according to neopterin / creatinine quartiles data expressed as number of patients with the event in each quartile ( % ) . the rate of death from all causes increased in higher neopterin / creatinine quartiles , and the differences were statistically significant between all three upper quartiles and the first quartile . the number of events more than trebled from the first to the fourth quartile ( 9.533.6% ) . for mace , the incidence rate was more than twice as high ( 11.125.0% ) in the fourth neopterin / creatinine quartile compared with the first one , and the difference was statistically significant . for renal graft loss the incidence rates was slightly higher in the fourth quartile , but no statistical significance was reached . meier failure estimates plots for all - cause mortality and mace showing time to event , or end of follow - up , by neopterin / creatinine quartile . in the univariable model , baseline neopterin expressed as neopterin - to - creatinine ratio , as well as potential risk factors and baseline demographics , the multivariable model is adjusted for the following baseline covariates : age , gender , smoking habit , diagnosis of coronary heart disease , ldl - cholesterol , systolic blood pressure , diabetes mellitus , serum creatinine , and proteinuria . hazard ratios for neopterin - to - creatinine ratio ( per 10 units in mol / mol ) with covariates in relation to outcomes in 628 stable renal transplant patients by univariable and multivariable cox regression analysis the multivariable analysis adjusts for relevant demographic covariates ( age , gender ) , known renal / cardiovascular risk factors ( smoking , coronary heart disease , ldl - cholesterol , systolic blood pressure , diabetes mellitus , creatinine , level of proteinuria ) , and the inflammation marker hscrp . in the univariable model , after adjustment for other established and potentially important risk factors , we found neopterin ( in mol / mol creatinine ) to have a significant positive association with all - cause of death ( hr 1.06 per 10 units , p = 0.002 , 95% ci 1.021.09 ) and mace ( hr 1.06 per 10 units , p = 0.009 , 95% ci 1.011.10 ) but no independent predictive value for graft loss . though reaching significance for mace in the univariable analyses , hscrp was not independently associated with any of the study endpoints in the multivariable analyses , nor was il-6 . using spearman s rank correlation , we found the correlation coefficient between neopterin and il-6 to be 0.26 , p < 0.001 , while for neopterin and crp it was 0.14 , p < 0.001 . among the remaining risk factors entered into the model , diabetes mellitus was strongly associated with all outcomes , as was current smoking for all outcomes but mace . as might have been expected , coronary heart disease was predictive of future mace and all - cause mortality , while serum creatinine and level of proteinuria was significantly associated with the renal endpoint . no relevant change in hr was seen , and the association with mace and all - cause death remained highly significant ( data not shown ) . in this analysis of a large cohort of ktr from the alert study , we have shown that the inflammatory marker neopterin is significantly associated with cardiovascular events and all cause mortality in ktr , even after adjustment for conventional and new risk factors . in patients with pre - dialysis chronic kidney disease ( ckd ) , serum neopterin is elevated and significantly correlated with markers of inflammation including hscrp , il-6 , and ifn-. we have shown previously that levels of il-6 and hscrp are associated with cardiovascular endpoints and all - cause mortality following kidney transplantation 4,16 . however , and of central importance , this study shows that in ktr the predictive power of neopterin was maintained after adjustment for hscrp and il-6 . in the multivariable analyses including neopterin , crp and in addition , though statistically significant , the correlations between neopterin and these two inflammation markers were weak ones . our epidemiological data on the predictive value of neopterin are in line with previous literature showing that neopterin rises quickly after macrophage activation 17 , is an excellent marker for longer term activation of cellular immunity during the maintenance phase , and appear to remain relatively stable over time 18 . in accordance with our results , studies have highlighted neopterin as a useful marker for long - term risk of all - cause and cardiovascular death in patients from diverse populations , including individuals undergoing coronary angiography 19 , patients with stable coronary artery disease 20 , newly diagnosed diabetics 21 , and dialysis - dependent ckd patients 22 . furthermore , it has recently been shown that elevated neopterin , but not crp level , predicts left ventricular dysfunction in patients with chronic stable angina pectoris 23 . a recent report from the hordaland study demonstrated that in elderly patients , without pre - existing coronary heart disease , higher levels of neopterin are associated with an increased risk of subsequent coronary events 24 . the significant association between neopterin and outcomes was maintained even after adjustment for markers of endothelial function ( sdma , adma ) . immunologic responses to allografts involve humoral and cellular components of both the adaptive and innate immune system , the t - cell playing a pivotal role in the initial recognition of anti - self 28 . the stronger association that we found between neopterin and the clinical endpoints than for other inflammatory markers may possibly reflect the dominance of t - cell and macrophage activation in the ongoing inflammatory status of allograft recipients . consistent with this , baseline neopterin values were substantially higher in our cohort compared with the general population 29 and patients with known cv disease 18 . in one of the earliest publications to assess levels of neopterin in ktr 32 , margreiter et al . they later extended their data to include urinary neopterin measurements in 294 kidney allograft recipients 33 . subsequent studies by others have confirmed that elevation of serum or urinary neopterin precedes the rise in creatinine by up to several days in patients with acute early complications 34,35 , and routine daily post - operative neopterin measurements have been suggested for the early detection of immunologic complications in kidney allograft recipients 36 . in the trial 33 conducted by reibnegger and colleagues , a high neopterin level in the early post - transplant period was associated with a higher risk of graft failure in the long term . in a smaller cohort of patients , 37 observed that elevated neopterin levels following transplantation were associated with inferior graft and patient survival , while a recent prospective study in 216 ktr showed an association between higher levels of neopterin and acute rejection in the first year of follow - up 38 . elevated neopterin was not significantly associated with renal graft loss after adjusting for level of proteinuria . 37 were primarily interested in early post - transplant macrophage activation associated with elevated neopterin , our study suggests that in clinically stable allograft recipients , serum neopterin is not independently associated with renal graft failure and does not add significantly to the prognostic information given by the degree of proteinuria . 39,40 reported significant associations between neopterin concentration at one yr post - transplant and the development of chronic rejection and chronic allograft dysfunction within two yr , but proteinuria was not included in their multivariable analysis , possibly explaining this discrepancy . a small study on children with primary nephrotic syndrome 41 showed a positive correlation between serum neopterin levels and proteinuria in patients with active disease . we are not proposing that enhanced macrophage activity is not an important factor in the development of a dysfunctional renal allograft , but the potential clinical value of neopterin with respect to graft function may be limited as long as proteinuria is considered a well established risk factor for poorer long - term renal outcomes in kidney allograft recipients 44,45 . reference ranges for neopterin are higher in the healthy elderly population ( > 75 yr ) . however , we did not find significant differences in baseline neopterin between different age groups , perhaps because kidney transplants are not performed for the oldest ckd patients ( recipients in our cohort were aged 23.674.8 yr ) , and because comorbidity , the immunological consequences of long - standing uremia and the anti - allograft immune response may overshadow the component of neopterin production related to aging . although the results show a strong association between neopterin and clinical outcomes , the data do not prove a causal relationship . as in most large prospective trials , a cohort selected for entry into a clinical trial , is not necessarily fully representative of the general renal transplant population , although the qualitative relationships between neopterin and the specified clinical outcomes are likely to apply , at least for caucasians . mean neopterin values were not different for the two randomization groups , and there was no significant skewing in the proportion of patients receiving fluvastatin in each quartile of neopterin - to - creatinine ratio . entering treatment group as a covariate in the multiple regression analysis did not change the hazard ratio for neopterin . in conclusion , in clinically stable renal transplant recipients there appears to be an independent association between serum neopterin concentration and long - term clinical outcomes of mace and all - cause mortality .

neisseria gonorrheae ( ng ) and neisseria meningitides are gram - negative bacterial pathogens responsible for gonorrhea and meningococcal meningitis , respectively . for these bacteria , phagocytosis , cellular invasion , is induced by the binding of opacity - associated ( opa ) proteins to host receptors . opa proteins are eight - stranded -barrel integral outer membrane proteins with four extracellular loops ( figure 1a ) . high sequence diversity is observed in regions of the extracellular loops of opa variants ( table s1 , supporting information ) , which is predominantly a result of recombination events between genes of the same isolate ( 70% ) and import of genes from other isolates ( 16% ) . the three variable regions within the extracellular loops , hypervariable 1 and 2 ( hv1 and hv2 ) and the semivariable ( sv ) regions , engage host receptors to induce phagocytosis and determine the specific host receptors engaged . the regions vary in length and do not comprise the entire extracellular loop : sv is 310 amino acids , hv1 is 2431 amino acids , and hv2 is 4551 amino acids . there are 26 sv , 96 hv1 , and 127 hv2 different sequences in the 338 distinct opa alleles sequenced ( http://www.neisseria.org ) . this sequence diversity likely plays a beneficial role in helping neisseria to evade host immune responses ; however , it poses a challenge in that highly varied loop sequences must engage a common set of receptors to mediate cellular invasion . the determinants of opa receptor interactions are of the utmost importance for understanding neisserial pathogenesis and the innate immune response . opa proteins also provide a means of foreign body cellular entry through specific human receptors that can be exploited synthetically for pharmaceutical and technological purposes . opahs bind to heparansulfate proteoglycans ( hspgs ) directly and indirectly to integrin receptors via an hspg - mediated interaction . the more abundant class , opacea , bind to the nonglycosylated face of the carcinoembryonic antigen - related cellular adhesion molecule ( ceacam ) ig n - domain . although all ceacam receptors contain this domain , opa proteins only bind to ceacam1 , 3 , 5 , and 6 , and most selectively bind to only a subset . the ceacam n - domain residues that interact with opacea have been identified : y34 and i91 are essential for all opacea interactions , and an additional seven nearby residues are implicated in binding depending on the particular opacea . the specificity - determining residues on opacea are predominantly in the hv1 and 2 regions , and the hv sequences are concomitant : chimeric opa proteins with an hv1 and hv2 region from two opa proteins that bind the same receptor do not bind . toward understanding the molecular recognition required to gain entry into human cells , we report the structure of opa60 , which binds ceacam1 , 3 , 5 , and 6 . structure determination of membrane proteins is challenging and even more so for proteins that have large portions of both soluble and membrane - embedded regions . thus , opa proteins presented some methodological obstacles in both nmr resonance assignments and structure calculation and refinement . to overcome these obstacles , we employed a hybrid method that used the restraints determined with solution nuclear magnetic resonance ( nmr ) spectroscopy in detergent micelles in conjunction with molecular dynamics ( md ) simulations in a lipid bilayer . this approach preserved the structural features that were well - determined spectroscopically but employed a more physical sampling method ( molecular dynamics versus simulated annealing ) and more detailed treatment of solvation and electrostatics to better define regions that either are flexible or remained underdetermined from the spectroscopic data alone . opa60 is a canonical eight - stranded -barrel with extensive ionic interactions inside the barrel . three of the extracellular loops are longer than those found in any -barrel structure previously determined . the hv regions within these loops are dynamic on the nanosecond time scale and are predominantly disordered . however , the loops are compact and interact with each other weakly such that long - lived specific intraloop interactions are not observed . the diverse and dynamic nature of the loop structural ensemble is likely required for highly variable opa loop sequences to bind a common receptor and also for a single opa protein to bind a variety of host receptors . ( a ) opa60 is an eight - stranded -barrel with long extracellular loops ( 60% of the protein ) . charged residues within the -barrel region are colored green and in the periplasmic turns colored blue . aromatic residues located near the headgroup region of the bilayer are colored yellow . the semivariable region ( sv ) is colored orange , and the hypervariable ( hv ) regions 1 and 2 are colored red . residues in the loops that have nmr assignments have gray circles and regions demonstrating transient helices in the md simulations have black outlined font . ( b ) the n , h trosy - hsqc is labeled with the nmr backbone assignment for opa60 in dpc micelles . ( c ) sample strips from the n - noesy spectrum indicating intra- ( solid lines ) and interstrand ( dashed lines ) noes observed . residues in -strand 4 ( black ) , -strand 3 ( blue ) and -strand 5 ( red ) are labeled . ( d ) nmr n , h trosy - hsqc spectra of opa60 in nanodiscs containing dmpc lipids with peaks labeled with the nmr backbone assignment . the spectra shown were recorded at 800 mhz and at 40 c ( b , c , and d ) or 10 c to resolve only loop resonances ( d ) . all nmr samples were deuterated with labile protons back exchanged and the protein concentrations were 750 m for dodecylphosphocholine samples and 500 m for nanodiscs preparations . the gene for opa60 with n- and c - terminal fusion tags was subcloned into pet28b from the orginal pex vector provided ( martine bos , ultrech university ) and transformed into bl21(de3 ) e. coli . cells were grown in d2o ( 99.8% ) minimal media containing 4 g / l c(99%)-glucose and 1 g / l n(99%)-ammonium chloride ( cambridge isotopes laboratory ) at 310 k until an od600 of 0.8 expression was induced with 1 mm isopropyl--thio - d - galactoside for 8 h. cells were lysed after resuspension in 50 mm tris hcl and 150 mm nacl ( lysis buffer ) . cell debris from the lysate was removed via centrifugation at 12000 g for 30 min . the pellet was resuspended in lysis buffer with the addition of 8 m urea overnight and centrifuged again at 12000 g for 30 min . the soluble fraction was added to a co - immobilized metal affinity chromatography column and washed with 15 cv of 20 mm sodium phosphate , ph 7.8 , 150 mm nacl , 20 mm imidazole , 8 m urea followed by a 5 cv elution ( 20 mm sodium phosphate , ph 7.0 , 150 mm nacal , 680 mm imidazole ) . the eluted protein fraction was concentrated to 200 m and rapidly diluted 20-fold with 20 mm tris hcl , ph 8.0 , 500 mm nacl , and 4.5 mm n - dodecylphosphocholine ( dpc ; anatrace ) . after 5 days of room temperature incubation , the protein was fully folded as assessed with sds page gel shift analysis . the sample was then concentrated and dialyzed against 3 4 l of 20 mm sodium phosphate , ph 6.2 , and 150 mm nacl for 1 h each . final nmr samples were concentrated to 400800 m and contained 110150 mm dodecylphosphocholine ( dpc ) as measured by comparing sample detergent intensities with standard concentrations . opa60 was reconstituted into nanodisks according to established protocols using plasmid for msp1d1h5 generously provided by gerhard wagner ( harvard university ) . msp1d1h5 was purified and assembled in 20 mm tris / hcl ph 7.5 , 100 mm nacl , and 5 mm edta buffer with the appropriate amount of dry lipid / detergent to obtain a mixture of msp1d1h5/dmpc / sodium cholate with a molar ratio of 1:50:100 . opa60 refolded in dpc was added to the mixture , and the opa60/msp1d1h5 ratio was adjusted to 1:4 . the mixture was incubated at 4 c for 1 h , and detergent was removed with 0.5 g of washed biobeads sm-2 ( biorad ) per ml of assembly mixture . h. biobeads were pelleted by centrifugation , and the decanted supernatant was concentrated and purified on a superdex 200 gel filtration column equilibrated with 20 mm sodium phosphate , ph 6.5 , 50 mm nacl , and 5 mm edta . fractions corresponding to the main peak were pooled and concentrated using an amicon centrifugal filter unit of 30 kda mwco ( millipore ) . the nmr sample consisted of 0.5 mm h , n opa60 in msp1d1h5 nanodiscs with d54 - 1,2-dimyristoyl - sn - glycero-3-phosphocholine ( dmpc ; avanti polar lipids ) , in gel filtration buffer supplemented with 10% ( v / v ) d2o . nmr spectra were collected on bruker avance spectrometers operating at proton frequencies of 600 and 800 mhz equipped with bruker 5 mm txi cryoprobes and recorded at 313 k. spectra were processed with topspin and assigned using cara ( cara.nmr.ch ) . the assignment strategy for opa60 is published and mapped onto the n , h - trosy - hsqc in figure s1 ( supporting information ) . through these strategies , complete nitrogen , hydrogen , c , c , and co resonances were assigned for residues 114 , 2931 , 5171 , 9597 , 109110 , 118140 , 157 , 159178 , 190212 , 231 , and 233238 ( figure 1b ) with only c , c , and co resonances for the seven assigned prolines and a lack of c assignment for two additional resonances ( y71 and i97 ) . further , the entire side chain carbon and hydrogen assignments were obtained for residues 159178 through tocsy and cosy assignment using a corresponding synthetic peptide . additionally , nine aromatic side chain protons ( y10 , f62 , w65 , f125 , f131 , y134 , f200 , y236 , and f238 ) were observed due to incomplete protein deuteration and dynamics of the side chain and assigned using the n - noesy spectrum . resonance assignments were achieved for 92% of the -barrel region ( as defined by the md refined structure ) and 27% of the extracellular loops . the nspa structure was not used for the assignment or identification of restraints . the talos+ program was used to obtain backbone dihedral angle restraints . assigned noe peak heights were measured and binned into strong , medium , or weak interactions . observed inter--strand noes are schematically indicated in figure 1a . thirty - one additional noes between backbone hn protons and aromatic side chain protons were also included . these were assigned upper limits of 3.5 , 5.0 , and 6.5 . in most cases , hydrogen bonding partners could be unambiguously assigned based on noe patterns ( a representative strip is shown in figure 1c ) , and two distance restraints were used with upper limits of 2.5 and 3.5 for hno and no , respectively . additionally , planar restraints ( 32 4 as a square potential ) were used to represent the lipid bilayer . the extracellular loops are very long , and without the bilayer restraint they sampled conformers that would be embedded into or transverse the bilayer ( figure s1 , supporting information ) . the restraint distance was chosen on the basis of ( 1 ) the hydrophobic thickness of porb a -barrel membrane protein from ng for which there is a crystal structure and ( 2 ) the residues that were observed to have noes with detergent headgroup choline protons ( figure s2 , supporting information ) . starting at 3000 k , 5000 steps of high - temperature annealing was used to fold the initial extended structure . twenty of the lowest overall violation energies of the 300 calculated structures with selected for further md simulations . all simulations were performed using gromacs 4.5 and the charmm36 forcefield for protein and lipid interactions . as detailed below , distance and dihedral restraints derived from the nmr data and used in xplor simulated annealing runs were enforced throughout the molecular dynamics simulations . simulations were run under npt conditions using the velocity - rescaling thermostat at 300 k with a time - constant of 0.1 ps and semi - isotropic pressure coupling using the parrinello all covalent bonds were constrained using lincs , and long - range electrostatics were computed every step using particle mesh ewald ( pme ) . a lipid bilayer of 512 dmpc molecules ( bilayer thickness , 34 1 ; hydrophobic thickness , 2326 ) was constructed using the charmm - gui membrane builder tool and solvated with approximately 40000 tip3p waters . ions were added to obtain a system with 150 mm nacl and no net charge . the dmpc bilayer was equilibrated prior to protein insertion with a resulting area per lipid headgroup of 0.60 nm , close to the experimentally determined value ( 0.606 0.005 nm ) . the system dimensions were approximately 12.5 nm ( sides ) and 11 nm ( height ) . each of the 20 lowest energy structures from xplor simulated annealing was independently inserted in the equilibrated membrane using the gromacs tool g_membed , removing approximately 1520 lipids in the process . each system was then energy minimized for 1000 steps using the steepest descents method . twenty production runs , one per protein structure , were then carried out for 100 ns using a time step of 2 fs . noe - based distance restraints and talos+-derived dihedral restraints as used in the xplor simulated annealing calculations were imposed using spring potentials with force constants of 1000 kj / mol / nm and 1000 kj / mol / rad , respectively . to perform the c density analysis , all trajectories were aligned to a single consistent reference structure using a rigid - body fit where the objective function was calculated only on the sheet and turn residues . then the density was calculated on a 3d - grid with the mdanalysis toolkit , using a grid - spacing of 0.1 nm . clustering was performed using the gromacs tool g_cluster , which was extended to include the k - means algorithm . the 20 most - populated , and therefore lowest free energy clusters , were selected , and the minimum energy structure from each was reported to form the hybrid refinement ensemble . the c rmsd within each cluster ranged from 1.52 to 4.22 and 1.87 to 5.33 for the entire protein and the extracellular loops , respectively ( table s2 , supporting information ) . the rmsd for the extracellular loops between clusters is much greater with the pairwise rmsd for the minimum energy structures ranging from 5.60 to 29.3 ( table s3 , supporting information ) . only contacts greater than 1.5 kt estimated free energy difference between the md and xplor structures additional analysis of contact between the hv1 and hv2 loops was performed by generating contact maps between these residues , where a contact was defined using a 6 interatomic distance cutoff . contact probabilities and lifetimes were computed using these contact maps , and highly contacting structures ( defined as > 50 simultaneous contacts ) were further analyzed via agglomerative single - linkage clustering using euclidean distance on the hv1-hv2 contact maps , yielding 10 clusters of hv1hv2 contacts . side chain restraints were not included in the md / nmr hybrid refinement . xplor ensemble without side chain noes had a mean global backbone rmsd for the -sheet residues of 1.04 0.15 . chemical shifts were calculated on all snapshots in the md simulations and for the xplor structures using the sparta+ software . the calculated shifts were then averaged for each atom , and the average values compared to the experimentally determined shifts ( which were corrected for deuterium and trosy induced shifts ) , where existing . in all cases , the errors reported are those used by sparta+ ( 0.92 ppm for c and 0.49 ppm for hn , respectively ) . relaxation rates were measured using two - dimensional n h trosy - based experiments recorded at 600 mhz and 313 k. nmr data were processed and fit with nmrpipe . h bond vector ci(t ) = < i ( 0 ) , i ( t ) > , averaged across all trajectories , where i is the n h bond vector for the ith residue . ( a ) -barrel and periplasmic turns are colored black ; extracellular loop 1 , green ; loop 2 , blue ; loop 3 , red ; loop 4 , magenta . ( b ) differences in carbon chemical shifts compared to random coil values are plotted ; ( c c ) = /3(ci1 + ci data for turns are colored red , for -strands blue , for loops green , and for the n - terminus gray . the chemical shifts have been deposited in the biomagresbank under the bmrb accession no . atomic coordinates for the xplor and md / xplor refined 20 conformers representing the structure of opa60 have been deposited in the pdb ( pdb i d : 2mlh and 2maf , respectively ) . opa60 is an eight - stranded -barrel with four extracellular loops ( figures 1a and 2a ) . the combination of a stable , membrane - inserted -barrel domain and long unstructured loops complicated assignment and structure determination . strategies for the assignment of the protein included trypsin cleavage , peptide synthesis , and assignment at various temperatures . hn noes , h - bond restraints , and backbone dihedrals calculated with talos ( table 1 ) . the backbone rmsd of the barrel region for the 20 lowest energy structures is 0.96 ( table 1 and figure 2a ) . the structure calculations were complicated by the long unstructured loops ; in the initial calculation , extracellular loops adopted unreasonable conformations that spanned the membrane embedded region with excursions to the periplasmic side of the protein ( figure s1 , supporting information ) . to address this problem , planar restraints were introduced at a distance of 32 4 ( see the experimental section for details ) . most of loops 1 , 2 , and 4 were not assigned ( figure 2b ) because the resonances were not observed ( although spectral overlap contributed ) . lowering the temperature broadened -barrel and some loop peaks beyond detection simplifying the spectra to only the most dynamic regions of the loops and facilitating 27 loop resonances to be assigned . to further assign the functionally important hv2 region , a synthetic 20 amino acid peptide was synthesized and had nearly complete spectral overlap with the full - length protein allowing 17 loop resonances to be assigned . there are two likely phenomena that contribute to the line broadening of the resonances that are not observed : ( i ) conformational exchange and ( ii ) structural heterogeneity . the former was speculated to contribute to the lack of assignments in ompx and ompa in dihexanoylphosphatidylcholine ( dhpc ) and dpc micelles , respectively . the missing resonances ( approximately half of the extracellular loops ) corresponded to residues that connect the ordered -barrel and the flexible central region of the extracellular loops . some of the missing loop resonances of ompx were resolved when the micelle was replaced with nanodiscs containing dmpc and 1,2-dimyristoyl - sn - glycero-3-phosphoglycerol ( dmpg ) . to better refine the structure , each of the 20 structures of the nmr ensemble was subjected to molecular dynamics simulations in a dmpc lipid bilayer . an interval of 100 ns of simulation was selected for the structure refinement because t1 and t2 relaxation data indicated the hv regions were dynamic on the nanosecond time scale . in addition , the chemical shifts and cd indicated the hv and extracellular loops , respectively , are random coil . although the solution nmr structure was determined in dpc , the simulations were sought in a more biologically relevant membrane environment . there are several pieces of evidence that suggest opa60 has the same structure in dmpc as in dpc . cd spectra of opa60 in dpc and dmpc small unilamellar vesicles ( liposomes ) indicate the protein structure is approximately 50% random coil 50% -strand ( figure s3 , supporting information ) . in addition , opa60 in nanodiscs with dmpc have -barrel chemical shifts that are superimposable with the dpc spectra ; however , a few barrel resonances corresponding to residues on strands 3 , 6 , and 8 are missing in the nanodisc spectrum ( figure 1d ) . to further refine the solution nmr structure , 100-ns md simulations were performed on each of the 20 lowest energy nmr structures embedded in a dmpc lipid bilayer ( bilayer thickness , 34 1 ; hydrophobic thickness , 2326 ) . the resulting ensemble from the md simulations has a backbone rmsd of 1.19 for the -barrel region ( figure 3a and table 1 ) . the md ensemble is composed of the minimum energy structure from each of the 20 lowest free energy clusters and captures 2937 of the 4000 snapshots ( table 1 ) . the c rmsd within each cluster ranged from 1.524.22 and 1.875.33 for the entire protein and the extracellular loops , respectively ( table s2 , supporting information ) . the rmsd for the extracellular loops between clusters is much greater than within clusters , with the pairwise rmsd for the minimum - energy structures ranging from 5.60 to 29.3 ( table s3 , supporting information ) . although sampling is not sufficient to achieve full convergence , principal components analysis of the loop conformations across all simulations yields good overlap in the subspace of the two largest principal components ( figure s4 , supporting information ) , indicating that at least in terms of the highest amplitude loop motions , individual simulations sampled overlapping rather than isolated regions of conformation space . thus , the ensemble from the 20 lowest energy clusters represents the loop structural diversity observed in the trajectories . the -barrel of opa60 is similar in sequence to nspa ( figure s5 , supporting information ) ; however , the overall fold from the md / nmr refinement is most similar to ompa and ompx ( table s4 , supporting information ) . similar to the 12 eight - stranded -barrels deposited in the protein data bank , the strands have a right - handed twist ( figure 3a ) and an aromatic belt around the circumference of the barrel at the lipid headgroup regions ( figure s6 , supporting information ) . a significant ionic network exists inside the barrel ( figure s7 , supporting information ) ; however , there is no observable pore through the length of the barrel . the ionic residues on the extracellular side of the barrel ( figure s7 , supporting information ) are excluded from solvent in many of the clusters , and those on the periplasmic surface ( figure s7 , supporting information ) are accessible to solvent in all the clusters . this ionic network may contribute to the significant stability observed for opa60 ; the barrel remains intact after cleavage with trypsin and boiling in sds loading buffer . basic residues ( figure s8 , supporting information ) are clustered in the extracellular loops near the barrel domain . these residues may interact with the negatively charged lipooligosaccharide outer leaflet of the outer membrane as was observed for fhua and one of the lps interactions identified with oprh . however , specific interactions between the barrel and lps were not detected by chemical shift perturbation when lps ( los , which is in the outer leaflet of the neisseria outer membrane , is not commercially available ) was titrated into the opa60dpc micelle ( data not shown ) . colored by density ( probability of a c atom within an grid cell ) from yellow ( 2 10 ) to blue . the gray planes show the average positions of the phosphorus atoms in the lipid head groups . contact map for the ensemble calculated with xplor in detergent ( a ) and for the md in lipid ( b ) . the contact map is rendered as a contour plot of contact probability , with evenly spaced contours from 10% to 100% contact probability in each ensemble colored from dark blue to dark red . contacts were defined as two atoms from respective residues approaching within 5 . comparison of the amide proton ( c ) and c ( d ) chemical shifts calculated from the xplor ensemble and the md ensemble . positive values indicate the chemical shifts calculated from the md ensemble agree better with the observed chemical shifts , and negative values indicate chemical shifts calculated from the xplor ensemble agree better with the observed chemical shifts . the md - refined ensemble has several more inter - residue contacts than the xplor refined ensemble ( figure 4a , b ) . these new contacts are primarily intraloop : 11 are across the periplasmic side of the -barrel and 17 extend the strands on the extracellular side ( only the 76 contacts that represented a greater than 1.5 kt estimated free energy difference between the md and xplor structures were considered ) . consequently , the most probable loop conformations in the md refined structures are much more compact than the xplor refined structures ( figure 3a ) with most of the loop density above the -barrel ( figure 3b ) . this decrease in loop volume can be quantified via the protein radius of gyration , which decreases from the initial structure over the course of 19 of the 20 trajectories ( figure s9 , supporting information ) . the accuracy of the structural representation of the xplor and md refined ensembles can be evaluated by comparing chemical shifts calculated from the ensemble structures via semiempirical shift prediction methods against the experimental chemical shifts ( exp ) . parts c and d of figure 4 compare the deviations between the experimentally measured values ( exp ) of c and hn opa60 chemical shifts and the xplor and md predictions . most residues do not show differences between xplor and md larger than the sparta+ reported prediction accuracy . however , for carbon shifts , which primarily depend on backbone dihedral angles , the md predictions agreed better with the experimental shifts indicating that the md ensemble is an accurate representation of the observed opa60 structure . the -barrel provides the scaffolding for the functional extracellular loops , which are disordered and sample a diverse ensemble of conformers . the nmr and md dynamics data ( figure 5 ) indicate that hv2 and three extracellular loops ( l1l3 ) , respectively , are dynamic on the nanosecond time scale . because the opa detergent complex has a large overall correlation time , t1 values are highly sensitive to backbone nanosecond motions . opa t1 values decrease significantly at the n - terminus , periplasmic turns , and extracellular loops 1 and 3 compared to the -strands ( figure 5a ) , indicating these regions have high amplitude motions in the nanosecond time scale . several of the -strands have a general trend of increased dynamics toward the n- and c - terminal ends of the strands ( although most have order parameters greater than 0.85 ) , which was previously reported for other -barrel membrane proteins investigated with nmr . t2 changes are much more difficult to interpret since values increase with nanosecond motions and decrease with s ms motions . nonetheless , the opa60 t2 values ( figure 5a ) are consistent with the interpretation of the t1 values . thus , on the basis of the nmr data , the hv2 region of opa has a high amplitude of motion on the nanosecond time scale . these nmr data are consistent with those observed with md , which provides a more comprehensive understanding of the motions of the loops . although sampling of loop conformations was not globally converged , the rank order of backbone dynamics showed good convergence . assessed at 20 ns , the spearman was 0.975 between the mean autocorrelation function value and the fifth percentile of sampled trajectories , while the between the mean and the 95th percentile was 0.988 . a gradient is observed for the md - derived backbone nh bond vector time autocorrelation function for each of the three longer extracellular loops ( l1l3 ; figure 5b ) , with loop regions furthest from the barrel more dynamic than the regions closest to the barrel . based on the md simulations , the sv , hv1 , and hv2 regions are moving within the nanosecond time regime and with a high amplitude of motion . ( a ) n t1 and n t2 relaxation values for opa60 plotted versus sequence and secondary structure . n t1 values that are less than 80% ( dotted line ) of the value predicted ( 1 s ) for a 20 ns overall correlation time have s values less than 0.85 ( e = 10 ps ) . data for turns are colored red ; -strands , blue ; and loops , green . ( b ) cartoon representation of opa60 with residues colored according to the n h orientational time autocorrelation function calculated from md trajectories . as might be expected from the extensive nanosecond - time scale dynamics , the loops do not maintain long - lived structural features . however , they do form considerable intraloop contacts , which are captured more readily by the hybrid refinement strategy than simulated annealing alone ( figure 4a , b and 6a ) . there are several contacts within each loop that are observed in the md refined structures . of the 76 contacts above 1.5 kt estimated free energy difference between the xplor and xplor / md ensembles , 31 are within each of the three loops ( l1l3 ) . contacts between hv1 and hv2 are of most interest since they are both required to bind to ceacam receptors . common contacts between the two regions are observed ( figure 6a ) ; however , these contacts have short lifetimes ( figure 6b ) . throughout the simulations these contacts are frequent but short - lived . based on this observation , the 4000 snapshots were reclustered based on the hv1 and hv2 regions and analyzed in terms of contacts and representative structures ( figure s10 , supporting information ) . recurrent contacts were observed between residues in the range 153165 ( hv1 ) and 8695 ( hv2 ) as well as 171180 ( hv1 ) and 9498 ( hv2 ) . not surprisingly , there are several hydrophobic residues that mediate these hv1hv2 interactions ( figure s10 , supporting information ) . the recurring contact patterns were observed in loop conformations that were globally quite different and across multiple independent md simulations from different starting structures , suggesting robust formation of transient yet frequent interactions . these observations are broadly consistent with the primary nmr data in that long - lived interactions or persistent structure in hv1 and hv2 were not observed on the basis of chemical shift ( figure 2b ) and the lack of nonsequential noes in assigned regions . ( b ) average contact lifetimes are plotted for each contact , with each contour line representing a 5-ns lifetime increment . comparison of these panels shows that hv1hv2 contacts are relatively frequent but short - lived . in addition to the contacts observed , the sv , hv1 , and hv2 regions each sample helical conformers in a small fraction of the 4000 snapshots of the 20 trajectories ( figure s11 , supporting information ) . other secondary structures , such as ppii and 310 helices , were less abundant in these regions ( figure s11 , supporting information ) . the existence of these lowly populated structures is difficult to probe with traditional nmr methods ; however , for populated secondary structure elucidation , carbon chemical shifts ( figure 2b ) are typically used . for the data obtained , only a few residues in the sv and hv2 regions indicate -helical structure ( positive values ) , but overall the values indicate the dominant population is random coil which is consistent with the md results . the lack of any long - lived discrete structure in the extracellular regions of opa60 is compatible with the degree of sequence variability that still confers binding to host receptors ( table s1 , supporting information ) . it would be surprising should such extreme variability result in a single stable structure . despite the structural plasticity of the extracellular loops , opa proteins must still bind a common set of receptors . depending on the hypervariable sequences in the extracellular loops , opa proteins bind selectively to the n - domain of ceacam1 , 3 , 5 , and/or 6 but do not bind the n - domains of ceacam4 , 7 , and 8 . using mutagenesis , residues y34 and i91 of ceacam n - domains ( figure s12 , supporting information ) were identified to be essential for the opa - receptor interaction . several other residues ( 27 , 28 , 29 , 32 , 39 , 44 , and 89 ) dictate the different opa ceacam selectivity reported ( figures s12 and s13 , supporting information ) . the total exposed surface area of these identified residues is approximately 440 and is composed of both hydrophobic and polar moieties , which can easily be complemented by the hydrophobic and polar groups in the hv regions of opa proteins ( table s1 , supporting information ) . beyond the enthaplic interactions , the dynamics and conformations of the extracellular loops are important to the molecular recognition event . the extracellular loops are intrinsically disordered yet are sampling a restricted volume such that there are interactions between the loops on the nanosecond time scale . md simulations further suggest recurrent yet transient interaction patterns between specific regions of the loops . thus , the loops adopt an intermediate state that is not folded but is also not lacking in interactions ; the state of the loops may be best described as premolten globule or both hv1 and hv2 are required ; the hv regions are concomitant since chimeric opa proteins with an hv1 and hv2 region from two opa proteins that bind the same receptor do not bind receptor . state may be a mechanism to retain disorder yet provide conformers in which hv1 and hv2 are in proximity and competent to interact with ceacam . the small ceacam binding surface ( figure s13 , supporting information ) and the requirement of both opa hypervariable regions suggest that a large folding event of the extracellular loops is unlikely upon binding suggesting that the binding mechanism is more likely conformational selection rather than induced fit . however , there is a plethora of commentary on the similarities and differences of these two binding mechanisms with the prevailing idea that binding reactions could have elements of both mechanisms . in addition , sequences are selected for function not mechanism ; therefore , the mechanism of different opa proteins may vary . since opa ceacam interactions differ among variants and receptors , the dynamic nature of the loops maximizes the likelihood that a sequence will engage the receptor by increasing conformer sampling and the potential binding modes for receptor engagement . we report the structure of opa60 , a neisserial outer membrane protein that induces host phagocytosis of the bacterium through specific receptor interactions . this eight - stranded -barrel protein possesses three extracellular loops ( greater than 34 residues ) that are longer than any -barrel structures yet reported and required a membrane restraint in the xplor structure calculation . to understand the structure and dynamics of the loops , we employed a hybrid xplor / md refinement where nmr - derived restraints were used in 20 100 ns md simulations to obtain a structure of opa60 in a dmpc membrane . the hybrid - refined structure is consistent with the initial xplor structure but has an increase in loop structure and compactness . although there is little secondary structure evident in either the md simulations or the primary nmr data , there are many short - lived contacts between the loops on the nanosecond time scale due to extension of the -strands . we hypothesize that the observed dynamic ensemble is critical for maximizing the conformations of a highly variable region of opa to engage receptors . that is , a high degree of plasticity is required to tolerate the diverse sequences in these regions and sample conformers competent to engage receptors . it remains to be shown , however , whether different opa variants engage a single receptor ( e.g. , ceacam1 ) via similar or different loop structures . the structure of several opa variants in complex with an identical receptor will elucidate whether the binding modes are indeed convergent or divergent .

the structure and dynamics of opa proteins , which we report herein , are responsible for the receptor - mediated engulfment of neisseria gonorrheae or neisseria meningitidis by human cells and can offer deep understanding into the molecular recognition of pathogen host receptor interactions . such interactions are vital to understanding bacterial pathogenesis as well as the mechanism of foreign body entry to a human cell , which may provide insights for the development of targeted pharmaceutical delivery systems . the size and dynamics of the extracellular loops of opa60 required a hybrid refinement approach wherein membrane and distance restraints were used to generate an initial nmr structural ensemble , which was then further refined using molecular dynamics in a dmpc bilayer . the resulting ensemble revealed that the extracellular loops , which bind host receptors , occupy compact conformations , interact with each other weakly , and are dynamic on the nanosecond time scale . we predict that this conformational sampling is critical for enabling diverse opa loop sequences to engage a common set of receptors .

Introduction Experimental Section Results and Discussion Conclusion

neisseria gonorrheae ( ng ) and neisseria meningitides are gram - negative bacterial pathogens responsible for gonorrhea and meningococcal meningitis , respectively . opa proteins are eight - stranded -barrel integral outer membrane proteins with four extracellular loops ( figure 1a ) . high sequence diversity is observed in regions of the extracellular loops of opa variants ( table s1 , supporting information ) , which is predominantly a result of recombination events between genes of the same isolate ( 70% ) and import of genes from other isolates ( 16% ) . the three variable regions within the extracellular loops , hypervariable 1 and 2 ( hv1 and hv2 ) and the semivariable ( sv ) regions , engage host receptors to induce phagocytosis and determine the specific host receptors engaged . this sequence diversity likely plays a beneficial role in helping neisseria to evade host immune responses ; however , it poses a challenge in that highly varied loop sequences must engage a common set of receptors to mediate cellular invasion . the determinants of opa receptor interactions are of the utmost importance for understanding neisserial pathogenesis and the innate immune response . opa proteins also provide a means of foreign body cellular entry through specific human receptors that can be exploited synthetically for pharmaceutical and technological purposes . although all ceacam receptors contain this domain , opa proteins only bind to ceacam1 , 3 , 5 , and 6 , and most selectively bind to only a subset . the ceacam n - domain residues that interact with opacea have been identified : y34 and i91 are essential for all opacea interactions , and an additional seven nearby residues are implicated in binding depending on the particular opacea . the specificity - determining residues on opacea are predominantly in the hv1 and 2 regions , and the hv sequences are concomitant : chimeric opa proteins with an hv1 and hv2 region from two opa proteins that bind the same receptor do not bind . toward understanding the molecular recognition required to gain entry into human cells , we report the structure of opa60 , which binds ceacam1 , 3 , 5 , and 6 . to overcome these obstacles , we employed a hybrid method that used the restraints determined with solution nuclear magnetic resonance ( nmr ) spectroscopy in detergent micelles in conjunction with molecular dynamics ( md ) simulations in a lipid bilayer . three of the extracellular loops are longer than those found in any -barrel structure previously determined . the hv regions within these loops are dynamic on the nanosecond time scale and are predominantly disordered . however , the loops are compact and interact with each other weakly such that long - lived specific intraloop interactions are not observed . the diverse and dynamic nature of the loop structural ensemble is likely required for highly variable opa loop sequences to bind a common receptor and also for a single opa protein to bind a variety of host receptors . ( a ) opa60 is an eight - stranded -barrel with long extracellular loops ( 60% of the protein ) . the sample was then concentrated and dialyzed against 3 4 l of 20 mm sodium phosphate , ph 6.2 , and 150 mm nacl for 1 h each . through these strategies , complete nitrogen , hydrogen , c , c , and co resonances were assigned for residues 114 , 2931 , 5171 , 9597 , 109110 , 118140 , 157 , 159178 , 190212 , 231 , and 233238 ( figure 1b ) with only c , c , and co resonances for the seven assigned prolines and a lack of c assignment for two additional resonances ( y71 and i97 ) . additionally , nine aromatic side chain protons ( y10 , f62 , w65 , f125 , f131 , y134 , f200 , y236 , and f238 ) were observed due to incomplete protein deuteration and dynamics of the side chain and assigned using the n - noesy spectrum . resonance assignments were achieved for 92% of the -barrel region ( as defined by the md refined structure ) and 27% of the extracellular loops . in most cases , hydrogen bonding partners could be unambiguously assigned based on noe patterns ( a representative strip is shown in figure 1c ) , and two distance restraints were used with upper limits of 2.5 and 3.5 for hno and no , respectively . additionally , planar restraints ( 32 4 as a square potential ) were used to represent the lipid bilayer . the extracellular loops are very long , and without the bilayer restraint they sampled conformers that would be embedded into or transverse the bilayer ( figure s1 , supporting information ) . the restraint distance was chosen on the basis of ( 1 ) the hydrophobic thickness of porb a -barrel membrane protein from ng for which there is a crystal structure and ( 2 ) the residues that were observed to have noes with detergent headgroup choline protons ( figure s2 , supporting information ) . as detailed below , distance and dihedral restraints derived from the nmr data and used in xplor simulated annealing runs were enforced throughout the molecular dynamics simulations . to perform the c density analysis , all trajectories were aligned to a single consistent reference structure using a rigid - body fit where the objective function was calculated only on the sheet and turn residues . clustering was performed using the gromacs tool g_cluster , which was extended to include the k - means algorithm . the 20 most - populated , and therefore lowest free energy clusters , were selected , and the minimum energy structure from each was reported to form the hybrid refinement ensemble . the c rmsd within each cluster ranged from 1.52 to 4.22 and 1.87 to 5.33 for the entire protein and the extracellular loops , respectively ( table s2 , supporting information ) . the rmsd for the extracellular loops between clusters is much greater with the pairwise rmsd for the minimum energy structures ranging from 5.60 to 29.3 ( table s3 , supporting information ) . contact probabilities and lifetimes were computed using these contact maps , and highly contacting structures ( defined as > 50 simultaneous contacts ) were further analyzed via agglomerative single - linkage clustering using euclidean distance on the hv1-hv2 contact maps , yielding 10 clusters of hv1hv2 contacts . side chain restraints were not included in the md / nmr hybrid refinement . ( b ) differences in carbon chemical shifts compared to random coil values are plotted ; ( c c ) = /3(ci1 + ci data for turns are colored red , for -strands blue , for loops green , and for the n - terminus gray . atomic coordinates for the xplor and md / xplor refined 20 conformers representing the structure of opa60 have been deposited in the pdb ( pdb i d : 2mlh and 2maf , respectively ) . strategies for the assignment of the protein included trypsin cleavage , peptide synthesis , and assignment at various temperatures . the backbone rmsd of the barrel region for the 20 lowest energy structures is 0.96 ( table 1 and figure 2a ) . the structure calculations were complicated by the long unstructured loops ; in the initial calculation , extracellular loops adopted unreasonable conformations that spanned the membrane embedded region with excursions to the periplasmic side of the protein ( figure s1 , supporting information ) . the missing resonances ( approximately half of the extracellular loops ) corresponded to residues that connect the ordered -barrel and the flexible central region of the extracellular loops . to better refine the structure , each of the 20 structures of the nmr ensemble was subjected to molecular dynamics simulations in a dmpc lipid bilayer . an interval of 100 ns of simulation was selected for the structure refinement because t1 and t2 relaxation data indicated the hv regions were dynamic on the nanosecond time scale . in addition , the chemical shifts and cd indicated the hv and extracellular loops , respectively , are random coil . in addition , opa60 in nanodiscs with dmpc have -barrel chemical shifts that are superimposable with the dpc spectra ; however , a few barrel resonances corresponding to residues on strands 3 , 6 , and 8 are missing in the nanodisc spectrum ( figure 1d ) . to further refine the solution nmr structure , 100-ns md simulations were performed on each of the 20 lowest energy nmr structures embedded in a dmpc lipid bilayer ( bilayer thickness , 34 1 ; hydrophobic thickness , 2326 ) . the resulting ensemble from the md simulations has a backbone rmsd of 1.19 for the -barrel region ( figure 3a and table 1 ) . the c rmsd within each cluster ranged from 1.524.22 and 1.875.33 for the entire protein and the extracellular loops , respectively ( table s2 , supporting information ) . the rmsd for the extracellular loops between clusters is much greater than within clusters , with the pairwise rmsd for the minimum - energy structures ranging from 5.60 to 29.3 ( table s3 , supporting information ) . although sampling is not sufficient to achieve full convergence , principal components analysis of the loop conformations across all simulations yields good overlap in the subspace of the two largest principal components ( figure s4 , supporting information ) , indicating that at least in terms of the highest amplitude loop motions , individual simulations sampled overlapping rather than isolated regions of conformation space . the ionic residues on the extracellular side of the barrel ( figure s7 , supporting information ) are excluded from solvent in many of the clusters , and those on the periplasmic surface ( figure s7 , supporting information ) are accessible to solvent in all the clusters . basic residues ( figure s8 , supporting information ) are clustered in the extracellular loops near the barrel domain . these residues may interact with the negatively charged lipooligosaccharide outer leaflet of the outer membrane as was observed for fhua and one of the lps interactions identified with oprh . however , specific interactions between the barrel and lps were not detected by chemical shift perturbation when lps ( los , which is in the outer leaflet of the neisseria outer membrane , is not commercially available ) was titrated into the opa60dpc micelle ( data not shown ) . positive values indicate the chemical shifts calculated from the md ensemble agree better with the observed chemical shifts , and negative values indicate chemical shifts calculated from the xplor ensemble agree better with the observed chemical shifts . these new contacts are primarily intraloop : 11 are across the periplasmic side of the -barrel and 17 extend the strands on the extracellular side ( only the 76 contacts that represented a greater than 1.5 kt estimated free energy difference between the md and xplor structures were considered ) . this decrease in loop volume can be quantified via the protein radius of gyration , which decreases from the initial structure over the course of 19 of the 20 trajectories ( figure s9 , supporting information ) . however , for carbon shifts , which primarily depend on backbone dihedral angles , the md predictions agreed better with the experimental shifts indicating that the md ensemble is an accurate representation of the observed opa60 structure . the -barrel provides the scaffolding for the functional extracellular loops , which are disordered and sample a diverse ensemble of conformers . the nmr and md dynamics data ( figure 5 ) indicate that hv2 and three extracellular loops ( l1l3 ) , respectively , are dynamic on the nanosecond time scale . opa t1 values decrease significantly at the n - terminus , periplasmic turns , and extracellular loops 1 and 3 compared to the -strands ( figure 5a ) , indicating these regions have high amplitude motions in the nanosecond time scale . several of the -strands have a general trend of increased dynamics toward the n- and c - terminal ends of the strands ( although most have order parameters greater than 0.85 ) , which was previously reported for other -barrel membrane proteins investigated with nmr . thus , on the basis of the nmr data , the hv2 region of opa has a high amplitude of motion on the nanosecond time scale . these nmr data are consistent with those observed with md , which provides a more comprehensive understanding of the motions of the loops . a gradient is observed for the md - derived backbone nh bond vector time autocorrelation function for each of the three longer extracellular loops ( l1l3 ; figure 5b ) , with loop regions furthest from the barrel more dynamic than the regions closest to the barrel . based on the md simulations , the sv , hv1 , and hv2 regions are moving within the nanosecond time regime and with a high amplitude of motion . as might be expected from the extensive nanosecond - time scale dynamics , the loops do not maintain long - lived structural features . however , they do form considerable intraloop contacts , which are captured more readily by the hybrid refinement strategy than simulated annealing alone ( figure 4a , b and 6a ) . recurrent contacts were observed between residues in the range 153165 ( hv1 ) and 8695 ( hv2 ) as well as 171180 ( hv1 ) and 9498 ( hv2 ) . these observations are broadly consistent with the primary nmr data in that long - lived interactions or persistent structure in hv1 and hv2 were not observed on the basis of chemical shift ( figure 2b ) and the lack of nonsequential noes in assigned regions . in addition to the contacts observed , the sv , hv1 , and hv2 regions each sample helical conformers in a small fraction of the 4000 snapshots of the 20 trajectories ( figure s11 , supporting information ) . the lack of any long - lived discrete structure in the extracellular regions of opa60 is compatible with the degree of sequence variability that still confers binding to host receptors ( table s1 , supporting information ) . despite the structural plasticity of the extracellular loops , opa proteins must still bind a common set of receptors . depending on the hypervariable sequences in the extracellular loops , opa proteins bind selectively to the n - domain of ceacam1 , 3 , 5 , and/or 6 but do not bind the n - domains of ceacam4 , 7 , and 8 . several other residues ( 27 , 28 , 29 , 32 , 39 , 44 , and 89 ) dictate the different opa ceacam selectivity reported ( figures s12 and s13 , supporting information ) . the total exposed surface area of these identified residues is approximately 440 and is composed of both hydrophobic and polar moieties , which can easily be complemented by the hydrophobic and polar groups in the hv regions of opa proteins ( table s1 , supporting information ) . beyond the enthaplic interactions , the dynamics and conformations of the extracellular loops are important to the molecular recognition event . the extracellular loops are intrinsically disordered yet are sampling a restricted volume such that there are interactions between the loops on the nanosecond time scale . thus , the loops adopt an intermediate state that is not folded but is also not lacking in interactions ; the state of the loops may be best described as premolten globule or both hv1 and hv2 are required ; the hv regions are concomitant since chimeric opa proteins with an hv1 and hv2 region from two opa proteins that bind the same receptor do not bind receptor . the small ceacam binding surface ( figure s13 , supporting information ) and the requirement of both opa hypervariable regions suggest that a large folding event of the extracellular loops is unlikely upon binding suggesting that the binding mechanism is more likely conformational selection rather than induced fit . in addition , sequences are selected for function not mechanism ; therefore , the mechanism of different opa proteins may vary . since opa ceacam interactions differ among variants and receptors , the dynamic nature of the loops maximizes the likelihood that a sequence will engage the receptor by increasing conformer sampling and the potential binding modes for receptor engagement . we report the structure of opa60 , a neisserial outer membrane protein that induces host phagocytosis of the bacterium through specific receptor interactions . this eight - stranded -barrel protein possesses three extracellular loops ( greater than 34 residues ) that are longer than any -barrel structures yet reported and required a membrane restraint in the xplor structure calculation . to understand the structure and dynamics of the loops , we employed a hybrid xplor / md refinement where nmr - derived restraints were used in 20 100 ns md simulations to obtain a structure of opa60 in a dmpc membrane . although there is little secondary structure evident in either the md simulations or the primary nmr data , there are many short - lived contacts between the loops on the nanosecond time scale due to extension of the -strands . we hypothesize that the observed dynamic ensemble is critical for maximizing the conformations of a highly variable region of opa to engage receptors .

as a cell of the innate immune system , macrophages play critical roles in both host defense against pathogens and proper tissue development . during embryonic development , a population of macrophages derived from yolk sac hematopoiesis can be found throughout the organism and are thought to contribute to the populations of tissue - resident macrophages in the adult . this process occurs prior to the induction of hematopoiesis in the bone marrow , strongly suggesting a unique origin and function for these embryonic macrophages [ 1 , 2 ] . additionally , embryonically derived , tissue - resident macrophages have been found in a diverse array of organs and tissues , including the mammary gland , and the maintenance of these populations does not require monocyte precursors . postnatally , however , the multistep differentiation program that leads to mature macrophages begins in the bone marrow with hematopoietic stem cells ( hscs ) . these c - kit / sca-1/lineage ( lin ) hscs give rise to two distinct multipotent progenitor populations : the c - kit / sca-1/lin / il-7r common lymphoid progenitor ( clp ) , which differentiate into b cells , t cells , nk cells , and a subset of dendritic cells ( dcs ) , and the c - kit / sca-1/lin / il-7r common myeloid progenitor ( cmp ) , which can populate the erythrocyte , megakaryocyte , myeloid - derived dc , granulocyte , and monocyte compartments [ 4 , 5 ] . more specific precursors of the monocyte / macrophage lineage have been identified , including the c - kit / lin / cx3cr1 monocyte - macrophage dc progenitors ( mdp ) that give rise to both monocytes and dendritic cells . recent work has also identified a cd135/ly6c committed progenitor derived from the mdp that is restricted to the monocyte - macrophage lineage . mature cd11b / cd115 monocytes can then enter the circulation in order to be distributed around the body . circulating monocytes are a heterogeneous population themselves , consisting of so - called patrolling monocytes and inflammatory monocytes . patrolling monocytes are responsible for crawling along the luminal side of the endothelium to monitor for danger - associated molecular patterns ( damps ) and , upon encountering such a signal , rapidly entering the tissue and beginning to recruit additional effector cells in order to start a productive immune response . a major function of inflammatory monocytes is to respond to sites of inflammation and tissue damage . monocytes are recruited to these sites by following a variety of chemokine gradients , the most well - characterized of which is chemokine ( c - c motif ) ligand 2 ( ccl2 ) [ 10 , 11 ] . upon arriving in the vasculature near the site of inflammation , monocytes begin a process of rolling adhesion in which selectin molecules on the surface of the endothelial cells bind to selectin ligands on the monocytes [ 12 , 13 ] . these interactions then allow tight binding to occur between vascular cell adhesion molecule 1 ( vcam1 ) on the endothelium and integrin molecules on the monocytes [ 1416 ] . finally , the monocytes are arrested and can exit the circulation and enter the inflamed tissue , a process known as diapedesis . once in the tissue , monocytes can be further differentiated to macrophages in the presence of colony stimulating factor-1 ( csf-1 ) to carry out effector functions involved in pathogen clearance , wound healing , and developmental regulation [ 17 , 18 ] . macrophages are a cell type with exquisite plasticity and are able to carry out a diverse array of functions . in order to accomplish this , macrophages respond to signals from cytokines , chemokines , growth factors , and pathogen - derived factors in the microenvironment . in the early stages of an infection , macrophages are activated by interferons produced by infected cells and by bacterial - derived compounds such as flagella , lipopolysaccharide ( lps ) , and unmethylated cpg motifs [ 1921 ] . these signals typically induce a proinflammatory response in macrophages to limit pathogen spread and recruit additional innate and adaptive immune cells to the site of infection . after the infection has been controlled and the pathogen cleared , macrophages are instrumental in the resolution of inflammation to prevent further tissue damage . cytokines such as interleukin-4 ( il-4 ) and il-13 can promote an anti - inflammatory response in macrophages to block additional activation of immune cells in the tissue and promote tissue remodeling and collagen deposition [ 22 , 23 ] . in addition to their roles in pathogen clearance and wound healing , macrophages can also respond to cytokines present in the tissue microenvironment during development , where complex and reciprocal interactions take place between epithelial and stromal cells . beginning early in embryogenesis , patterning of the mammary glands occurs with the specification of the sites of the developing glands [ 2426 ] . as development continues , epithelial cells invaginate into the surrounding mesenchyme and form the mammary bud . just prior to birth , the cells begin to proliferate and allow the bud to invade into the adjacent fat pad . once this has occurred , the mammary epithelial cells ( mecs ) begin a process of ductal morphogenesis to generate a rudimentary ductal tree [ 27 , 28 ] . a prominent structure in pubertal mammary gland development is the terminal end bud ( teb ) , the site of actively proliferating epithelial cells . these organized structures are found at the distal end of the mammary ducts and contain cap cells and body cells , which give rise to cells of the myoepithelial and luminal lineages , respectively [ 29 , 30 ] . as the cells proliferate , the tebs advance through the fat pad until they reach the edge , at which time they regress to form the terminal ducts . at this point , side branching occurs to create secondary and tertiary ducts from the main ducts to fill the entire fat pad laterally . the mammary gland undergoes large - scale expansions and regressions during repeated estrous cycles , with new epithelial buds sprouting from the ducts and subsequently disappearing as estrogen and progesterone levels rise and fall [ 31 , 32 ] . during pregnancy , however , these hormone - induced changes stop being cyclical and the gland enters a state of preparation for lactation . alveolar buds form in response to prolactin and develop into mature alveoli to produce milk [ 33 , 34 ] . after weaning , the mammary gland must return to its resting , prepregnancy state through a tightly regulated process of programmed cell death called involution . at this time , the mammary gland begins to expand and regress again during estrous cycles and is ready to expand again in response to another pregnancy . during postnatal development , previous work has shown that cytokines il-4 and il-13 are critical for promoting the differentiation and maturation of luminal epithelial cells . additionally , the requirement of estrogen receptor ( er ) and progesterone receptor ( pr ) signaling in pubertal development has been demonstrated through elegant tissue recombination studies . while embryonic development is unaffected , mammary glands of er-null mice fail to elongate through the fat pad during pubertal development and lack defined tebs . despite this lack of outgrowth , er-null epithelium is still responsive to progesterone and form alveoli during pregnancy . the requirement of er signaling is limited to the epithelial cells , as transplantation of wild - type mecs into an er-null fat pad results in normal ductal morphogenesis . additional studies have shown a differing role for pr signaling , with transplantation of pr - null mecs into wild - type fat pad resulting in the formation of a normal ductal tree . as expected , however , pr - null mecs fail to respond to progesterone during pregnancy and do not form alveolar structures . intriguingly , transplantation of wild - type mecs into a pr - null fat results in a modest defect in ductal outgrowth , suggesting a role for pr signaling in stromal cells regulating mec proliferation in a paracrine manner . notably , er and pr signaling promote mec proliferation in a paracrine manner , with previous reports demonstrating that proliferating cells are not contained within the er or pr compartments [ 3941 ] . hormone signaling is a tightly regulated process , with any deviations above or below the optimal levels resulting in similar defects . exposure to exogenous estrogen treatment results in decreased ductal elongation , similar to results seen in er-null mec transplants ; however , estrogen treatment also leads to increased lateral branching . thus , keeping hormone levels and signaling within a specified range is of critical importance for maintaining mammary gland integrity . as a cell type that serves to act as a first line of defense against foreign substances and pathogens , it is only logical to have macrophages dispersed throughout the body . but in addition to their role as immunological surveyors , macrophages also play critical roles in regulating mammary gland development . previous studies have indicated that macrophages are found in close association with mecs at many well - characterized stages of mammary gland development . immunostaining of mammary glands for the macrophage marker f4/80 indicates the presence of macrophages surrounding the body cells of the teb [ 43 , 44 ] . these macrophages are poised to phagocytose cellular debris from mecs undergoing apoptosis while generating the hollow lumen of the mammary ducts . at maturity , macrophages can be found lining the mammary ducts where they promote epithelial cell proliferation and differentiation through production of growth factors , chemokines , and inflammatory mediators . during lactation , f4/80 macrophages have been observed in close proximity to the alveoli and are a major cellular component of milk [ 43 , 44 , 46 ] . once lactation is completed and weaning occurs , the mammary gland undergoes involution to return to its prepregnant state , involving large amounts of apoptosis and extracellular matrix ( ecm ) remodeling . again , macrophages are major contributors to this process , phagocytosing apoptotic cellular debris and producing matrix remodeling factors to facilitate the transition back to the fully involuted state [ 47 , 48 ] . numerous studies have been undertaken using genetic and biochemical approaches to deplete macrophages during mammary gland development . mice homozygous for a null mutation in csf-1 , the critical factor required for macrophage differentiation , show significant impairment in ductal elongation during mammary gland development . this defect can be rescued through the use of a tetracycline - inducible transgene to reexpress csf-1 . architecturally , organization of collagen i into long fibers around the neck of the tebs is impaired in csf-1-deficient mice while total collagen i deposition is unaffected , implicating a specific role for macrophages in regulating collagen organization but not collagen biosynthesis . the contributions of macrophages to estrous - cycle induced changes were described elegantly using the cd11b - dtr inducible mouse model of macrophage depletion . macrophages are found at different frequencies in the mammary gland during the estrous cycle , reaching a maximum during diestrus . depletion of macrophages resulted in a nearly 50% reduction in alveolar bud formation in response to progesterone treatment and an overall decrease in mec proliferation . additional work using sublethal irradiation has demonstrated that cells of the hematopoietic lineage are required for the formation of tebs during pubertal development and that macrophages modulate their immunostimulatory profile over the course of the estrous cycle [ 45 , 52 ] . while these studies clearly demonstrate that a role for macrophages is regulating mammary gland development , the mechanism by which this occurs remains unclear . one possible mechanism is that macrophages in the microenvironment respond to the same cytokines and growth factors required for epithelial cell development and respond in a unique way . il-4 and il-13 have been implicated in mammary epithelial cell differentiation and are found at measureable amounts in the developing mammary gland . when exposed to these cytokines , macrophages respond by producing a host of anti - inflammatory factors and tissue remodeling agents known to be needed during mammary gland development . studies of macrophages in infection models have illustrated that tissue - resident macrophages are more predisposed to an anti - inflammatory response compared to monocyte - derived macrophages recruited from the circulation [ 53 , 54 ] . transforming growth factor - beta ( tgf- ) and members of the matrix metalloproteinases ( mmp ) family are produced by macrophages at high levels in response to il-4/il-13 stimulation in vitro [ 19 , 55 ] . in the setting of the mammary gland in vivo , mmps are required to degrade and remodel the ecm to allow further ductal elongation to occur through the fat pad , while tgf- plays a suppressive role to limit the extent of ductal branching [ 5659 ] . thus , it is possible that il-4 and il-13 play dual roles in the microenvironment : promoting mec differentiation and stimulating tissue - resident macrophage function . while ductal elongation is driven primarily by ovarian - produced estrogen , studies in breast cancer have shown that macrophages themselves are capable of producing estrogen locally through the expression of the estrogen synthesizing enzyme aromatase . there is a relative lack of knowledge to date regarding the role of macrophage - produced estrogen , but it is tempting to speculate that macrophages associated with the tebs or lining the mammary ducts could regulate development and proliferation directly by creating pools of locally concentrated estrogen . further studies are warranted to determine if macrophages express aromatase in vivo and how the resulting rise in estrogen levels in the mammary gland affects development . in addition , the increased estrogen and proliferative signals in the mammary gland may also help establish a protumorigenic environment , in which the mecs are primed for the tumor initiation when exposed to an oncogenic insult . understanding how changes that take place in the mammary gland during development can affect tumor initiation at a later point in life is critical in developing preventative strategies through life - style changes and therapeutic intervention . recent evidence has supported the long - postulated idea that chronic inflammation enhances the risk of developing cancer [ 6164 ] . furthermore , diseases with systemic inflammatory components are major risk factors for certain types of cancer , including breast cancer [ 61 , 65 ] . in patients with crohn 's disease , increased expression of the proinflammatory cytokine tumor necrosis factor - alpha ( tnf ) recruits inflammatory macrophages and leads to the production of additional proinflammatory factors , initiating a feed - forward loop which leads to tissue damage and predisposition to oncogenic initiation . one of the most common diseases associated with cancer risk is obesity , with 34.9% of adults in the united states being classified as obese . patients with obesity often have elevated serum levels of proinflammatory molecules , such as il-6 , which induce a systemic chronic inflammatory state . in the mammary gland microenvironment specifically , obesity is directly linked with increased il-6 signaling and increased macrophage recruitment compared to normal - weight mammoplasty specimens . in a resting state , the amount of proinflammatory and anti - inflammatory signals are maintained in a state of equilibrium ( figure 1 ) . however , in pathologic settings such as obesity , inflammatory homeostasis is lost and the balance is tipped in favor of proinflammatory factors . in these cases , the increased abundance of proinflammatory factors relative to anti - inflammatory factors affects cells in the microenvironment . once macrophages are exposed to proinflammatory factors they upregulate the production of additional proinflammatory factors , creating a feed - forward loop that further upsets inflammatory homeostasis . it is interesting to speculate why obese patients with increased levels of il-6 have a predisposition to developing er breast cancers specifically . studies focused on endometrial carcinoma have revealed a paracrine signaling axis whereby cancer cells produce il-6 to stimulate stromal cells to upregulate aromatase and produce estrogen , thus inducing a cycle of increased cancer cell proliferation and il-6 production . it remains to be seen if a similar axis exists in breast cancer , but with their role in regulating mammary gland development , it is not difficult to hypothesize that macrophages may upregulate aromatase expression in response to il-6 in the context of obesity , thus providing a mechanistic explanation of the propensity for obese women to develop er breast tumors . in addition to pathologic inflammatory conditions , acute inflammatory responses in the context of normal tissue processes can have profound impacts on the microenvironment . in the 5-year period following childbirth women are susceptible to developing postpartum breast cancer with a particularly poor prognosis . elegant xenograft studies in mice have revealed that the microenvironment of the involuting mammary gland significantly enhances tumor growth compared to nulliparous mammary glands . most recently , an overall profile was created to determine the relative abundance of immune cells during the process of involution compared to nulliparous and lactating glands . while modest changes were observed in dc recruitment at all time points of involution , a near 10-fold increase in macrophage recruitment is observed during the first week of involution and remains elevated at 4 weeks after weaning . this increased macrophage recruitment was accompanied by increased cd4 and cd8 t cell recruitment and an increased presence of foxp3 regulatory t cells . mechanistically , the microenvironment of the involuting mammary gland induces macrophages to take on an immunosuppressive profile by producing il-10 and suppressing t cell activation . this acute disruption of inflammatory homeostasis results in the formation of a protumorigenic niche through direct suppression of adaptive immunity . a better understanding of the critical balance between proinflammatory and anti - inflammatory factors is clearly needed in order to develop new therapeutic regimens for the treatment and even prevention of breast cancer . in addition to their contributions to normal mammary gland development , macrophages are well - established constituents of the breast tumor microenvironment . increased macrophage density in pretreatment biopsies of breast cancer patients correlates with reduced recurrence - free and overall survival [ 7476 ] . therefore , efforts have focused on understanding the mechanisms through which macrophages contribute to breast cancer growth and progression and these topics have been reviewed extensively [ 19 , 55 , 7779 ] . myeloid cells in the tumor microenvironment , in particular macrophages , have been shown to contribute to tumor growth and progression in a variety of ways . tumor - associated macrophages ( tams ) secrete soluble factors , such as vascular endothelial growth factor ( vegf ) , which induce angiogenesis and partially relieve the hypoxic stress within fast - growing tumors . in addition to promoting angiogenesis , tams support tumor cell survival , migration , and invasion through the secretion of growth factors such as egf and fgfs and chemokines such as cxcl1/2 [ 8184 ] . of note , tams not only secrete factors but also facilitate the release of protumorigenic factors from the ecm , a topic discussed later in this review . in recent studies using intravital imaging techniques , lohela et al . demonstrated that prolonged depletion of myeloid - derived cells in a model of breast cancer resulted in delayed tumor growth , decreased angiogenesis , and fewer lung metastases . furthermore , production of growth factors and ecm remodeling by tams have been implicated in promoting breast cancer resistance to chemotherapeutic agents such as paclitaxel , doxorubicin , and etoposide ( reviewed in ) . finally , numerous studies have provided evidence of tams interacting with cells of the adaptive immune system , mainly cd4 and cd8 t lymphocytes , and both directly and indirectly suppressing their antitumor effects [ 8789 ] . understanding macrophage functions in the context of normal tissue development can provide insights into the functions of macrophages during tumor growth and progression . specifically , there are parallels between the mechanisms of macrophage recruitment and macrophage - mediated alterations in ecm in both the normal mammary gland and the tumor microenvironment . furthermore , it is becoming clear that the balance between proinflammatory and anti - inflammatory factors is key to the regulation of macrophage function within the tumor microenvironment . therefore , further discussion will focus on macrophage recruitment , polarization , and regulation of ecm within the tumor microenvironment . as mentioned above , ccl2 and csf-1 likewise , these factors have been implicated in recruitment of macrophages to both primary and metastatic tumor sites . using genetic approaches , seminal studies demonstrated that csf-1 is critical for macrophage recruitment and differentiation in tumor microenvironment of mmtv - pymt mice . these studies demonstrated that reduced macrophage infiltration significantly reduced the ability of the tumor cells to metastasize to the lung . tumor cell - derived csf-1 has also been linked to the proliferation of a protumor subset of cd11bf4/80 macrophages in the mmtv - neu transgenic model of mammary tumor growth . in these studies , administration of the csf-1r inhibitor gw2580 to tumor bearing mice drastically reduced the numbers of cd11bf4/80 macrophages in s phase . these , and other recent studies , suggest that in addition to recruitment of monocytes from the bloodstream , certain tam populations are able to proliferate within the tumor microenvironment [ 9193 ] . taken together , these studies indicate that therapies aimed at targeting the accumulation and/or proliferation of tams may improve clinical outcomes for breast cancer patients , and as a result csf-1r inhibitors and blocking antibodies have entered clinical trials for various cancer types , including breast cancer . in a recent report , ries et al . described a significant depletion of cd68/cd168 macrophages in a small cohort of breast cancer patients and among those receiving the highest protocol dose , analysis revealed a switch of lymphocyte infiltrates from cd4 t cells before treatment to cd8 t cells after treatment . this study provides proof - of - principal that blockade of the csf-1/csf-1r pathway results in fewer macrophages recruited to human breast tumors , and this change in myeloid recruitment affects the overall composition of the tumor microenvironment . another key chemokine that has been implicated in macrophage recruitment to the tumor microenvironment is ccl2/mcp-1 . numerous studies have found that tumor cell - derived ccl2 promotes macrophage recruitment both in vitro and in vivo [ 9597 ] . in recent studies , both ccl2 and ccl5/rantes were found to correlate with increased macrophage recruitment in human patient samples , and specifically in er samples . using estrogen - supplemented oophorectomized mice bearing mmtv - pymt mammary tumors , further studies demonstrated that inhibition of either ccl2 or ccl5 using blocking antibodies resulted in reduced macrophage infiltration and reduced tumor growth . in addition to promoting recruitment of macrophages to the primary tumor site , ccl2 has also been implicated in indirectly promoting the seeding and growth of tumor cells in the metastatic site . specifically , ccl2 was found to recruit a distinct population of macrophages termed metastasis - associated macrophages , defined as cd11bly6c , to the lung metastatic site . once localized to this site , ccr2 activation stimulates macrophages to secrete an additional chemokine , ccl3 , which contributes to tumor cell - macrophage interactions and retention in the metastatic site through activation of ccr1 . taken together , these studies suggest that blocking macrophage recruitment through inhibition of chemokine signaling may effectively reduce macrophage contributions during tumor growth and progression . however , some challenges have been associated with targeting chemokines including the induction of compensatory mechanisms in response to chemokine inhibition . in a recent study evaluating ccl2 blockade , bonapace et al . found that while blocking ccl2 reduced lung metastasis , which was maintained upon continuous ccl2 inhibition , cessation of ccl2 neutralization led to increased metastasis and accelerated death . assessment of combinatorial therapies , which included targeting additional cytokines , such as il-6 , that were increased in the lungs upon treatment cessation , alleviated the increase in metastasis . thus , these studies suggest that targeting chemokines , such as ccl2 , as a therapeutic strategy should be approached with caution and could possibly require combination - based approaches for success . in addition to csf-1 and ccl2 , other chemokines have also been linked to macrophage recruitment in the primary tumor site . using an inducible model of mammary tumorigenesis , we identified cx3cl1 as a mediator of macrophage recruitment to early stage mammary hyperplasias . more recent studies have linked cx3cl1 expression with poor outcome in breast cancer patients , although whether high cx3cl1 is linked to macrophage recruitment in human breast cancer samples remains to be determined . boyle et al . recently reported that ccl20-ccr6 axis is important for regulating macrophage recruitment into mammary tumors of mmtv - pymt mice . in these studies , growth of mammary tumors in ccr6-knockout mice led to reduced mammary tumor initiation and growth . further analysis of these tumors revealed a reduction in immune cell infiltration along with changes in macrophage polarization as shown by reduced expression of il-4r and cd206 . importantly , reconstitution of tams into ccr6-knockout mice bearing orthotopically transplanted mmtv - pymt tumors restored tumor growth demonstrating the importance of this chemokine axis for mammary tumor growth . in addition to general recruitment to the tumor microenvironment , a subpopulation of macrophages is also known to accumulate in hypoxic regions within tumors . recruitment of macrophages into hypoxic regions is mediated through soluble factors such as vegf , endothelin-2 , and angiopoietin-2 [ 104 , 105 ] . semaphorins , such as sema3a , were recently linked to recruitment of macrophages to hypoxic regions via a neuropilin-1-dependent mechanism . additional recent studies have also found that hypoxic cancer cells produce chemoattractants that promote macrophage recruitment , including oncostatin m and eotaxin , which also act to polarize macrophages to a protumor phenotype and are required for tumor progression . taken together , these studies demonstrate that macrophage recruitment into the tumor microenvironment can be driven by many different factors , highlighting the complexity of the mechanisms driving macrophage infiltration . although less extensively studied compared with tumor cell - derived chemokines , stromal cells , including carcinoma associated fibroblasts ( cafs ) , mesenchymal stem cells ( mscs ) , and endothelial cells , also produce chemokines that can potentially recruit macrophages into the microenvironment . stimulation of cafs and mscs with tumor cell - derived conditioned media leads to upregulation of various chemokines , including ccl2 , cxcl8 , and ccl5 . furthermore , yoshimura et al . demonstrated that stromal cell - derived ccl2 contributes to macrophage recruitment to 4t1 tumors and that loss of stromal cell ccl2 leads to decreased lung metastasis . recent genetic studies have demonstrated a critical role for bmp signaling in the regulation of chemokines from fibroblasts . specifically , loss of bmpr2 from fibroblasts led to increased metastasis of mmtv - pymt tumors corresponding with increased chemokine expression and increased infiltration of myeloid cells . in addition to chemoattractants derived from tumor and stromal cells , there is evidence that tumor - associated ecm may also contribute to macrophage recruitment . furthermore , it has been proposed that proteolysis of collagen i promotes macrophage recruitment into the involuting mammary gland , which is characterized as a tumor - promoting environment . another ecm component linked to macrophage recruitment is hyaluronan , which is a glycosaminoglycan consisting of repeating disaccharide subunits of glucuronic acid and n - acetylglucosamine . macrophages are often associated with a hyaluronan - containing matrix within the tumor environment , and studies have suggested that hyaluronan can act directly on macrophages to regulate their migration . specifically , hyaluronan has been shown to promote macrophage chemotaxis using in vitro chemotaxis assays . consistent with these findings , in vivo studies have demonstrated that reduction of hyaluronan in the mammary tumor stroma correlates with decreased macrophage infiltration . taken together , the numerous studies focusing on macrophage recruitment demonstrate that macrophage infiltration into the tumor microenvironment can potentially be mediated by a variety of factors ( figure 2 ) . further studies are warranted to understand the relative contributions of tumor cell versus stromal cell derived chemokines and ecm components to macrophage recruitment during tumor growth and progression . once recruited to the tumor microenvironment , macrophages respond to the plethora of stimuli within the microenvironment and differentiate into various effector subsets . currently , the most widely accepted classification of macrophage polarization is based on descriptions of classical ( m1 ) versus alternative ( m2 ) polarization , which were developed as a result of initial studies investigating macrophage responses to helper t cells 1 ( th1 ) and helper t cell 2 ( th2 ) derived molecules . classically activated macrophages develop in response to interferon - gamma ( ifn ) and pathogen - derived toll - like receptor ligands [ 19 , 117 ] . this response is characterized by the production of cytotoxic factors such as reactive oxygen species and nitric oxide , increased rates of phagocytosis , and enhanced antigen presentation on the cell surface . alternatively activated macrophages , on the other hand , develop as part of the wound healing program and as such are thought to antagonize inflammation . m2 macrophages are induced by the th2 cytokines il-4 and il-13 , as well as in response to il-10 , immunoglobulins , and glucocorticoids [ 55 , 118 ] . these cells , in turn , secrete factors that promote angiogenesis , upregulate expression of scavenging receptors , and produce enzymes to remodel the surrounding extracellular matrix . as interest and work in the field of macrophage biology has expanded , the nomenclature describing the activation status of macrophages has become complex and often confusing . in an attempt to streamline the methods used to generate and describe the cells used by the different research groups , murray et al . published a comprehensive set of recommendations which will undoubtedly simplify future analysis and comparison of macrophage subsets . based on their functions within the tumor microenvironment , several studies have demonstrated that tams express higher levels of scavenging receptors , angiogenic factors , and proteases , similar to m2 macrophages . furthermore , tam polarization to the m2-like phenotype in the mmtv - pymt model has been attributed to il-4-producing th2 cells within the tumor microenvironment . however , there is evidence that macrophages exhibit different phenotypes during different stages of tumor initiation and progression . during early stages of transformation , recently recruited macrophages are exposed to a wide variety of proinflammatory signals derived from the epithelial cells and the surrounding stroma and often express m1-related factors that have protumorigenic properties , such as il-1 and il-6 [ 120 , 121 ] . as a component of the proinflammatory response , production of reactive oxygen and nitrogen species could also potentially enhance the rate of epithelial cell mutation and thus accelerate tumorigenesis . in established tumors , macrophages exhibit alternatively activated functions including the production of immunosuppressive factors , such as il-10 and tgf- , which are capable of actively suppressing the antitumor immune response [ 79 , 88 , 89 ] . these macrophages also produce growth factors and remodel the matrix , supporting tumor cell growth and enhancing invasion . therefore , tam phenotypes are now thought to include a combination of markers typically assigned to the m1 and m2 phenotypes . macrophages within the tumor microenvironment towards the m1/classically activated phenotype , care must be taken to ensure that the potentially protumorigenic functions of these macrophages are suppressed . recent sophisticated analyses utilizing genomewide studies and rna - sequencing have revealed that macrophage phenotypes in vivo are far more heterogeneous and complex than initially expected . xue et al . performed a detailed transcriptome analysis of primary human monocytes stimulated with 28 different signals , the results of which suggest a spectrum model where 9 different macrophage activation programs were identified in response to different combinations of stimuli . analysis of the enriched gene sets in human macrophages derived from smokers and copd patients revealed activation programs within these primary macrophages that were significantly different from the hypothesized phenotypes . in smokers ' samples , a complex network of stimuli including glucocorticoids , free fatty acids , and il-4 were detected , while in copd patient samples the previously published il-4/il-13 associated gene signatures were not reproduced and instead a profound loss of inflammatory genes was reported . these results demonstrate the complexity of activating signals responsible for the phenotypes of macrophages in human pathologies , and they suggest that a simple bipolar m1/m2 paradigm may not be sufficient to describe macrophages associated with disease states . based on the observation that the microenvironment of lung disease is capable of producing a spectrum of macrophage activation states , it seems likely that this heterogeneity would also be observed in the tumor microenvironment . indeed , while performing gene - expression profiling on tams and mammary tissue macrophages from tumor bearing mmtv - pymt mice , franklin et al instead , they reported tam differentiation to be dependent on signaling of the transcription factor rbpj , a key regulator of canonical notch signaling . in addition , recent evidence suggests that individual tumors may contain several different subsets of macrophages and those might differ in their functions . movahedi et al . reported the presence of two distinct tam populations in mammary ts / a tumors , distinguishable most easily by the level of mhcii expression on their surface . mhcii macrophages were shown to reside mainly in hypoxic tumor regions and expressed markers associated with m2 polarization . the mhcii subset , however , expressed m1-signature genes such as cox2 , nos2 , and il-12 . these cells were shown to secrete proinflammatory cytokines and chemokines such as il-6 , ccl5 , and cxcl3 , which could in turn serve to further recruit additional proinflammatory cells to the tumor margins . however , both macrophage subsets were shown to be poor antigen presenting cells and were able to suppress t cell proliferation , indicating that both subsets might be capable of contributing to protumor immunosuppression . interestingly , ruffell et al . observed a similar localization of mhcii and mhcii tams in mammary tumors derived from mmtv - pymt mice ; however , the ability of tams to suppress cd8 t cell proliferation was limited to the mhcii subset of cells . these findings indicate that some tam properties are most likely universal ( recruitment , localization ) , whereas other properties ( specific interactions with other infiltrating cells ) might be dependent on the tumor model under investigation . in a recent study examining macrophage localization within human breast tumors , high cd68 macrophage staining within gaps of ductal tumor structures correlated with reduced lymph node metastasis . taken together , these data suggest that tams represent a macrophage population that is distinct from both m1 and m2 macrophages as they are canonically described in the setting of infection , but there is most likely a spectrum of tams whose phenotype and function depend on tumor type and location within the tumor . one of the identified mechanisms through which macrophages may regulate ductal elongation during mammary gland development is through organization of ecm , such as collagen . while some functions of tams in the tumor microenvironment , including promotion of tumor cell migration and invasion , angiogenesis , and suppression of adaptive immune responses , have been extensively examined , the contributions of macrophages to the modulation of ecm remain relatively understudied . macrophages actively contribute to the changes in ecm through the production of ecm components and through the release of factors that cleave ecm . consequently , ecm components and their fragments can act directly on macrophages to promote their recruitment , retention , and function . one of the mechanisms through which alternatively activated macrophages contribute to resolution of inflammation is through producing and remodeling the ecm . macrophages have been found to produce fibronectin and collagen , including high levels of type vi collagen , which is increased in alternatively activated macrophages and promotes monocyte adhesion . while studies focusing on the contributions of macrophages to ecm deposition in the context of breast cancer are limited , it is worth noting that collagen vi is found at the invasive edge of breast tumors , where macrophages are known to localize , and promotes epithelial - mesenchymal transition of cancer cells . thus , studies aimed at determining whether macrophages at the leading edge contribute to these high levels of collagen vi are warranted . in addition to producing ecm , macrophages express high levels of proteases that can contribute to the cleavage and remodeling of ecm . gene profiling of tams demonstrates increased expression of proteases including mmps , adams , and cathepsins . macrophage - derived proteases can contribute to protumor alterations in the stroma in a number of ways including facilitating ecm breakdown for subsequent invasion and migration , liberation of tumor - promoting factors from the ecm , and generation of bioactive ecm fragments . for example , macrophage - derived mmps have been linked to the release of angiogenic factors , such as vegf and fgfs , in the tumor microenvironment . in addition , studies demonstrated that alternatively activated macrophages are directly involved in collagen turnover , specifically through uptake and degradation of collagen by cx3cr1-positive cells involving the mannose receptor . uptake of collagen requires mmp activity , potentially linking macrophage regulation of collagen to both cleavage and uptake . hyaluronan is generated as a high molecular weight glycosaminoglycan that can be broken down into fragments that are characterized as inflammatory and protumorigenic . hyaluronan cleavage occurs through enzymatic degradation by hyaluronidases ( hyals ) or by mechanisms involving reactive oxygen and nitrogen species . macrophages have been found to express hyaluronidases and can thus potentially contribute to the breakdown of hyaluronan during inflammation and tumor progression . in turn , it has been suggested that hyaluronan can direct macrophage function . exposure of macrophages to hyaluronan , either purified or tumor - derived , leads to increased expression of various inflammatory mediators including il-1 and il-10 . in recent studies , tumor - derived microvesicles were found to induce il-10 expression in macrophages using a hyaluronan - dependent mechanism through the pi3k / akt / mtor pathway . together , these studies suggest a link between hyaluronan and regulation of macrophage function , possibly through enhancing immunosuppressive function . together , these observations suggest that macrophages are likely to be important regulators of ecm production and remodeling in the tumor microenvironment , and further studies are warranted to define the specific functional consequences of these actions . in conclusion , it is clear that inflammation is a complex process that has evolved to resolve damage to the body caused by pathogens or disease . in the normal mammary gland , tissue - resident macrophages play a vital role in the regulation of development and maintenance of tissue homeostasis . pro- and anti - inflammatory factors produced in the microenvironment act not only on epithelial cells , but also on macrophages and lead to the further disruption of inflammatory homeostasis and the creation of a protumorigenic niche that is primed for oncogenic initiation . tumor cells acquire the capacity to harness the functions of inflammatory cells , such as macrophages , to aid in their growth and progression . experimental studies have demonstrated that macrophages interact with cancer cells and their phenotype and function evolve as the tumor itself evolves . however , recent studies demonstrating the complexity of macrophage polarization and the impact of macrophage localization within the tumor microenvironment suggest that the contributions of macrophages to breast cancer growth and progression are likely to be quite complex . therefore , it will be critical to obtain a better understanding of the mechanisms that drive macrophage recruitment , polarization , and function within the tumor microenvironment at different stages of breast cancer formation and progression .

macrophages are critical mediators of inflammation and important regulators of developmental processes . as a key phagocytic cell type , macrophages evolved as part of the innate immune system to engulf and process cell debris and pathogens . macrophages produce factors that act directly on their microenvironment and also bridge innate immune responses to the adaptive immune system . resident macrophages are important for acting as sensors for tissue damage and maintaining tissue homeostasis . it is now well - established that macrophages are an integral component of the breast tumor microenvironment , where they contribute to tumor growth and progression , likely through many of the mechanisms that are utilized during normal wound healing responses . because macrophages contribute to normal mammary gland development and breast cancer growth and progression , this review will discuss both resident mammary gland macrophages and tumor - associated macrophages with an emphasis on describing how macrophages interact with their surrounding environment during normal development and in the context of cancer .

1. Introduction to Macrophages 2. Resident Macrophages in the Mammary Gland 3. Macrophages in the Tumor Microenvironment 4. Summary

as a cell of the innate immune system , macrophages play critical roles in both host defense against pathogens and proper tissue development . during embryonic development , a population of macrophages derived from yolk sac hematopoiesis can be found throughout the organism and are thought to contribute to the populations of tissue - resident macrophages in the adult . this process occurs prior to the induction of hematopoiesis in the bone marrow , strongly suggesting a unique origin and function for these embryonic macrophages [ 1 , 2 ] . additionally , embryonically derived , tissue - resident macrophages have been found in a diverse array of organs and tissues , including the mammary gland , and the maintenance of these populations does not require monocyte precursors . patrolling monocytes are responsible for crawling along the luminal side of the endothelium to monitor for danger - associated molecular patterns ( damps ) and , upon encountering such a signal , rapidly entering the tissue and beginning to recruit additional effector cells in order to start a productive immune response . a major function of inflammatory monocytes is to respond to sites of inflammation and tissue damage . upon arriving in the vasculature near the site of inflammation , monocytes begin a process of rolling adhesion in which selectin molecules on the surface of the endothelial cells bind to selectin ligands on the monocytes [ 12 , 13 ] . once in the tissue , monocytes can be further differentiated to macrophages in the presence of colony stimulating factor-1 ( csf-1 ) to carry out effector functions involved in pathogen clearance , wound healing , and developmental regulation [ 17 , 18 ] . macrophages are a cell type with exquisite plasticity and are able to carry out a diverse array of functions . in order to accomplish this , macrophages respond to signals from cytokines , chemokines , growth factors , and pathogen - derived factors in the microenvironment . in the early stages of an infection , macrophages are activated by interferons produced by infected cells and by bacterial - derived compounds such as flagella , lipopolysaccharide ( lps ) , and unmethylated cpg motifs [ 1921 ] . these signals typically induce a proinflammatory response in macrophages to limit pathogen spread and recruit additional innate and adaptive immune cells to the site of infection . after the infection has been controlled and the pathogen cleared , macrophages are instrumental in the resolution of inflammation to prevent further tissue damage . in addition to their roles in pathogen clearance and wound healing , macrophages can also respond to cytokines present in the tissue microenvironment during development , where complex and reciprocal interactions take place between epithelial and stromal cells . a prominent structure in pubertal mammary gland development is the terminal end bud ( teb ) , the site of actively proliferating epithelial cells . additional studies have shown a differing role for pr signaling , with transplantation of pr - null mecs into wild - type fat pad resulting in the formation of a normal ductal tree . as a cell type that serves to act as a first line of defense against foreign substances and pathogens , it is only logical to have macrophages dispersed throughout the body . but in addition to their role as immunological surveyors , macrophages also play critical roles in regulating mammary gland development . previous studies have indicated that macrophages are found in close association with mecs at many well - characterized stages of mammary gland development . these macrophages are poised to phagocytose cellular debris from mecs undergoing apoptosis while generating the hollow lumen of the mammary ducts . at maturity , macrophages can be found lining the mammary ducts where they promote epithelial cell proliferation and differentiation through production of growth factors , chemokines , and inflammatory mediators . during lactation , f4/80 macrophages have been observed in close proximity to the alveoli and are a major cellular component of milk [ 43 , 44 , 46 ] . again , macrophages are major contributors to this process , phagocytosing apoptotic cellular debris and producing matrix remodeling factors to facilitate the transition back to the fully involuted state [ 47 , 48 ] . numerous studies have been undertaken using genetic and biochemical approaches to deplete macrophages during mammary gland development . mice homozygous for a null mutation in csf-1 , the critical factor required for macrophage differentiation , show significant impairment in ductal elongation during mammary gland development . macrophages are found at different frequencies in the mammary gland during the estrous cycle , reaching a maximum during diestrus . additional work using sublethal irradiation has demonstrated that cells of the hematopoietic lineage are required for the formation of tebs during pubertal development and that macrophages modulate their immunostimulatory profile over the course of the estrous cycle [ 45 , 52 ] . while these studies clearly demonstrate that a role for macrophages is regulating mammary gland development , the mechanism by which this occurs remains unclear . one possible mechanism is that macrophages in the microenvironment respond to the same cytokines and growth factors required for epithelial cell development and respond in a unique way . il-4 and il-13 have been implicated in mammary epithelial cell differentiation and are found at measureable amounts in the developing mammary gland . when exposed to these cytokines , macrophages respond by producing a host of anti - inflammatory factors and tissue remodeling agents known to be needed during mammary gland development . studies of macrophages in infection models have illustrated that tissue - resident macrophages are more predisposed to an anti - inflammatory response compared to monocyte - derived macrophages recruited from the circulation [ 53 , 54 ] . in the setting of the mammary gland in vivo , mmps are required to degrade and remodel the ecm to allow further ductal elongation to occur through the fat pad , while tgf- plays a suppressive role to limit the extent of ductal branching [ 5659 ] . thus , it is possible that il-4 and il-13 play dual roles in the microenvironment : promoting mec differentiation and stimulating tissue - resident macrophage function . while ductal elongation is driven primarily by ovarian - produced estrogen , studies in breast cancer have shown that macrophages themselves are capable of producing estrogen locally through the expression of the estrogen synthesizing enzyme aromatase . there is a relative lack of knowledge to date regarding the role of macrophage - produced estrogen , but it is tempting to speculate that macrophages associated with the tebs or lining the mammary ducts could regulate development and proliferation directly by creating pools of locally concentrated estrogen . further studies are warranted to determine if macrophages express aromatase in vivo and how the resulting rise in estrogen levels in the mammary gland affects development . in addition , the increased estrogen and proliferative signals in the mammary gland may also help establish a protumorigenic environment , in which the mecs are primed for the tumor initiation when exposed to an oncogenic insult . understanding how changes that take place in the mammary gland during development can affect tumor initiation at a later point in life is critical in developing preventative strategies through life - style changes and therapeutic intervention . furthermore , diseases with systemic inflammatory components are major risk factors for certain types of cancer , including breast cancer [ 61 , 65 ] . in patients with crohn 's disease , increased expression of the proinflammatory cytokine tumor necrosis factor - alpha ( tnf ) recruits inflammatory macrophages and leads to the production of additional proinflammatory factors , initiating a feed - forward loop which leads to tissue damage and predisposition to oncogenic initiation . one of the most common diseases associated with cancer risk is obesity , with 34.9% of adults in the united states being classified as obese . in the mammary gland microenvironment specifically , obesity is directly linked with increased il-6 signaling and increased macrophage recruitment compared to normal - weight mammoplasty specimens . it remains to be seen if a similar axis exists in breast cancer , but with their role in regulating mammary gland development , it is not difficult to hypothesize that macrophages may upregulate aromatase expression in response to il-6 in the context of obesity , thus providing a mechanistic explanation of the propensity for obese women to develop er breast tumors . in addition to pathologic inflammatory conditions , acute inflammatory responses in the context of normal tissue processes can have profound impacts on the microenvironment . in the 5-year period following childbirth women are susceptible to developing postpartum breast cancer with a particularly poor prognosis . elegant xenograft studies in mice have revealed that the microenvironment of the involuting mammary gland significantly enhances tumor growth compared to nulliparous mammary glands . mechanistically , the microenvironment of the involuting mammary gland induces macrophages to take on an immunosuppressive profile by producing il-10 and suppressing t cell activation . a better understanding of the critical balance between proinflammatory and anti - inflammatory factors is clearly needed in order to develop new therapeutic regimens for the treatment and even prevention of breast cancer . in addition to their contributions to normal mammary gland development , macrophages are well - established constituents of the breast tumor microenvironment . therefore , efforts have focused on understanding the mechanisms through which macrophages contribute to breast cancer growth and progression and these topics have been reviewed extensively [ 19 , 55 , 7779 ] . myeloid cells in the tumor microenvironment , in particular macrophages , have been shown to contribute to tumor growth and progression in a variety of ways . tumor - associated macrophages ( tams ) secrete soluble factors , such as vascular endothelial growth factor ( vegf ) , which induce angiogenesis and partially relieve the hypoxic stress within fast - growing tumors . of note , tams not only secrete factors but also facilitate the release of protumorigenic factors from the ecm , a topic discussed later in this review . demonstrated that prolonged depletion of myeloid - derived cells in a model of breast cancer resulted in delayed tumor growth , decreased angiogenesis , and fewer lung metastases . finally , numerous studies have provided evidence of tams interacting with cells of the adaptive immune system , mainly cd4 and cd8 t lymphocytes , and both directly and indirectly suppressing their antitumor effects [ 8789 ] . understanding macrophage functions in the context of normal tissue development can provide insights into the functions of macrophages during tumor growth and progression . specifically , there are parallels between the mechanisms of macrophage recruitment and macrophage - mediated alterations in ecm in both the normal mammary gland and the tumor microenvironment . furthermore , it is becoming clear that the balance between proinflammatory and anti - inflammatory factors is key to the regulation of macrophage function within the tumor microenvironment . these studies demonstrated that reduced macrophage infiltration significantly reduced the ability of the tumor cells to metastasize to the lung . tumor cell - derived csf-1 has also been linked to the proliferation of a protumor subset of cd11bf4/80 macrophages in the mmtv - neu transgenic model of mammary tumor growth . in these studies , administration of the csf-1r inhibitor gw2580 to tumor bearing mice drastically reduced the numbers of cd11bf4/80 macrophages in s phase . taken together , these studies indicate that therapies aimed at targeting the accumulation and/or proliferation of tams may improve clinical outcomes for breast cancer patients , and as a result csf-1r inhibitors and blocking antibodies have entered clinical trials for various cancer types , including breast cancer . this study provides proof - of - principal that blockade of the csf-1/csf-1r pathway results in fewer macrophages recruited to human breast tumors , and this change in myeloid recruitment affects the overall composition of the tumor microenvironment . another key chemokine that has been implicated in macrophage recruitment to the tumor microenvironment is ccl2/mcp-1 . in addition to promoting recruitment of macrophages to the primary tumor site , ccl2 has also been implicated in indirectly promoting the seeding and growth of tumor cells in the metastatic site . specifically , ccl2 was found to recruit a distinct population of macrophages termed metastasis - associated macrophages , defined as cd11bly6c , to the lung metastatic site . once localized to this site , ccr2 activation stimulates macrophages to secrete an additional chemokine , ccl3 , which contributes to tumor cell - macrophage interactions and retention in the metastatic site through activation of ccr1 . taken together , these studies suggest that blocking macrophage recruitment through inhibition of chemokine signaling may effectively reduce macrophage contributions during tumor growth and progression . in addition to general recruitment to the tumor microenvironment , a subpopulation of macrophages is also known to accumulate in hypoxic regions within tumors . taken together , these studies demonstrate that macrophage recruitment into the tumor microenvironment can be driven by many different factors , highlighting the complexity of the mechanisms driving macrophage infiltration . in addition to chemoattractants derived from tumor and stromal cells , there is evidence that tumor - associated ecm may also contribute to macrophage recruitment . furthermore , it has been proposed that proteolysis of collagen i promotes macrophage recruitment into the involuting mammary gland , which is characterized as a tumor - promoting environment . macrophages are often associated with a hyaluronan - containing matrix within the tumor environment , and studies have suggested that hyaluronan can act directly on macrophages to regulate their migration . further studies are warranted to understand the relative contributions of tumor cell versus stromal cell derived chemokines and ecm components to macrophage recruitment during tumor growth and progression . once recruited to the tumor microenvironment , macrophages respond to the plethora of stimuli within the microenvironment and differentiate into various effector subsets . currently , the most widely accepted classification of macrophage polarization is based on descriptions of classical ( m1 ) versus alternative ( m2 ) polarization , which were developed as a result of initial studies investigating macrophage responses to helper t cells 1 ( th1 ) and helper t cell 2 ( th2 ) derived molecules . alternatively activated macrophages , on the other hand , develop as part of the wound healing program and as such are thought to antagonize inflammation . based on their functions within the tumor microenvironment , several studies have demonstrated that tams express higher levels of scavenging receptors , angiogenic factors , and proteases , similar to m2 macrophages . furthermore , tam polarization to the m2-like phenotype in the mmtv - pymt model has been attributed to il-4-producing th2 cells within the tumor microenvironment . however , there is evidence that macrophages exhibit different phenotypes during different stages of tumor initiation and progression . during early stages of transformation , recently recruited macrophages are exposed to a wide variety of proinflammatory signals derived from the epithelial cells and the surrounding stroma and often express m1-related factors that have protumorigenic properties , such as il-1 and il-6 [ 120 , 121 ] . as a component of the proinflammatory response , production of reactive oxygen and nitrogen species could also potentially enhance the rate of epithelial cell mutation and thus accelerate tumorigenesis . macrophages within the tumor microenvironment towards the m1/classically activated phenotype , care must be taken to ensure that the potentially protumorigenic functions of these macrophages are suppressed . based on the observation that the microenvironment of lung disease is capable of producing a spectrum of macrophage activation states , it seems likely that this heterogeneity would also be observed in the tumor microenvironment . indeed , while performing gene - expression profiling on tams and mammary tissue macrophages from tumor bearing mmtv - pymt mice , franklin et al instead , they reported tam differentiation to be dependent on signaling of the transcription factor rbpj , a key regulator of canonical notch signaling . one of the identified mechanisms through which macrophages may regulate ductal elongation during mammary gland development is through organization of ecm , such as collagen . while some functions of tams in the tumor microenvironment , including promotion of tumor cell migration and invasion , angiogenesis , and suppression of adaptive immune responses , have been extensively examined , the contributions of macrophages to the modulation of ecm remain relatively understudied . macrophages actively contribute to the changes in ecm through the production of ecm components and through the release of factors that cleave ecm . consequently , ecm components and their fragments can act directly on macrophages to promote their recruitment , retention , and function . one of the mechanisms through which alternatively activated macrophages contribute to resolution of inflammation is through producing and remodeling the ecm . while studies focusing on the contributions of macrophages to ecm deposition in the context of breast cancer are limited , it is worth noting that collagen vi is found at the invasive edge of breast tumors , where macrophages are known to localize , and promotes epithelial - mesenchymal transition of cancer cells . in addition to producing ecm , macrophages express high levels of proteases that can contribute to the cleavage and remodeling of ecm . macrophage - derived proteases can contribute to protumor alterations in the stroma in a number of ways including facilitating ecm breakdown for subsequent invasion and migration , liberation of tumor - promoting factors from the ecm , and generation of bioactive ecm fragments . for example , macrophage - derived mmps have been linked to the release of angiogenic factors , such as vegf and fgfs , in the tumor microenvironment . hyaluronan is generated as a high molecular weight glycosaminoglycan that can be broken down into fragments that are characterized as inflammatory and protumorigenic . macrophages have been found to express hyaluronidases and can thus potentially contribute to the breakdown of hyaluronan during inflammation and tumor progression . together , these observations suggest that macrophages are likely to be important regulators of ecm production and remodeling in the tumor microenvironment , and further studies are warranted to define the specific functional consequences of these actions . in conclusion , it is clear that inflammation is a complex process that has evolved to resolve damage to the body caused by pathogens or disease . in the normal mammary gland , tissue - resident macrophages play a vital role in the regulation of development and maintenance of tissue homeostasis . pro- and anti - inflammatory factors produced in the microenvironment act not only on epithelial cells , but also on macrophages and lead to the further disruption of inflammatory homeostasis and the creation of a protumorigenic niche that is primed for oncogenic initiation . tumor cells acquire the capacity to harness the functions of inflammatory cells , such as macrophages , to aid in their growth and progression . experimental studies have demonstrated that macrophages interact with cancer cells and their phenotype and function evolve as the tumor itself evolves . however , recent studies demonstrating the complexity of macrophage polarization and the impact of macrophage localization within the tumor microenvironment suggest that the contributions of macrophages to breast cancer growth and progression are likely to be quite complex . therefore , it will be critical to obtain a better understanding of the mechanisms that drive macrophage recruitment , polarization , and function within the tumor microenvironment at different stages of breast cancer formation and progression .

the biomedical community has traditionally used two types of ceramic implants for bone tissue regeneration technologies : inert materials such as alumina , zirconia , and carbon ( hulber 1993 ; wise et al 1995 ) and bioactive ceramics ( vallet - reg 2001a ) , which interact with the physiological environment when implanted leading to their integration in the living tissue ( hench 1984 ; kokubo et al 1990 ; langer et al 1993 ; hench 1998 ) . depending on the patient needs , an appropriate ceramic material should be selected to manufacture a determined implant . when hard tissues , natural composite materials , are aimed , the synthetic approach of these composite materials is a way to mimic nature in the laboratory ( vallet - reg et al 2006a ) . in the past few years bioactive ceramics like calcium phosphates ( yamamuro et al 1990a ) , bioactive glasses and glass - ceramics ( yamamuro et al 1990b ) have been deeply investigated . organic - inorganic hybrid materials have been also widely proposed as good candidates in the biomaterials field because they combine the properties of ceramics and organic polymers on the nanometric scale ( tsuru et al 1997 ; chen et al 1999 ; sanchez et al 2005 ; colilla et al 2006 ; vallet - reg et al 2006a ) . star gels have been recently reported as alternative materials for bone tissue regeneration because they combine the good mechanical properties of certain organic - inorganic hybrids with the bioactivity of conventional glasses ( manzano et al 2006 ; vallet - reg et al 2006b ) . however , much research effort has been committed to the investigation of ordered mesoporous silica materials in the biomedical field for two main reasons : their capability to regenerate bone tissue ( vallet - reg et al 2006c , 2006d ) combined with their drug delivery possibilities ( vallet - reg et al 2006e , 2007a ) . when these silica - based ordered mesoporous materials are exposed to the physiological environment , a series of chemical reactions take place in the material - living tissue interface , which lead to the material incorporation into the living tissue . those processes are confirmed through the nucleation and growth on the bioceramic surface of a layer of carbonated hydroxyapatite which is analogous to the mineral phase of bone tissues ( vallet - reg et al 2004a ) . recently , much research effort has been devoted to the design of mesoporous matrices with appropriated structural , textural and chemical properties in order to accelerate the formation of the apatite - like layer ( izquierdo - barba et al 2005a ; vallet - reg et al 2005 ) . the open texture and outstanding textural properties of mesoporous materials have inspired the usage of this type of materials as controlled delivery systems of biologically active molecules ( vallet - reg et al 2001b ) . in fact , their high pore volume permits to host a large amount of a determined biologically active molecule . also their ordered pore network allows a fine control of the molecule load and release kinetics . since molecules adsorption into the mesopores is a surface phenomenon , their high surface area will also favor the adsorption of a large amount of molecules into their mesopores . and the last , but no the least , is the possibility of functionalizing the silanol - containing surface with a selected organic group depending on the molecule to be adsorbed to allow a better control over molecule loading and release . this double perspective of the mesoporous materials , tissue regeneration and drug delivery , has promoted the research of these materials for biomedical applications in the last few years ( vallet - reg et al 2008 ; izquierdo - barba et al 2008 ) . in fact , the outstanding properties of silica - based ordered mesoporous materials make them suitable to be used as starting materials for the further design of scaffolds for bone tissue engineering . the regular repeating mesoporous structures of these silica - based mesoporous materials has motivated the adsorption in their pores and subsequent release of a large variety of biologically active species such as proteins , polypeptides or amino acids . ( yiu et al 2001a ; deere et al 2002 ; balas et al 2008 ) . within their possible biomedical applications , the case of protein delivery systems using serum albumins should be highlighted ( vallet - reg et al 2007b ) because these proteins represent one of the major components in plasma proteins in humans . albumin is usually composed of a single - chain of 582 amino acids with an average length of 10 nm and width of 6 nm ( peters 1995 ; sugio et al 1999 ; rcsb 2008 ) . besides contributing to colloid osmotic blood pressure and to the maintenance of the blood ph , the most outstanding property of albumin is its ability to bind reversibly an incredible variety of ligands . as a consequence , this multifunctional transport protein can be used as carrier or reservoir of certain polypeptides with special interest in osseous regeneration technologies . therefore , albumin could store , protect , and deliver determined peptides useful for bone regeneration to specific places . moreover , albumins are also capable of binding a wide variety of drugs than can be delivered to sites of pharmacological action ( carter et al 1994 ) . several proteins have been demonstrated to retain their functionality without being denatured inside the frameworks of silica - based mesoporous materials ( deere et al 2002 ; cheng et al 2003 ; lei et al 2004 ; vinu et al 2004 ; hartmann 2005 ; katiyar et al 2005 ; lee et al 2006 ; slowing et al 2007 ) . these findings open up many expectations for the research in the adsorption of proteins and other biologically active agents into mesoporous silicas . the adsorption and release characteristics of each porous material depend on the physicochemical properties of the individual material . among those properties , it should be highlighted the pore diameter , surface area , and pore volume , which are critically reviewed here . to achieve this task , it is necessary to look into the synthesis conditions of these materials , which modulate the physicochemical properties of each structure . silica - based ordered mesoporous materials are synthesized using a surfactant templating method ( figure 1 ) and present ordered porous structures with narrow pore size distributions and some of them with thick walls , which enhance their stability . they also show large surface areas and large pore volumes . in the early 1990s , japanese researchers ( yanagisawa et al 1990 ; inagaki et al 1993 ) and mobil corporation research and development scientists ( kresge et al 1992 ) for the first time reported the synthesis of novel periodic mesostructured materials , known as ksw - n and m41s family , respectively . these materials are characterized by regular arrays of uniform channels , whose dimensions can be tailored through the choice of surfactants , additives and synthesis conditions in the so - called liquid crystal templating mechanism ( beck et al 1992 ) . this process is based on the formation of liquid crystals in mixtures of polar solvents and surfactants with a non - polar tail group . the formation of the liquid crystals is as follows : an increasing amount of surfactant molecules is dissolved in an aqueous solution , and when surfactant concentration reaches the critical micellar concentration ( cmc ) , the surfactant molecules cluster together as micelles . these micelles are formed because the hydrophobic tails of the surfactant tend to congregate while their hydrophilic heads are procuring protection in water ( figure 1 ) . the final mesostructure of the material will depend on the organization of the surfactant molecules into the micellar liquid crystals which act as templates for the formation of the mesoporous materials . these liquid crystal structures depend on the composition and chemical nature of the surfactant , and also on the solution mixture conditions , such as surfactant concentration , ph , temperature , the presence of additives , etc . in the final step of the synthetic process , once the silica source has condensed around the micelles , the surfactant is removed by thermal degradation or solvent extraction . this surfactant removal gives rise to a network of cavities within the silica framework that governs the physicochemical properties of the material . different mesoporous structures have been developed in the last few years as a consequence of different modifications in the synthetic procedure . among them , the most representative and employed materials are mcm - n ( mobil composition of matter ) series ( beck et al 1992 ; kresge et al 1992 ; firouzi et al 1997 ; zhang et al 1997 ; kruk et al 2001 ; kaneda et al 2002 ) , sba - n ( santa barbara materials ) series ( zhao et al 1998 ; ravikovitch et al 2002a , 2002b ) , msu - n ( michigan state university ) series ( bagshaw et al 1995 ) , kit - n ( korean advanced institute of science and technology ) series ( ryoo et al 1996 ) , fsm - n ( folded sheet material ) series ( inagaki et al 1996 ) , and ams - n ( anionic surfactant templated mesoporous silica ) series ( che et al 2003 ) . in general , silica - based ordered mesoporous materials display exceptional properties , such as stable mesoporous structure , high surface area ( ca 1000 m / g ) , large pore volume ( ca 1 cm / g ) , regular and tunable pore size ( 250 nm ) , homogeneous pore morphology , non - toxic and biocompatible behavior , and the possibility of undergoing chemical functionalization of pore walls with different organic groups . in fact , these inorganic mesoporous materials offer the possibility of tuning the chemical properties of their surfaces to achieve the desired properties ( hoffmann et al 2006 ) . two major methods of producing functionalized materials have been widely used : direct functionalization ( antochshuk et al 2000 ) , which involves the addition of a trialkoxysilane with the selected functional group to the reaction mixture during the synthesis process ; and post - synthesis functionalization ( liu et al 1998 ) , which involves the grafting of the functional group onto the mesoporous material after surfactant removal . the direct functionalization method , also known as the co - condensation process , presents some advantages like a homogeneous dispersion of the functional group , retention of the width of the pores of the original framework , and the presence of available silanol groups for the adsorbates . however , there are some drawbacks like the possibility of absence of the functional groups on the surface of the material , the possible loss of structure and order of the final structure , and the need of removing the surfactant by solvent extraction rather than by calcination , which could lead to an incomplete removal of the surfactant molecules . on the other hand , the post - synthesis functionalization method presents the advantages of retention of the order and structure , and the sureness that the functional groups will be located on the surface of the pore walls . though this pathway means an extra step in the synthetic process and a reduction in the pore diameter , and also it might present a heterogeneous distribution of the grafted groups , it brings a noticeable change in the adsorption characteristics of the silica surface as well as in its polarity . the chemical modification of the pore walls would be selected depending on the molecule to be adsorbed , taking into account the desired load and release kinetics . since 2001 , when mcm-41 was proposed for the first time as controlled delivery system ( vallet - reg et al 2001b ) , different mesoporous matrices have been tested as delivery systems of several drugs , such as ibuprofen ( muoz et al 2003 ; izquierdo - barba et al 2005b ; manzano et al 2008 ) , gentamicin ( xue et al 2004 ; doadrio et al 2006 ) , amoxicillin ( vallet - reg et al 2004b ) , erythromycin ( izquierdo - barba et al 2005b ; doadrio et al 2006 ) , vancomycin ( yang et al 2005 ) , naproxen ( cavallaro et al 2004 ) , aspirin ( zeng et al 2005 ; zhu et al 2005 ) , diflunisal ( yang et al 2005 ) , captopril ( qu et al 2006 ) , and alendronate ( balas et al 2006 ; nieto et al 2008 ) . moreover , mesoporous silicas have been also evaluated as carriers of other biologically active species , such as proteins , eg , bovine serum albumin [ bsa ] ( song et al 2005 ; vallet - reg et al 2007b ) and certain amino acids , eg , l - tryptophan ( l - trp ) ( balas et al 2008 ) . the textural properties of silica - based ordered mesoporous materials seem to govern the loading and release of biologically active molecules . therefore , the influence of pore diameter , surface area , and pore volume in molecule adsorption and delivery kinetics will be tackled and critically discussed in the following sections . moreover , organic modification of silica walls with different functional groups to achieve a better control over molecule loading and release rate will be also described . the adsorption of molecules into silica - based ordered mesoporous materials is a size - selective process and consequently , the mesopore diameter determines the size of the molecule to be hosted . that is , if the molecule is smaller in size than the pore opening , it would have access to the large internal surface area and mesoporous volume of the material . on the contrary , usually , the molecule - loading process is carried out by soaking the mesoporous carrier in a highly concentrated molecule solution . hence , the adsorptive properties of mesoporous materials will determine the amount of adsorbate loaded . therefore , considering that the size of common drugs falls into the nanometer scale and that mesopore diameters can be tuned from 1.5 nm to several tens of nanometers , it seems reasonable to think that mesoporous matrices are able to host a wide range of biologically active molecules , ranging from small drugs to macromolecules such as large - size proteins . pore diameter has been probed to act as a drug - release rate modulator . when mcm-41 was reported for the first time as a drug delivery system ( vallet - reg et al 2001b ) , several cationic surfactants with different chain lengths thus , the surfactant with the longest chain led to the mcm-41 with the largest pore diameter . ibuprofen was chosen as the model drug and drug loading and release studies were carried out . loading tests showed that the mcm-41 with the largest pore size released 68% of the loaded drug after 24 h into a simulated body fluid . on the contrary , the mcm-41 with the smallest pore diameter released only 55% of the loaded drug after the same time period . further studies confirmed the influence of pore size on drug delivery rate ( horcajada et al 2004 ) . in this research , cationic surfactants with hydrocarbon chains of different lengths were used as structure directing agents , leading to mcm-41 materials with diverse mesopore diameters , ranging from 2.5 nm ( short - chain surfactants ) to 3.6 nm ( long - chain surfactants ) , as displayed in table 1 . the resulting release parameters , which are summarized in table 1 , revealed that the greater the mesopore diameter , the larger the amount of ibuprofen released . pore diameter is a limiting factor in molecule adsorption when the confinement of really large molecules , such as proteins and other biologically active molecules , is targeted . therefore , the adsorption of globular proteins on mesoporous mcm-41 ( with pore diameters ranging from 2 to 5 nm ) has been reported in the literature ( deere et al 2002 ) . in general , proteins with hydrodynamic dimensions larger than the mesopore diameter were adsorbed on the outer surface of mcm-41 ( katiyar et al 2005 ) . for this reason , when the confinement of proteins into mesoporous matrices is targeted , large pore mesoporous materials should be employed . wright ( yiu et al 2001b ) investigated the influence of protein dimensions on the adsorption into sba-15 mesoporous molecular sieve , whose silica walls were organically modified using thiol groups . for this purpose , a series of proteins with molecular weights ranging from 12000 to 76000 u were used to investigate adsorption on sba-15 materials . the structures of the employed proteins together with their dimensions in nanometers ( rcsb 2008 ) are displayed in figure 2 . further adsorption studies revealed that the proteins with the smallest sizes , ie , cytochrome c , lysozyme , myoglobin , and -lactoglobulin , showed significant adsorption . on the contrary , the proteins with the largest sizes , ie , conalbumin , serum albumin and ovalbumin were excluded from the internal surfaces of thiol - functionalized sba-15 . this fact agrees with the sieving expected from the 5.1 nm pore size of thiol - functionalized sba-15 when compared to the proteins dimensions . moreover , the exclusion of ovalbumin ( 4.0 5.0 7.0 nm ) , with dimensions quite close to thiol - functionalized sba-15 matrix , indicates that this size selectivity is rigorous and that there are very few pores appreciably larger than the average pore size . it should be remarked that the maximum amounts of protein loaded , expressed as a volume percent of the available internal pore volume ( 5% for -lactoglobulin , 15% for myoglobin , and 43% for cytochrome c ) showed that the protein molecules were adsorbed within the mesopores and not only on the external surfaces . from the above results it can be deduced that when the confinement of large size proteins is the aim , large pore mesoporous matrices are needed . this fact inspired the idea of using sba-15 as a mesoporous carrier employing different hydrothermal treatments during the synthesis to wide the pore diameter of sba-15 ( vallet - reg et al 2007b ) . hence , pore diameters ranging from 8.2 nm to 11.4 nm were obtained for sba-15 materials submitted to hydrothermal treatments periods ranging from 1 to 7 days ( figure 3a ) . moreover , bsa loading dependence on sba-15 diameter was observed , as displayed in figure 3b . hence , the amount of bsa loaded was 15% , 23% , 24% , and 27% ( weight percentage ) for sba-15 exhibiting pore diameters of 8.2 , 9.5 , 10.5 , and 11.4 nm , respectively . with the aim of promoting host - guest sba-15-protein interaction , an organic modification of silica surface was performed using aminopropyl groups and following the post - synthesis method ( vallet - reg et al 2007b ) . thus , the amino groups of sba-15 modified materials would undergo attracting electrostatic interactions with the carboxylic fraction of amide groups from the protein . as mentioned before the bsa molecule is just on the limit of the mesopore dimensions , and thus , after amino - functionalization the amount of bsa loaded decreased compared to unmodified matrices ( see table in figure 3c ) . however , the bsa loading on amino - modified sba-15 matrices underwent a behavior comparable to unmodified - matrices in terms of loading increment as the pore size increased . the amino functionalization of sba-15 had a noticeable influence on bsa delivery kinetics ( figure 3d ) . the bsa release from pure silica mesopore surfaces essentially showed a burst profile , where more than 90% of the adsorbed protein was released within the initial 24 h of tests . the rest of the adsorbed protein was linearly released up to complete delivery in 192 h in all tested materials , regardless of the hydrothermal treatment carried out for synthesis . however , amino - modified sba-15 materials showed an incomplete release of the protein from the mesopores in all cases . this partial protein retention was attributed to the strong attracting interaction of silica walls amine groups with the protein . after 192 h , the released protein ranged from 25% ( sba-15 - 7d - nh2 ) up to 60% ( sba-15 - 3d - nh2 ) of the initially loaded amount of protein . as an illustrative example , figure 3d shows bsa release profiles of sba-15 submitted to 7 days of hydrothermal treatment . therefore , the pore size has been revealed as a key factor governing the adsorption of proteins in mesoporous materials . the pore size should be noticeably larger than the protein to allow the diffusion of the protein into the mesopores . however , once the protein is adsorbed , the ruling factor on the release kinetics has been shown to be the organic functionalization , due to the host - guest attracting interactions . the adsorption of biologically active molecules into meso - porous matrices is governed by the chemical interaction between silanol groups covering the silica surface and the functional groups of the guest molecule . therefore , surface area is expected to determine the amount of molecules confined into the silica matrix . this fact was probed when mcm-41 mesoporous matrices exhibiting different surfaces areas , sbet , ranging from 768 to 1157 m / g , which were synthesized using surfactants with diverse length - chains , were employed as ibuprofen delivery systems ( horcajada et al 2004 ) . the amount of ibuprofen loaded was found to depend on the surface area , as graphically displayed in figure 4 . as expected , there was an increase in ibuprofen loading amount with the enlargement in the surface area values . surface area influence was also shown when alendronate , a potent bisphosphonate used in osteoporosis treatments , was confined into mcm-41 and sba-15 mesoporous matrices ( balas et al 2006 ) . both mesoporous matrices exhibited the same structure ( 2d hexagonal and p6 mm symmetry ) but different surface areas , sbet of 1157 and 719 m / g for mcm-41 and sba-15 , respectively . when both matrices were loaded with alendronate under the same conditions , the maximum amounts of drug loaded were 14% and 8% for mcm-41 and sba-15 , respectively . this fact showed the clear dependence of maximum drug load on the matrix surface area in agreement with results reported later in the literature ( qu et al 2006 ) . mcm-41 and sba-15 were functionalized using amino groups with the aim of increasing the attracting host - guest interactions , and alendronate loading and release studies were carried out ( balas et al 2006 ) . the amount of alendronate loaded followed the same trend that unmodified materials , ie mcm-41-nh2 loaded more alendronate ( 37% ) than sba-15-nh2 ( 22% ) as a result of the higher surface area of mcm-41 ( table 2 ) . in addition , it should be noticed that the amount of alendronate loaded in modified materials was almost 3 times larger than those of unmodified materials . this fact can be explained by the stronger attracting interactions between phosphonate groups and amino groups of modified materials compared to the weaker interaction taking place between phosphonate groups and silanol groups from unmodified matrices ( figure 5 ) . regarding alendronate release , it should be highlighted that in all cases an initial burst effect was observed . this fast release of the drug could be due to several reasons : alendronate that could be adsorbed in the outer surface of the matrix or by the existent alendronate gradient between mesoporous matrix and delivery medium . therefore , after 24 h of assay , 28% of the total amount of alendronate adsorbed was delivered from mcm-41-nh2 , whereas at the same time this percentage was 58% for unmodified mcm-41 ( table 2 ) . on the other hand , 11% of the total alendronate loaded was released after 24 h of assay from sba-15-nh2 matrix , whereas 55% of alendronate loaded was delivered after this time from sba-15 . after such burst effect , the alendronate was released to the medium in a sustained manner following first order kinetics for unmodified and modified mcm-41 materials and zero order or linear kinetics for unmodified and modified sba-15 materials . moreover , the increase in the total drug delivery time in functionalized materials compared with unmodified matrices ( table 2 ) can be ascribed to the stronger interactions between phosphonate groups from alendronate and amino groups covering the pore walls . this work evidences that the amount of alendronate adsorbed and drug delivery rate can be controlled by appropriately modifying the mesoporous carriers with amino groups . in this sense , organic functionalization allows a higher control over drug loading and release kinetics . thus , surface area was found to be an important factor in the loading capacity of these mesoporous materials . it was found that the higher the surface area , the higher the drug adsorption . adsorption of biologically active molecules is a surface phenomenon that takes place by attracting interactions between the silanol groups in the pore walls ( or functional groups in the case of functionalized materials ) and functional groups of the guest molecule . thus , the amount of molecules adsorbed will depend on the pore diameter and surface area as the limiting factor . however , when the confinement of really large molecules is aimed , such as large - size and large - volume proteins , pore volume seems to play a key role in molecule adsorption . recently , in an effort to promote the loading of large biomolecules , mesostructured cellular foams ( mesocellular foams , mcfs ) have been employed as host matrices for the adsorption of different enzymes and proteins ( han et al 1999 ; zhang et al 2007 ) . the synthesis of mcfs type materials is carried out by employing triblock copolymers and introducing a swelling agent , such as 1,3,5-trimethylbenzene ( tmb ) into the structure directing template ( schmidt - winkel et al 1999 ) . tem images of mcf compared to mcm-41 and sba-15 mesoporous materials are displayed in figure 6 . the characteristic two - dimensional hexagonal arrays of pores of mcm-41 and sba-15 mesoporous matrices can be observed . moreover , mcf exhibits three - dimensional , continuous , ultra - large pore mesoporous structures with large spherical cells interconnected by uniform windows ( see arrows in figure 6 ) . n2 adsorption measurements revealed that mcm-41 and sba-15 mesoporous materials exhibit type iv isotherms , typical of ordered mesoporous materials . the shape of the hysteresis loops points to cylindrical mesopores with very narrow pore size distributions ( figure 6 ) ( gregg et al 1982 ) . in the case of mcfs , the sharp rise in the adsorption / desorption isotherms at relative pressures close to 1 points to the existence of large mesopores in these materials ( gregg et al 1982 ) . the pore diameter of mcm-41 and sba-15 materials are ca 3 and 9 nm , respectively . the diameter of spherical cells and windows can be modulated by adjusting the amount of swelling agents and the synthesis temperature ( schmidt - winkel et al 1999 ) . when the confinement of large - size bsa is targeted , mesoporous matrices exhibiting large pore diameters are needed . thus , sba-15 and mcf before and after functionalization using amino groups where tested as delivery systems for bsa . the amount of bsa loaded in mcf materials was higher ( 24% ) than in sba-15 matrices ( 15% ) , due to the higher pore volume in the former ( table 3 ) . it should be highlighted that the surface area is not the determinant factor that governs protein adsorption , because it exhibits the opposite trend to protein loading ( table 3 ) . the amount of bsa loaded after functionalization followed the same trend that unmodified materials , ie , mcf - nh2 loaded more bsa ( 27% ) than sba-15-nh2 ( 10% ) , as a result of the higher pore volume of the former . the increase in bsa loading after functionalization of mcf matrix can be attributed to the higher attracting electrostatic interactions of amino groups with the amide groups of protein . however , as it was previously mentioned , organic functionalization always leads to a decrease in pore diameter ( table 3 ) . the pore diameter of sba-15 ( 8.5 nm ) is just on the limit of bsa size and therefore , after functionalization the pore diameter decreased to 6.9 nm and consequently , a decrease in the amount of bsa adsorbed was observed . on the other hand , as it can be observed in figure 7 , unmodified matrices exhibited an initial burst effect when almost 60% of the protein was quickly released to the delivery medium and then the loaded protein was delivered in a controlled fashion . on the contrary , the initial burst in amino - modified matrices was drastically reduced to ca 10% , and more than 80% of the loaded bsa was released to the medium in a sustained manner . after 24 h of assay , 74% of the total bsa loaded in sba-15 was released to the medium , whereas after the same time only 27% was released to the medium from sba-15-nh2 matrix . moreover , after 24 h of delivery test 62% of the loaded bsa was released from unmodified mcf material and this percentage decreased to 22% after functionalization with amino groups . this research work demonstrates that bsa can be successfully adsorbed into large - volume mesoporous matrices exhibiting the appropriate pore diameter to allow protein confinement . therefore , when the loading of large - size and large - volume biologically active molecules is targeted , pore diameter acts as the limiting factor and pore volume determines the amount of molecule hosted . the adsorption of molecules into silica - based ordered mesoporous materials is a size - selective process and consequently , the mesopore diameter determines the size of the molecule to be hosted . that is , if the molecule is smaller in size than the pore opening , it would have access to the large internal surface area and mesoporous volume of the material . on the contrary , usually , the molecule - loading process is carried out by soaking the mesoporous carrier in a highly concentrated molecule solution . hence , the adsorptive properties of mesoporous materials will determine the amount of adsorbate loaded . therefore , considering that the size of common drugs falls into the nanometer scale and that mesopore diameters can be tuned from 1.5 nm to several tens of nanometers , it seems reasonable to think that mesoporous matrices are able to host a wide range of biologically active molecules , ranging from small drugs to macromolecules such as large - size proteins . pore diameter has been probed to act as a drug - release rate modulator . when mcm-41 was reported for the first time as a drug delivery system ( vallet - reg et al 2001b ) , several cationic surfactants with different chain lengths were employed , which led to mcm-41 materials with different pore sizes . thus , the surfactant with the longest chain led to the mcm-41 with the largest pore diameter . ibuprofen was chosen as the model drug and drug loading and release studies were carried out . loading tests showed that the mcm-41 with the largest pore size released 68% of the loaded drug after 24 h into a simulated body fluid . on the contrary , the mcm-41 with the smallest pore diameter released only 55% of the loaded drug after the same time period . further studies confirmed the influence of pore size on drug delivery rate ( horcajada et al 2004 ) . in this research , cationic surfactants with hydrocarbon chains of different lengths were used as structure directing agents , leading to mcm-41 materials with diverse mesopore diameters , ranging from 2.5 nm ( short - chain surfactants ) to 3.6 nm ( long - chain surfactants ) , as displayed in table 1 . the resulting release parameters , which are summarized in table 1 , revealed that the greater the mesopore diameter , the larger the amount of ibuprofen released . pore diameter is a limiting factor in molecule adsorption when the confinement of really large molecules , such as proteins and other biologically active molecules , is targeted . therefore , the adsorption of globular proteins on mesoporous mcm-41 ( with pore diameters ranging from 2 to 5 nm ) has been reported in the literature ( deere et al 2002 ) . in general , proteins with hydrodynamic dimensions larger than the mesopore diameter were adsorbed on the outer surface of mcm-41 ( katiyar et al 2005 ) . for this reason , when the confinement of proteins into mesoporous matrices is targeted , large pore mesoporous materials should be employed . wright ( yiu et al 2001b ) investigated the influence of protein dimensions on the adsorption into sba-15 mesoporous molecular sieve , whose silica walls were organically modified using thiol groups . for this purpose , a series of proteins with molecular weights ranging from 12000 to 76000 u were used to investigate adsorption on sba-15 materials . the structures of the employed proteins together with their dimensions in nanometers ( rcsb 2008 ) are displayed in figure 2 . further adsorption studies revealed that the proteins with the smallest sizes , ie , cytochrome c , lysozyme , myoglobin , and -lactoglobulin , showed significant adsorption . on the contrary , the proteins with the largest sizes , ie , conalbumin , serum albumin and ovalbumin were excluded from the internal surfaces of thiol - functionalized sba-15 . this fact agrees with the sieving expected from the 5.1 nm pore size of thiol - functionalized sba-15 when compared to the proteins dimensions . moreover , the exclusion of ovalbumin ( 4.0 5.0 7.0 nm ) , with dimensions quite close to thiol - functionalized sba-15 matrix , indicates that this size selectivity is rigorous and that there are very few pores appreciably larger than the average pore size . it should be remarked that the maximum amounts of protein loaded , expressed as a volume percent of the available internal pore volume ( 5% for -lactoglobulin , 15% for myoglobin , and 43% for cytochrome c ) showed that the protein molecules were adsorbed within the mesopores and not only on the external surfaces . from the above results it can be deduced that when the confinement of large size proteins is the aim , large pore mesoporous matrices are needed . this fact inspired the idea of using sba-15 as a mesoporous carrier employing different hydrothermal treatments during the synthesis to wide the pore diameter of sba-15 ( vallet - reg et al 2007b ) . hence , pore diameters ranging from 8.2 nm to 11.4 nm were obtained for sba-15 materials submitted to hydrothermal treatments periods ranging from 1 to 7 days ( figure 3a ) . moreover , bsa loading dependence on sba-15 diameter was observed , as displayed in figure 3b . hence , the amount of bsa loaded was 15% , 23% , 24% , and 27% ( weight percentage ) for sba-15 exhibiting pore diameters of 8.2 , 9.5 , 10.5 , and 11.4 nm , respectively . with the aim of promoting host - guest sba-15-protein interaction , an organic modification of silica surface was performed using aminopropyl groups and following the post - synthesis method ( vallet - reg et al 2007b ) . thus , the amino groups of sba-15 modified materials would undergo attracting electrostatic interactions with the carboxylic fraction of amide groups from the protein . as mentioned before the bsa molecule is just on the limit of the mesopore dimensions , and thus , after amino - functionalization the amount of bsa loaded decreased compared to unmodified matrices ( see table in figure 3c ) . however , the bsa loading on amino - modified sba-15 matrices underwent a behavior comparable to unmodified - matrices in terms of loading increment as the pore size increased . the amino functionalization of sba-15 had a noticeable influence on bsa delivery kinetics ( figure 3d ) . the bsa release from pure silica mesopore surfaces essentially showed a burst profile , where more than 90% of the adsorbed protein was released within the initial 24 h of tests . the rest of the adsorbed protein was linearly released up to complete delivery in 192 h in all tested materials , regardless of the hydrothermal treatment carried out for synthesis . however , amino - modified sba-15 materials showed an incomplete release of the protein from the mesopores in all cases . this partial protein retention was attributed to the strong attracting interaction of silica walls amine groups with the protein . after 192 h , the released protein ranged from 25% ( sba-15 - 7d - nh2 ) up to 60% ( sba-15 - 3d - nh2 ) of the initially loaded amount of protein . as an illustrative example , figure 3d shows bsa release profiles of sba-15 submitted to 7 days of hydrothermal treatment . therefore , the pore size has been revealed as a key factor governing the adsorption of proteins in mesoporous materials . the pore size should be noticeably larger than the protein to allow the diffusion of the protein into the mesopores . however , once the protein is adsorbed , the ruling factor on the release kinetics has been shown to be the organic functionalization , due to the host - guest attracting interactions . the adsorption of biologically active molecules into meso - porous matrices is governed by the chemical interaction between silanol groups covering the silica surface and the functional groups of the guest molecule . therefore , surface area is expected to determine the amount of molecules confined into the silica matrix . this fact was probed when mcm-41 mesoporous matrices exhibiting different surfaces areas , sbet , ranging from 768 to 1157 m / g , which were synthesized using surfactants with diverse length - chains , were employed as ibuprofen delivery systems ( horcajada et al 2004 ) . the amount of ibuprofen loaded was found to depend on the surface area , as graphically displayed in figure 4 . as expected , there was an increase in ibuprofen loading amount with the enlargement in the surface area values . surface area influence was also shown when alendronate , a potent bisphosphonate used in osteoporosis treatments , was confined into mcm-41 and sba-15 mesoporous matrices ( balas et al 2006 ) . both mesoporous matrices exhibited the same structure ( 2d hexagonal and p6 mm symmetry ) but different surface areas , sbet of 1157 and 719 m / g for mcm-41 and sba-15 , respectively . when both matrices were loaded with alendronate under the same conditions , the maximum amounts of drug loaded were 14% and 8% for mcm-41 and sba-15 , respectively . this fact showed the clear dependence of maximum drug load on the matrix surface area in agreement with results reported later in the literature ( qu et al 2006 ) . mcm-41 and sba-15 were functionalized using amino groups with the aim of increasing the attracting host - guest interactions , and alendronate loading and release studies were carried out ( balas et al 2006 ) . the amount of alendronate loaded followed the same trend that unmodified materials , ie mcm-41-nh2 loaded more alendronate ( 37% ) than sba-15-nh2 ( 22% ) as a result of the higher surface area of mcm-41 ( table 2 ) . in addition , it should be noticed that the amount of alendronate loaded in modified materials was almost 3 times larger than those of unmodified materials . this fact can be explained by the stronger attracting interactions between phosphonate groups and amino groups of modified materials compared to the weaker interaction taking place between phosphonate groups and silanol groups from unmodified matrices ( figure 5 ) . regarding alendronate release , it should be highlighted that in all cases an initial burst effect was observed . this fast release of the drug could be due to several reasons : alendronate that could be adsorbed in the outer surface of the matrix or by the existent alendronate gradient between mesoporous matrix and delivery medium . therefore , after 24 h of assay , 28% of the total amount of alendronate adsorbed was delivered from mcm-41-nh2 , whereas at the same time this percentage was 58% for unmodified mcm-41 ( table 2 ) . on the other hand , 11% of the total alendronate loaded was released after 24 h of assay from sba-15-nh2 matrix , whereas 55% of alendronate loaded was delivered after this time from sba-15 . after such burst effect , the alendronate was released to the medium in a sustained manner following first order kinetics for unmodified and modified mcm-41 materials and zero order or linear kinetics for unmodified and modified sba-15 materials . moreover , the increase in the total drug delivery time in functionalized materials compared with unmodified matrices ( table 2 ) can be ascribed to the stronger interactions between phosphonate groups from alendronate and amino groups covering the pore walls . this work evidences that the amount of alendronate adsorbed and drug delivery rate can be controlled by appropriately modifying the mesoporous carriers with amino groups . in this sense , organic functionalization allows a higher control over drug loading and release kinetics . thus , surface area was found to be an important factor in the loading capacity of these mesoporous materials . it was found that the higher the surface area , the higher the drug adsorption . as previously commented , adsorption of biologically active molecules is a surface phenomenon that takes place by attracting interactions between the silanol groups in the pore walls ( or functional groups in the case of functionalized materials ) and functional groups of the guest molecule . thus , the amount of molecules adsorbed will depend on the pore diameter and surface area as the limiting factor . however , when the confinement of really large molecules is aimed , such as large - size and large - volume proteins , pore volume seems to play a key role in molecule adsorption . recently , in an effort to promote the loading of large biomolecules , mesostructured cellular foams ( mesocellular foams , mcfs ) have been employed as host matrices for the adsorption of different enzymes and proteins ( han et al 1999 ; zhang et al 2007 ) . the synthesis of mcfs type materials is carried out by employing triblock copolymers and introducing a swelling agent , such as 1,3,5-trimethylbenzene ( tmb ) into the structure directing template ( schmidt - winkel et al 1999 ) . tem images of mcf compared to mcm-41 and sba-15 mesoporous materials are displayed in figure 6 . the characteristic two - dimensional hexagonal arrays of pores of mcm-41 and sba-15 mesoporous matrices can be observed . moreover , mcf exhibits three - dimensional , continuous , ultra - large pore mesoporous structures with large spherical cells interconnected by uniform windows ( see arrows in figure 6 ) . n2 adsorption measurements revealed that mcm-41 and sba-15 mesoporous materials exhibit type iv isotherms , typical of ordered mesoporous materials . the shape of the hysteresis loops points to cylindrical mesopores with very narrow pore size distributions ( figure 6 ) ( gregg et al 1982 ) . in the case of mcfs , the sharp rise in the adsorption / desorption isotherms at relative pressures close to 1 points to the existence of large mesopores in these materials ( gregg et al 1982 ) . pore size distributions of different mesoporous matrices are also shown in figure 6 . the pore diameter of mcm-41 and sba-15 materials are ca 3 and 9 nm , respectively . the diameter of spherical cells and windows can be modulated by adjusting the amount of swelling agents and the synthesis temperature ( schmidt - winkel et al 1999 ) . when the confinement of large - size bsa is targeted , mesoporous matrices exhibiting large pore diameters are needed . thus , sba-15 and mcf before and after functionalization using amino groups where tested as delivery systems for bsa . the amount of bsa loaded in mcf materials was higher ( 24% ) than in sba-15 matrices ( 15% ) , due to the higher pore volume in the former ( table 3 ) . it should be highlighted that the surface area is not the determinant factor that governs protein adsorption , because it exhibits the opposite trend to protein loading ( table 3 ) . the amount of bsa loaded after functionalization followed the same trend that unmodified materials , ie , mcf - nh2 loaded more bsa ( 27% ) than sba-15-nh2 ( 10% ) , as a result of the higher pore volume of the former . the increase in bsa loading after functionalization of mcf matrix can be attributed to the higher attracting electrostatic interactions of amino groups with the amide groups of protein . however , as it was previously mentioned , organic functionalization always leads to a decrease in pore diameter ( table 3 ) . the pore diameter of sba-15 ( 8.5 nm ) is just on the limit of bsa size and therefore , after functionalization the pore diameter decreased to 6.9 nm and consequently , a decrease in the amount of bsa adsorbed was observed . on the other hand , as it can be observed in figure 7 , unmodified matrices exhibited an initial burst effect when almost 60% of the protein was quickly released to the delivery medium and then the loaded protein was delivered in a controlled fashion . on the contrary , the initial burst in amino - modified matrices was drastically reduced to ca 10% , and more than 80% of the loaded bsa was released to the medium in a sustained manner . after 24 h of assay , 74% of the total bsa loaded in sba-15 was released to the medium , whereas after the same time only 27% was released to the medium from sba-15-nh2 matrix . moreover , after 24 h of delivery test 62% of the loaded bsa was released from unmodified mcf material and this percentage decreased to 22% after functionalization with amino groups . this research work demonstrates that bsa can be successfully adsorbed into large - volume mesoporous matrices exhibiting the appropriate pore diameter to allow protein confinement . therefore , when the loading of large - size and large - volume biologically active molecules is targeted , pore diameter acts as the limiting factor and pore volume determines the amount of molecule hosted . in this work it has been shown that available pore volume and surface play a key role in the protein loading capacity of silica - based ordered mesoporous materials . however , if the pore opening is not wide enough , access of the protein molecules to much of this surface area and pore volume will be restricted . ideally , if large biomolecules such as certain proteins are targeted to be adsorbed in ordered mesoporous materials , these matrices should present several characteristics : pore size large enough to allow diffusion into the pores , surface area as high as possible to allow a large retention percentage , and pore volume as high as possible to offer available space into the mesopores to be filled by the protein . recent advances in nanotechnology offer the possibility of appropriately tailoring the structural and textural properties of mesoporous silicas depending on the desired application . the outstanding features of silica - based ordered mesoporous materials open up promising expectations in the biomedical field because they can be employed as starting materials for the further design of scaffolds for bone tissue regeneration .

research in the development of new bioceramics with local drug delivery capability for bone regeneration technologies is receiving great interest by the scientific biomedical community . among bioceramics , silica - based ordered mesoporous materials are excellent candidates as bone implants due to two main reasons : first , the bioactive behavior of such materials in contact with simulated body fluids , ie , a carbonate hydroxyapatite similar to the mineral phase of bone is formed onto the materials surfaces . second , their capability of acting as delivery systems of a large variety of biologically active molecules , including drugs to treat bone infection , inflammation or diseases , and molecules that promote bone tissue regeneration , such as peptides , proteins , growth factors , and other osteogenic agents . the recent chemical and technological advances in the nanometer scale has allowed the design of mesoporous silicas with tailored structural and textural properties aimed at achieving a better control over molecule loading and release kinetics . moreover organic modification of mesoporous silica walls has been revealed as a key strategy to modulate molecule adsorption and delivery rates .

Introduction Silica-based ordered mesoporous materials: General remarks Tailoring the performance of mesoporous controlled delivery systems Pore diameter: An adsorptive size selectivity factor and release rate modulator Influence of surface area Influence of pore volume Conclusions

the biomedical community has traditionally used two types of ceramic implants for bone tissue regeneration technologies : inert materials such as alumina , zirconia , and carbon ( hulber 1993 ; wise et al 1995 ) and bioactive ceramics ( vallet - reg 2001a ) , which interact with the physiological environment when implanted leading to their integration in the living tissue ( hench 1984 ; kokubo et al 1990 ; langer et al 1993 ; hench 1998 ) . star gels have been recently reported as alternative materials for bone tissue regeneration because they combine the good mechanical properties of certain organic - inorganic hybrids with the bioactivity of conventional glasses ( manzano et al 2006 ; vallet - reg et al 2006b ) . however , much research effort has been committed to the investigation of ordered mesoporous silica materials in the biomedical field for two main reasons : their capability to regenerate bone tissue ( vallet - reg et al 2006c , 2006d ) combined with their drug delivery possibilities ( vallet - reg et al 2006e , 2007a ) . when these silica - based ordered mesoporous materials are exposed to the physiological environment , a series of chemical reactions take place in the material - living tissue interface , which lead to the material incorporation into the living tissue . those processes are confirmed through the nucleation and growth on the bioceramic surface of a layer of carbonated hydroxyapatite which is analogous to the mineral phase of bone tissues ( vallet - reg et al 2004a ) . recently , much research effort has been devoted to the design of mesoporous matrices with appropriated structural , textural and chemical properties in order to accelerate the formation of the apatite - like layer ( izquierdo - barba et al 2005a ; vallet - reg et al 2005 ) . the open texture and outstanding textural properties of mesoporous materials have inspired the usage of this type of materials as controlled delivery systems of biologically active molecules ( vallet - reg et al 2001b ) . in fact , their high pore volume permits to host a large amount of a determined biologically active molecule . since molecules adsorption into the mesopores is a surface phenomenon , their high surface area will also favor the adsorption of a large amount of molecules into their mesopores . and the last , but no the least , is the possibility of functionalizing the silanol - containing surface with a selected organic group depending on the molecule to be adsorbed to allow a better control over molecule loading and release . this double perspective of the mesoporous materials , tissue regeneration and drug delivery , has promoted the research of these materials for biomedical applications in the last few years ( vallet - reg et al 2008 ; izquierdo - barba et al 2008 ) . in fact , the outstanding properties of silica - based ordered mesoporous materials make them suitable to be used as starting materials for the further design of scaffolds for bone tissue engineering . the regular repeating mesoporous structures of these silica - based mesoporous materials has motivated the adsorption in their pores and subsequent release of a large variety of biologically active species such as proteins , polypeptides or amino acids . besides contributing to colloid osmotic blood pressure and to the maintenance of the blood ph , the most outstanding property of albumin is its ability to bind reversibly an incredible variety of ligands . therefore , albumin could store , protect , and deliver determined peptides useful for bone regeneration to specific places . several proteins have been demonstrated to retain their functionality without being denatured inside the frameworks of silica - based mesoporous materials ( deere et al 2002 ; cheng et al 2003 ; lei et al 2004 ; vinu et al 2004 ; hartmann 2005 ; katiyar et al 2005 ; lee et al 2006 ; slowing et al 2007 ) . these findings open up many expectations for the research in the adsorption of proteins and other biologically active agents into mesoporous silicas . silica - based ordered mesoporous materials are synthesized using a surfactant templating method ( figure 1 ) and present ordered porous structures with narrow pore size distributions and some of them with thick walls , which enhance their stability . these materials are characterized by regular arrays of uniform channels , whose dimensions can be tailored through the choice of surfactants , additives and synthesis conditions in the so - called liquid crystal templating mechanism ( beck et al 1992 ) . these liquid crystal structures depend on the composition and chemical nature of the surfactant , and also on the solution mixture conditions , such as surfactant concentration , ph , temperature , the presence of additives , etc . among them , the most representative and employed materials are mcm - n ( mobil composition of matter ) series ( beck et al 1992 ; kresge et al 1992 ; firouzi et al 1997 ; zhang et al 1997 ; kruk et al 2001 ; kaneda et al 2002 ) , sba - n ( santa barbara materials ) series ( zhao et al 1998 ; ravikovitch et al 2002a , 2002b ) , msu - n ( michigan state university ) series ( bagshaw et al 1995 ) , kit - n ( korean advanced institute of science and technology ) series ( ryoo et al 1996 ) , fsm - n ( folded sheet material ) series ( inagaki et al 1996 ) , and ams - n ( anionic surfactant templated mesoporous silica ) series ( che et al 2003 ) . in general , silica - based ordered mesoporous materials display exceptional properties , such as stable mesoporous structure , high surface area ( ca 1000 m / g ) , large pore volume ( ca 1 cm / g ) , regular and tunable pore size ( 250 nm ) , homogeneous pore morphology , non - toxic and biocompatible behavior , and the possibility of undergoing chemical functionalization of pore walls with different organic groups . two major methods of producing functionalized materials have been widely used : direct functionalization ( antochshuk et al 2000 ) , which involves the addition of a trialkoxysilane with the selected functional group to the reaction mixture during the synthesis process ; and post - synthesis functionalization ( liu et al 1998 ) , which involves the grafting of the functional group onto the mesoporous material after surfactant removal . the chemical modification of the pore walls would be selected depending on the molecule to be adsorbed , taking into account the desired load and release kinetics . since 2001 , when mcm-41 was proposed for the first time as controlled delivery system ( vallet - reg et al 2001b ) , different mesoporous matrices have been tested as delivery systems of several drugs , such as ibuprofen ( muoz et al 2003 ; izquierdo - barba et al 2005b ; manzano et al 2008 ) , gentamicin ( xue et al 2004 ; doadrio et al 2006 ) , amoxicillin ( vallet - reg et al 2004b ) , erythromycin ( izquierdo - barba et al 2005b ; doadrio et al 2006 ) , vancomycin ( yang et al 2005 ) , naproxen ( cavallaro et al 2004 ) , aspirin ( zeng et al 2005 ; zhu et al 2005 ) , diflunisal ( yang et al 2005 ) , captopril ( qu et al 2006 ) , and alendronate ( balas et al 2006 ; nieto et al 2008 ) . moreover , mesoporous silicas have been also evaluated as carriers of other biologically active species , such as proteins , eg , bovine serum albumin [ bsa ] ( song et al 2005 ; vallet - reg et al 2007b ) and certain amino acids , eg , l - tryptophan ( l - trp ) ( balas et al 2008 ) . the textural properties of silica - based ordered mesoporous materials seem to govern the loading and release of biologically active molecules . therefore , the influence of pore diameter , surface area , and pore volume in molecule adsorption and delivery kinetics will be tackled and critically discussed in the following sections . moreover , organic modification of silica walls with different functional groups to achieve a better control over molecule loading and release rate will be also described . the adsorption of molecules into silica - based ordered mesoporous materials is a size - selective process and consequently , the mesopore diameter determines the size of the molecule to be hosted . hence , the adsorptive properties of mesoporous materials will determine the amount of adsorbate loaded . therefore , considering that the size of common drugs falls into the nanometer scale and that mesopore diameters can be tuned from 1.5 nm to several tens of nanometers , it seems reasonable to think that mesoporous matrices are able to host a wide range of biologically active molecules , ranging from small drugs to macromolecules such as large - size proteins . when mcm-41 was reported for the first time as a drug delivery system ( vallet - reg et al 2001b ) , several cationic surfactants with different chain lengths thus , the surfactant with the longest chain led to the mcm-41 with the largest pore diameter . ibuprofen was chosen as the model drug and drug loading and release studies were carried out . pore diameter is a limiting factor in molecule adsorption when the confinement of really large molecules , such as proteins and other biologically active molecules , is targeted . therefore , the adsorption of globular proteins on mesoporous mcm-41 ( with pore diameters ranging from 2 to 5 nm ) has been reported in the literature ( deere et al 2002 ) . further adsorption studies revealed that the proteins with the smallest sizes , ie , cytochrome c , lysozyme , myoglobin , and -lactoglobulin , showed significant adsorption . on the contrary , the proteins with the largest sizes , ie , conalbumin , serum albumin and ovalbumin were excluded from the internal surfaces of thiol - functionalized sba-15 . it should be remarked that the maximum amounts of protein loaded , expressed as a volume percent of the available internal pore volume ( 5% for -lactoglobulin , 15% for myoglobin , and 43% for cytochrome c ) showed that the protein molecules were adsorbed within the mesopores and not only on the external surfaces . therefore , the pore size has been revealed as a key factor governing the adsorption of proteins in mesoporous materials . however , once the protein is adsorbed , the ruling factor on the release kinetics has been shown to be the organic functionalization , due to the host - guest attracting interactions . the adsorption of biologically active molecules into meso - porous matrices is governed by the chemical interaction between silanol groups covering the silica surface and the functional groups of the guest molecule . mcm-41 and sba-15 were functionalized using amino groups with the aim of increasing the attracting host - guest interactions , and alendronate loading and release studies were carried out ( balas et al 2006 ) . the amount of alendronate loaded followed the same trend that unmodified materials , ie mcm-41-nh2 loaded more alendronate ( 37% ) than sba-15-nh2 ( 22% ) as a result of the higher surface area of mcm-41 ( table 2 ) . this fast release of the drug could be due to several reasons : alendronate that could be adsorbed in the outer surface of the matrix or by the existent alendronate gradient between mesoporous matrix and delivery medium . moreover , the increase in the total drug delivery time in functionalized materials compared with unmodified matrices ( table 2 ) can be ascribed to the stronger interactions between phosphonate groups from alendronate and amino groups covering the pore walls . in this sense , organic functionalization allows a higher control over drug loading and release kinetics . adsorption of biologically active molecules is a surface phenomenon that takes place by attracting interactions between the silanol groups in the pore walls ( or functional groups in the case of functionalized materials ) and functional groups of the guest molecule . however , when the confinement of really large molecules is aimed , such as large - size and large - volume proteins , pore volume seems to play a key role in molecule adsorption . in the case of mcfs , the sharp rise in the adsorption / desorption isotherms at relative pressures close to 1 points to the existence of large mesopores in these materials ( gregg et al 1982 ) . thus , sba-15 and mcf before and after functionalization using amino groups where tested as delivery systems for bsa . the amount of bsa loaded in mcf materials was higher ( 24% ) than in sba-15 matrices ( 15% ) , due to the higher pore volume in the former ( table 3 ) . the amount of bsa loaded after functionalization followed the same trend that unmodified materials , ie , mcf - nh2 loaded more bsa ( 27% ) than sba-15-nh2 ( 10% ) , as a result of the higher pore volume of the former . on the contrary , the initial burst in amino - modified matrices was drastically reduced to ca 10% , and more than 80% of the loaded bsa was released to the medium in a sustained manner . therefore , when the loading of large - size and large - volume biologically active molecules is targeted , pore diameter acts as the limiting factor and pore volume determines the amount of molecule hosted . the adsorption of molecules into silica - based ordered mesoporous materials is a size - selective process and consequently , the mesopore diameter determines the size of the molecule to be hosted . hence , the adsorptive properties of mesoporous materials will determine the amount of adsorbate loaded . therefore , considering that the size of common drugs falls into the nanometer scale and that mesopore diameters can be tuned from 1.5 nm to several tens of nanometers , it seems reasonable to think that mesoporous matrices are able to host a wide range of biologically active molecules , ranging from small drugs to macromolecules such as large - size proteins . when mcm-41 was reported for the first time as a drug delivery system ( vallet - reg et al 2001b ) , several cationic surfactants with different chain lengths were employed , which led to mcm-41 materials with different pore sizes . pore diameter is a limiting factor in molecule adsorption when the confinement of really large molecules , such as proteins and other biologically active molecules , is targeted . therefore , the adsorption of globular proteins on mesoporous mcm-41 ( with pore diameters ranging from 2 to 5 nm ) has been reported in the literature ( deere et al 2002 ) . further adsorption studies revealed that the proteins with the smallest sizes , ie , cytochrome c , lysozyme , myoglobin , and -lactoglobulin , showed significant adsorption . on the contrary , the proteins with the largest sizes , ie , conalbumin , serum albumin and ovalbumin were excluded from the internal surfaces of thiol - functionalized sba-15 . it should be remarked that the maximum amounts of protein loaded , expressed as a volume percent of the available internal pore volume ( 5% for -lactoglobulin , 15% for myoglobin , and 43% for cytochrome c ) showed that the protein molecules were adsorbed within the mesopores and not only on the external surfaces . therefore , the pore size has been revealed as a key factor governing the adsorption of proteins in mesoporous materials . however , once the protein is adsorbed , the ruling factor on the release kinetics has been shown to be the organic functionalization , due to the host - guest attracting interactions . the adsorption of biologically active molecules into meso - porous matrices is governed by the chemical interaction between silanol groups covering the silica surface and the functional groups of the guest molecule . mcm-41 and sba-15 were functionalized using amino groups with the aim of increasing the attracting host - guest interactions , and alendronate loading and release studies were carried out ( balas et al 2006 ) . this fast release of the drug could be due to several reasons : alendronate that could be adsorbed in the outer surface of the matrix or by the existent alendronate gradient between mesoporous matrix and delivery medium . moreover , the increase in the total drug delivery time in functionalized materials compared with unmodified matrices ( table 2 ) can be ascribed to the stronger interactions between phosphonate groups from alendronate and amino groups covering the pore walls . in this sense , organic functionalization allows a higher control over drug loading and release kinetics . as previously commented , adsorption of biologically active molecules is a surface phenomenon that takes place by attracting interactions between the silanol groups in the pore walls ( or functional groups in the case of functionalized materials ) and functional groups of the guest molecule . however , when the confinement of really large molecules is aimed , such as large - size and large - volume proteins , pore volume seems to play a key role in molecule adsorption . in the case of mcfs , the sharp rise in the adsorption / desorption isotherms at relative pressures close to 1 points to the existence of large mesopores in these materials ( gregg et al 1982 ) . the amount of bsa loaded in mcf materials was higher ( 24% ) than in sba-15 matrices ( 15% ) , due to the higher pore volume in the former ( table 3 ) . the amount of bsa loaded after functionalization followed the same trend that unmodified materials , ie , mcf - nh2 loaded more bsa ( 27% ) than sba-15-nh2 ( 10% ) , as a result of the higher pore volume of the former . the pore diameter of sba-15 ( 8.5 nm ) is just on the limit of bsa size and therefore , after functionalization the pore diameter decreased to 6.9 nm and consequently , a decrease in the amount of bsa adsorbed was observed . on the contrary , the initial burst in amino - modified matrices was drastically reduced to ca 10% , and more than 80% of the loaded bsa was released to the medium in a sustained manner . in this work it has been shown that available pore volume and surface play a key role in the protein loading capacity of silica - based ordered mesoporous materials . ideally , if large biomolecules such as certain proteins are targeted to be adsorbed in ordered mesoporous materials , these matrices should present several characteristics : pore size large enough to allow diffusion into the pores , surface area as high as possible to allow a large retention percentage , and pore volume as high as possible to offer available space into the mesopores to be filled by the protein . recent advances in nanotechnology offer the possibility of appropriately tailoring the structural and textural properties of mesoporous silicas depending on the desired application . the outstanding features of silica - based ordered mesoporous materials open up promising expectations in the biomedical field because they can be employed as starting materials for the further design of scaffolds for bone tissue regeneration .

it is estimated that yearly 134,420 indian women are newly diagnosed with cancer of the cervix and each year the mortality due to this disease is estimated to be around 72,825 indian women . most of the cases ( 85% ) present in advanced cases and late stages . out of these not all cases achieve a complete cure owing to various reasons such as advanced stage , poor differentiated status of histology of the tumor , treatment failures and treatment gaps . the present study aims to find out the socio - demographic causes , which lead to non - compliance to treatment even after registration in radiotherapy ( rt ) department of a state government rural medical college hospital . this department has got a co-60 machine for external beam radiotherapy ( ebrt ) and refers patients on a government referral basis to kolkata ( 567 km away ) for brachytherapy . the present study aims to find out the socio - demographic causes , which lead to non - compliance to treatment even after registration in radiotherapy ( rt ) department of a state government rural medical college hospital . this department has got a co-60 machine for external beam radiotherapy ( ebrt ) and refers patients on a government referral basis to kolkata ( 567 km away ) for brachytherapy . this study is a retrospective analysis during the period 2011 - 2012 conducted at north bengal medical college ( nbmch ) . all patients were first evaluated by clinical , radiological and laboratory investigations at gynecology and obstetrics department and then by rt department at nbmch . all patients were planned for treatment by rt with co-60 machine ( theratronics international ontariocanada , machine id-1117 , serial no -191 ) . brachytherapy was given at 7 gy per fraction for 3 weekly by high dose rate ( hdr ) at rt department , kolkata . concurrent chemotherapy was given to a select group of patients by cisplatin weekly at 40 mg / m after assessing , creatinine clearance and other blood parameters such as complete hemogram , random blood sugar and liver function tests . all patients diagnosed with biopsy proven cervical adenocarcinoma or squamous cell carcinoma or adenosquamous carcinomacases of international federation of gynecological and obstetrics ( figo ) stage ib to ivapost - operative carcinoma cervix casespatients receiving external radiation at nbmch followed by brachytherapy at kolkata . all patients diagnosed with biopsy proven cervical adenocarcinoma or squamous cell carcinoma or adenosquamous carcinoma cases of international federation of gynecological and obstetrics ( figo ) stage ib to iva post - operative carcinoma cervix cases patients receiving external radiation at nbmch followed by brachytherapy at kolkata . patients having metastatic diseasepatients having double primary cancerspatients having other comorbid conditions such as diabetes , hypertension , ischemic heart disease , tuberculosis and autoimmune disorder . patients having metastatic disease patients having double primary cancers patients having other comorbid conditions such as diabetes , hypertension , ischemic heart disease , tuberculosis and autoimmune disorder . in this retrospective study , the treatment record files of patients who were finally selected according to the exclusion and inclusion criteria , were analyzed with respect to their treatment details . it was noted whether these patients had completed radiation , which comprised of taking the course of ebrt at nbmch and then brachytherapy at kolkata . with respect to the treatment adherence and completion , the selected patients were divided into three groups : 1 group ( group a ) consisted of those patients who did not take or complete external ebrt after their registration at nbmch2 group ( group b ) consisted of those patients who completed ebrt but did not take or complete intracavitary brachytherapy ( icrt)3 group ( group c ) consisted of those patients who completed ebrt and icrt . 1 group ( group a ) consisted of those patients who did not take or complete external ebrt after their registration at nbmch 2 group ( group b ) consisted of those patients who completed ebrt but did not take or complete intracavitary brachytherapy ( icrt ) 3 group ( group c ) consisted of those patients who completed ebrt and icrt . the factors such as age , parity , menopausal status , monthly income level , level of education , distance from home , which meant time taken and expenses to travel for ebrt were analyzed . the analysis was performed using simple tests of percentage evaluation and representative bar diagrams and pie charts . all patients diagnosed with biopsy proven cervical adenocarcinoma or squamous cell carcinoma or adenosquamous carcinomacases of international federation of gynecological and obstetrics ( figo ) stage ib to ivapost - operative carcinoma cervix casespatients receiving external radiation at nbmch followed by brachytherapy at kolkata . all patients diagnosed with biopsy proven cervical adenocarcinoma or squamous cell carcinoma or adenosquamous carcinoma cases of international federation of gynecological and obstetrics ( figo ) stage ib to iva post - operative carcinoma cervix cases patients receiving external radiation at nbmch followed by brachytherapy at kolkata . patients having metastatic diseasepatients having double primary cancerspatients having other comorbid conditions such as diabetes , hypertension , ischemic heart disease , tuberculosis and autoimmune disorder . patients having metastatic disease patients having double primary cancers patients having other comorbid conditions such as diabetes , hypertension , ischemic heart disease , tuberculosis and autoimmune disorder . in this retrospective study , the treatment record files of patients who were finally selected according to the exclusion and inclusion criteria , were analyzed with respect to their treatment details . it was noted whether these patients had completed radiation , which comprised of taking the course of ebrt at nbmch and then brachytherapy at kolkata . with respect to the treatment adherence and completion , the selected patients were divided into three groups : 1 group ( group a ) consisted of those patients who did not take or complete external ebrt after their registration at nbmch2 group ( group b ) consisted of those patients who completed ebrt but did not take or complete intracavitary brachytherapy ( icrt)3 group ( group c ) consisted of those patients who completed ebrt and icrt . 1 group ( group a ) consisted of those patients who did not take or complete external ebrt after their registration at nbmch 2 group ( group b ) consisted of those patients who completed ebrt but did not take or complete intracavitary brachytherapy ( icrt ) 3 group ( group c ) consisted of those patients who completed ebrt and icrt . the factors such as age , parity , menopausal status , monthly income level , level of education , distance from home , which meant time taken and expenses to travel for ebrt were analyzed . the analysis was performed using simple tests of percentage evaluation and representative bar diagrams and pie charts . the lowest age of the presentation was 28 years and the highest age of the presentation was 80 years . clearly it shows that the majority ( 55.55% ) cases are in the age group of 40 - 50 years . majority of the cases were post - menopausal ( 56.94% ) and had a family burden of more than three children ( 59.02% ) . only 6.25% cases were self - employed and the rest had to depend on the family and husband for their livelihood . patients were subdivided into three groups [ figure 6 ] according to their compliance to treatment . 38.88% of the cases had completed the entire treatment ( group c ) , which comprised of both external radiation and brachytherapy . however , 25% cases did not complete external radiation and 36.11% cases could complete external radiation , but did not comply with brachytherapy in kolkata . the analysis was carried out to find out the possible important causes of these treatment failures . comparison of the three groups was performed with respect to six different socio - demographic parameters [ figures 712 ] . group analysis age wise group analysis of literacy group wise parity analysis group wise analysis of menopausal status group wise analysis of socio - economic status group wise analysis of distance it was found that elderly women ( < 50 years of age and post - menopausal ) belonged to groups a and b , i.e. , were defaulters . 52.78% cases were elderly and could not complete ebrt , i.e. , group a [ figure 7 ] . group a also had the highest number of illiterate women ( 57.69% cases [ figure 8 ] . group a had the maximum number of women who had more than three children at home ( 76.92% cases ) [ figure 9 ] . socio - economic status was based on whether the family had a below the poverty line ( bpl ) card or not . among the defaulters , it was surprisingly seen that in group b ( i.e. , those who could not go to kolkata for brachytherapy ) 69.23% cases [ figure 11 ] were not bpl card holders or not poor . distance assessment was based on the fact that near = up to 100 km and distant < 100 km . it was seen that 63.89% cases of group a and 69.23% cases of group b were not living far , but were the majority of the defaulters [ figure 12 ] . a matched comparison of all the factors was performed to visualize the highest percentage of cases in each group [ figure 13 ] . it was found that 76.92% cases of group a had the highest percentage of cases and this was pertaining to the issue of having many children . 69.23% cases of group b ( i.e. , brachytherapy failures ) was the most common and second highest recorded number of cases with respect age 40 - 50 years , post - menopausal state , of poor socio - economic status and also surprisingly had home nearby . matched comparison of all the variables in three groups a total of 56 cases had completed treatment . however , we found that even there were two subsets of patients who could complete the entire treatment within 8 weeks and who took a prolonged time to complete the treatment . further , analysis was performed to find out the issues pertaining to prolonged or delayed treatment [ figures 1419 ] . 87.5% cases of group c1 , i.e. , those who completed the entire treatment in 8 weeks were not poor . 56.25% of cases of group c1 were middle aged , had less number of children ( para = 2 ) and were post - menopausal . however , among those who had prolonged time to complete treatment , 95% had < two children . a matched analysis of both the subgroups c1 and c2 was done [ figure 19 ] and the data was visualized . age comparison of c subgroups education status comparison of c subgroups comparison of parity status of c subgroups comparison of menopausal state of c subgroups comparison of distance of c subgroups carcinoma of the cervix is a leading cause of cancer in india and most of the cases present in figo iib to iiib . the standard treatment of locally advanced cases of cervical carcinoma is rt with concurrent chemotherapy followed by brachytherapy . to achieve best local tumor control the entire treatment , in locally advanced cases classically ebrt is given first and this takes 4 - 5 weeks to complete . after completion of ebrt , brachytherapy is given weekly once for 2 - 3 fractions ( doses ) by hdr and thus it take 2 - 3 weeks to complete . usually , a gap of 2 - 3 weeks is given between completion of external radiation and the start of brachytherapy depending upon the severity of acute toxicity of radiation . thus , the entire treatment depends on many factors such as patient 's general condition , tolerance to treatment , availability of radiation facility of both ebrt and hdr and radioresponsiveness of the tumor . however , every patient has to stay near the hospital or radiation department for the entire 8 weeks of treatment and this is a very important logistical issue , which is often not emphasized or addressed and this leads to treatment breaks . the incidence of cervical cancer is greatest among women of lower socio - economic backgrounds . unquantifiable cultural and individual logistical hurdles such as limited financial , social and personal resources are often significant barriers for receiving treatment . , fear , language , transportation , religious and cultural beliefs may also play a role . , , , , , , these barriers may disrupt patient 's desires to complete their treatment , causing prolonged radiation therapy schedules . , , consequently cervical cancer has been reported to cause approximately 25% of cancer deaths in certain minority populations . the rt department in nbmch is the only place having a co-60 machine in the government set - up in the entire north bengal in the state of west bengal , india and cancer patients come from six adjoining districts of north bengal ( malda , uttar and dakshin dinajpur , jalpaiguri , coochbehar , darjeeling ) , sikkim , western districts of assam ( dhubri , kokrajhar ) , bihar and also from neighboring countries such as nepal , bangladesh and bhutan for radiation treatment . hence , the burden of cancer cases registration in rt out - patient department ( opd ) is very high per year and is increasing every year . moreover , after completion of ebrt these patients are referred to kolkata which is approximately 567 km away from siliguri ( the place where nbmch is situated ) for brachytherapy . all patients are given forms for railway concession for travel from their home place in north bengal to kolkata so that they can go for completion of the treatment . this study was an attempt to find out the possible causes for these failures and to find out steps for ensuring better treatment compliance than the present scenario . in this study , it has been seen that 25% of cases ( group a ) did not complete ebrt even after registration at rt opd . in this group , majority of the cases in this group were elderly ( 52.78% cases of age < 50 years ) and post - menopausal ( 58.33% cases ) . hence , it may be speculated that the problem of old age and family burden was definitely a major contributing factor to not complying to ebrt even after starting it . this problem was also seen in the group b ( which had completed ebrt but did not go to kolkata for brachytherapy ) , i.e. , three out of the six assessment factors ( age of 40 - 50 years , poor socio - economic status and post - menopausal status ) had 69.23% of cases in each . even in this group women who had more than three children ( 60.86% ) had the maximum problem of not going for brachytherapy 567 km away . indeed there was therefore a major link between issues of elderly age , family burden and poor compliance to treatment . most of the husbands are daily wagers and have children at home who also need care and food to eat . these women mainly look after the general household and the kitchen at home and hence their absence from home for a long time is general difficult to manage for the husband and their children , which ultimately leads to the fact that these women are somehow compelled to return home even if the rt was started . since these women are weak with disease and can not do or help in the household work , they are often a burden to the family of this population . in most of the cases ( 93.75% ) , the woman has to depend on husband or any other family member for constant company for travel , stay and complying to needs during ebrt , which is not always possible . hence , they prefer to stay back at home due to lack of a persistent caregiver . almost two - third of the patients live outside the main town and the average time taken to travel is 5 - 6 h by train or by road . this includes the daily bus fare of atleast two persons up and down , which is not less than rs . 200 daily , schedule and timing of the conveyance mode which is subjected to weather conditions , road conditions , railways arrival and departure timings coinciding with the hospital opd hours , holidays and strikes , road and rail blocks . this leads to the fact that inspite of the initial registration at rt opd these patients find it difficult to travel multiple times for further investigative work - up and getting rt dates before the start of treatment . hence , this was probably the reason why inspite of the near distance ( > 100 km ) from their respective homes to nbmch in 63.89% cases of group a and 69.23% cases of ( group b ) there was poor compliance to treatment . similar issues of distance and transportation related problems were addressed in studies by ramondetta et al . from houston , texas and mundt et al . from chicago and it was shown that a combination of the lack of adequate methods of transportation and social support may contribute to the high cost of treatment in patients with cervical cancer . , poor socio - economic status was the second common factor in both groups of failures . during ebrt each patient has to stay near the rt department for 1 - 1 months as the hospital does not have any hostel for the patient and family to stay during long - term treatment . hence each , patient has to take room / house for rent at an average expense of rs . 1200/month for lodging only excluding daily other expenses for food , clothing , emergency medicines and general household expenses . in order to do this each lady has to either bring her husband or any willing relative of her own in order to accompany her during the ebrt treatment and this doubles up the expenses of food , clothing and other daily needs and this is around rs . , the total cost of stay for the patient and her family is approximately rs . however , poor socio - economic status was a major problem to travel for brachytherapy ( 69.23% ) rather than coming for ebrt at first ( 52.78% cases ) . surprisingly it was found that 64.29% of cases who had completed the entire treatment ( group c ) had a poor socio - economic status , i.e. , ebrt followed by getting brachytherapy 567 km away . hence , subgroup analysis of group c was performed to critically further analyze all the six evaluable socio - demographic factors respectively in subgroup c1 ( those who completed treatment within 8 weeks ) and c2 ( < 8 weeks to complete ) . only 28.57% cases of group c could complete the treatment within 8 weeks and 87.5% of cases out of these group c1 were not of bpl category . 71.42% cases were in group c2 and 45% cases of group c2 were of bpl category , which reflected in the broad group c as the majority of cases . hence , the critical analysis of group c did showed that a high association of good treatment compliance with good socio - economic status in a select subgroup of patients . nearly 68.75% cases of group c1 lived near nbmch compared to 55% cases of group c2 who lived far away when the factor of distance was compared . this also suggested that people living near had to travel less and hence could complete the treatment in time inspite of having a poor socio - economic status . women were of middle age ( 40 - 50 years ) mostly and did not have more than two children at home in both groups c1 and c2 . this indirectly reflected that good treatment in young or middle aged women who at the same time did not have a great burden of children . literacy was not a very strong decisive factor as reflected from this study as most of the cases in any group were either illiterate or not better than just having primary education and there was no major difference between the number of illiterate ( 41.66% ) women and those who had merely primary education ( 43.05% cases only ) . factors like advanced age related poor treatment compliance and outcome have been addressed in the study by brun et al . in january 2005 , trimble et al . had shown that in the us many women with cervical cancer are not treated properly and the factors associated included lack of access to the health - care system , access to a medical specialist , lack of third - party coverage and lack of the necessary social support to help patients be compliant with their medical treatment . the authors emphasized that we must implement research to identify the obstacles that may prevent adherence to treatment recommendations . similar such studies were performed by authors like formenti et al . at los angeles , carpenter and cowell at texas , and suggested several possible reasons like poor understanding of the disease process and the importance of treatment , as well as concerns about transportation , childcare , employment for high rates of non - compliance among inner - city minority patients treated with radiation for cervical cancer . there has been a lot of emphasis on socio - demographic issues regarding cervical cancer screening program and the current study is our initial attempt in india to address these issues and find out the possible suggestions to ensure proper treatment compliance . however , larger more detailed studies will be needed to clarify the reasons for these failures and to dene any solution for better treatment outcome . the limitations of this study this study was an initial attempt and probably the first reported study of the issue of non - compliance to rt treatment of cervical cancer in a peripheral rural medical college hospital . the relative time period was short moreover many other factors like linguistics barrier , involvement of other forms of treatment - like homeopathy , ayurvedic or even local tantric or indigenous methods of shedding spells or casts were not included- these could also have been a hindrance to the compliance of treatment . however , this study needs to be carried out for a more prolonged time and include more number of patients so that a proper re - evaluation of more number of important factors leading to non - compliance can be analyzed statistically . this study strongly suggests that a family burden of more than three children is a major determinant of poor treatment compliance . the best outcome is directly linked with a good socio - economic status though a bpl category does not necessarily mean that she will not comply to treatment . hence our suggestions encourage small family size : effort should be made to reduce the undue delay in the investigative work up in radiology and pathology department and in getting radiation commencement dates . this will ensure lesser number of unwanted travel and expensesevery effort should be made to increase the facilities of arranging the patient 's hostel near to the rt department at a cheap and subsidized rate from the government so that they can stay with their family during the entire treatment and avoid undue excess expenditure in a limited source of incomeeven though , the bpl category patients get free treatment facilities for radiation and chemotherapy ; this should be extended in an uncomplicated manner to all patients irrespective of their financial status with regards not only to treatment , but to travel both by rail and specially by road as people hear have to travel long distances by road alonesmall incentives like giving a small cash token amount of money after completing ebrt and brachytherapy can be given as a reward so as to encourage more treatment compliance and goal directed attitude among the patient and the caregivers quite like the concept of mid - day meal for ensuring good formal education among children and also like getting cash money after getting tubal ligation carried out in camps and hospitallast but not the least every effort made to increase the number of rt centers with basic external beam and brachytherapy facilities in this part of the country so that people have to travel less from their homes . effort should be made to reduce the undue delay in the investigative work up in radiology and pathology department and in getting radiation commencement dates . this will ensure lesser number of unwanted travel and expenses every effort should be made to increase the facilities of arranging the patient 's hostel near to the rt department at a cheap and subsidized rate from the government so that they can stay with their family during the entire treatment and avoid undue excess expenditure in a limited source of income even though , the bpl category patients get free treatment facilities for radiation and chemotherapy ; this should be extended in an uncomplicated manner to all patients irrespective of their financial status with regards not only to treatment , but to travel both by rail and specially by road as people hear have to travel long distances by road alone small incentives like giving a small cash token amount of money after completing ebrt and brachytherapy can be given as a reward so as to encourage more treatment compliance and goal directed attitude among the patient and the caregivers quite like the concept of mid - day meal for ensuring good formal education among children and also like getting cash money after getting tubal ligation carried out in camps and hospital last but not the least every effort made to increase the number of rt centers with basic external beam and brachytherapy facilities in this part of the country so that people have to travel less from their homes .

introduction : carcinoma cervix is a leading cause of cancer in india . however , majority of the patients face a problem of not being able to complete the treatment.aim:this study was an attempt to find out the important causes of this non - compliance to treatment in a rural medical college hospital where majority of the cancer cases are of cervical cancer.results:out of 144 patients studied over 2 years 88 cases could not complete the treatment . the study revealed that due old age 58.33% cases were defaulters , having many children at home meant a burden to 76.92% cases and 63.89% cases had a problem of not been able to travel a far distance of more than 100 km from home to hospital for treatment.conclusion:these were the important factors of non - compliance and suggested more important than the issues of literacy and poor socio - economic status .

INTRODUCTION Aim MATERIALS AND METHODS Inclusion criteria Exclusion criteria RESULTS DISCUSSION CONCLUSIONS

it is estimated that yearly 134,420 indian women are newly diagnosed with cancer of the cervix and each year the mortality due to this disease is estimated to be around 72,825 indian women . most of the cases ( 85% ) present in advanced cases and late stages . out of these not all cases achieve a complete cure owing to various reasons such as advanced stage , poor differentiated status of histology of the tumor , treatment failures and treatment gaps . the present study aims to find out the socio - demographic causes , which lead to non - compliance to treatment even after registration in radiotherapy ( rt ) department of a state government rural medical college hospital . the present study aims to find out the socio - demographic causes , which lead to non - compliance to treatment even after registration in radiotherapy ( rt ) department of a state government rural medical college hospital . this study is a retrospective analysis during the period 2011 - 2012 conducted at north bengal medical college ( nbmch ) . all patients were first evaluated by clinical , radiological and laboratory investigations at gynecology and obstetrics department and then by rt department at nbmch . all patients were planned for treatment by rt with co-60 machine ( theratronics international ontariocanada , machine id-1117 , serial no -191 ) . all patients diagnosed with biopsy proven cervical adenocarcinoma or squamous cell carcinoma or adenosquamous carcinomacases of international federation of gynecological and obstetrics ( figo ) stage ib to ivapost - operative carcinoma cervix casespatients receiving external radiation at nbmch followed by brachytherapy at kolkata . all patients diagnosed with biopsy proven cervical adenocarcinoma or squamous cell carcinoma or adenosquamous carcinoma cases of international federation of gynecological and obstetrics ( figo ) stage ib to iva post - operative carcinoma cervix cases patients receiving external radiation at nbmch followed by brachytherapy at kolkata . in this retrospective study , the treatment record files of patients who were finally selected according to the exclusion and inclusion criteria , were analyzed with respect to their treatment details . it was noted whether these patients had completed radiation , which comprised of taking the course of ebrt at nbmch and then brachytherapy at kolkata . with respect to the treatment adherence and completion , the selected patients were divided into three groups : 1 group ( group a ) consisted of those patients who did not take or complete external ebrt after their registration at nbmch2 group ( group b ) consisted of those patients who completed ebrt but did not take or complete intracavitary brachytherapy ( icrt)3 group ( group c ) consisted of those patients who completed ebrt and icrt . 1 group ( group a ) consisted of those patients who did not take or complete external ebrt after their registration at nbmch 2 group ( group b ) consisted of those patients who completed ebrt but did not take or complete intracavitary brachytherapy ( icrt ) 3 group ( group c ) consisted of those patients who completed ebrt and icrt . the factors such as age , parity , menopausal status , monthly income level , level of education , distance from home , which meant time taken and expenses to travel for ebrt were analyzed . all patients diagnosed with biopsy proven cervical adenocarcinoma or squamous cell carcinoma or adenosquamous carcinomacases of international federation of gynecological and obstetrics ( figo ) stage ib to ivapost - operative carcinoma cervix casespatients receiving external radiation at nbmch followed by brachytherapy at kolkata . all patients diagnosed with biopsy proven cervical adenocarcinoma or squamous cell carcinoma or adenosquamous carcinoma cases of international federation of gynecological and obstetrics ( figo ) stage ib to iva post - operative carcinoma cervix cases patients receiving external radiation at nbmch followed by brachytherapy at kolkata . in this retrospective study , the treatment record files of patients who were finally selected according to the exclusion and inclusion criteria , were analyzed with respect to their treatment details . with respect to the treatment adherence and completion , the selected patients were divided into three groups : 1 group ( group a ) consisted of those patients who did not take or complete external ebrt after their registration at nbmch2 group ( group b ) consisted of those patients who completed ebrt but did not take or complete intracavitary brachytherapy ( icrt)3 group ( group c ) consisted of those patients who completed ebrt and icrt . the factors such as age , parity , menopausal status , monthly income level , level of education , distance from home , which meant time taken and expenses to travel for ebrt were analyzed . the lowest age of the presentation was 28 years and the highest age of the presentation was 80 years . clearly it shows that the majority ( 55.55% ) cases are in the age group of 40 - 50 years . majority of the cases were post - menopausal ( 56.94% ) and had a family burden of more than three children ( 59.02% ) . only 6.25% cases were self - employed and the rest had to depend on the family and husband for their livelihood . patients were subdivided into three groups [ figure 6 ] according to their compliance to treatment . 38.88% of the cases had completed the entire treatment ( group c ) , which comprised of both external radiation and brachytherapy . however , 25% cases did not complete external radiation and 36.11% cases could complete external radiation , but did not comply with brachytherapy in kolkata . the analysis was carried out to find out the possible important causes of these treatment failures . comparison of the three groups was performed with respect to six different socio - demographic parameters [ figures 712 ] . group analysis age wise group analysis of literacy group wise parity analysis group wise analysis of menopausal status group wise analysis of socio - economic status group wise analysis of distance it was found that elderly women ( < 50 years of age and post - menopausal ) belonged to groups a and b , i.e. , were defaulters . 52.78% cases were elderly and could not complete ebrt , i.e. group a had the maximum number of women who had more than three children at home ( 76.92% cases ) [ figure 9 ] . socio - economic status was based on whether the family had a below the poverty line ( bpl ) card or not . among the defaulters , it was surprisingly seen that in group b ( i.e. , those who could not go to kolkata for brachytherapy ) 69.23% cases [ figure 11 ] were not bpl card holders or not poor . distance assessment was based on the fact that near = up to 100 km and distant < 100 km . it was seen that 63.89% cases of group a and 69.23% cases of group b were not living far , but were the majority of the defaulters [ figure 12 ] . it was found that 76.92% cases of group a had the highest percentage of cases and this was pertaining to the issue of having many children . , brachytherapy failures ) was the most common and second highest recorded number of cases with respect age 40 - 50 years , post - menopausal state , of poor socio - economic status and also surprisingly had home nearby . matched comparison of all the variables in three groups a total of 56 cases had completed treatment . however , we found that even there were two subsets of patients who could complete the entire treatment within 8 weeks and who took a prolonged time to complete the treatment . further , analysis was performed to find out the issues pertaining to prolonged or delayed treatment [ figures 1419 ] . , those who completed the entire treatment in 8 weeks were not poor . however , among those who had prolonged time to complete treatment , 95% had < two children . age comparison of c subgroups education status comparison of c subgroups comparison of parity status of c subgroups comparison of menopausal state of c subgroups comparison of distance of c subgroups carcinoma of the cervix is a leading cause of cancer in india and most of the cases present in figo iib to iiib . the standard treatment of locally advanced cases of cervical carcinoma is rt with concurrent chemotherapy followed by brachytherapy . to achieve best local tumor control the entire treatment , in locally advanced cases classically ebrt is given first and this takes 4 - 5 weeks to complete . after completion of ebrt , brachytherapy is given weekly once for 2 - 3 fractions ( doses ) by hdr and thus it take 2 - 3 weeks to complete . thus , the entire treatment depends on many factors such as patient 's general condition , tolerance to treatment , availability of radiation facility of both ebrt and hdr and radioresponsiveness of the tumor . however , every patient has to stay near the hospital or radiation department for the entire 8 weeks of treatment and this is a very important logistical issue , which is often not emphasized or addressed and this leads to treatment breaks . the incidence of cervical cancer is greatest among women of lower socio - economic backgrounds . , , , , , , these barriers may disrupt patient 's desires to complete their treatment , causing prolonged radiation therapy schedules . , , consequently cervical cancer has been reported to cause approximately 25% of cancer deaths in certain minority populations . hence , the burden of cancer cases registration in rt out - patient department ( opd ) is very high per year and is increasing every year . all patients are given forms for railway concession for travel from their home place in north bengal to kolkata so that they can go for completion of the treatment . this study was an attempt to find out the possible causes for these failures and to find out steps for ensuring better treatment compliance than the present scenario . in this study , it has been seen that 25% of cases ( group a ) did not complete ebrt even after registration at rt opd . in this group , majority of the cases in this group were elderly ( 52.78% cases of age < 50 years ) and post - menopausal ( 58.33% cases ) . hence , it may be speculated that the problem of old age and family burden was definitely a major contributing factor to not complying to ebrt even after starting it . , three out of the six assessment factors ( age of 40 - 50 years , poor socio - economic status and post - menopausal status ) had 69.23% of cases in each . even in this group women who had more than three children ( 60.86% ) had the maximum problem of not going for brachytherapy 567 km away . indeed there was therefore a major link between issues of elderly age , family burden and poor compliance to treatment . most of the husbands are daily wagers and have children at home who also need care and food to eat . these women mainly look after the general household and the kitchen at home and hence their absence from home for a long time is general difficult to manage for the husband and their children , which ultimately leads to the fact that these women are somehow compelled to return home even if the rt was started . since these women are weak with disease and can not do or help in the household work , they are often a burden to the family of this population . in most of the cases ( 93.75% ) , the woman has to depend on husband or any other family member for constant company for travel , stay and complying to needs during ebrt , which is not always possible . hence , they prefer to stay back at home due to lack of a persistent caregiver . almost two - third of the patients live outside the main town and the average time taken to travel is 5 - 6 h by train or by road . 200 daily , schedule and timing of the conveyance mode which is subjected to weather conditions , road conditions , railways arrival and departure timings coinciding with the hospital opd hours , holidays and strikes , road and rail blocks . this leads to the fact that inspite of the initial registration at rt opd these patients find it difficult to travel multiple times for further investigative work - up and getting rt dates before the start of treatment . hence , this was probably the reason why inspite of the near distance ( > 100 km ) from their respective homes to nbmch in 63.89% cases of group a and 69.23% cases of ( group b ) there was poor compliance to treatment . similar issues of distance and transportation related problems were addressed in studies by ramondetta et al . from houston , texas and mundt et al . from chicago and it was shown that a combination of the lack of adequate methods of transportation and social support may contribute to the high cost of treatment in patients with cervical cancer . , poor socio - economic status was the second common factor in both groups of failures . hence each , patient has to take room / house for rent at an average expense of rs . 1200/month for lodging only excluding daily other expenses for food , clothing , emergency medicines and general household expenses . , the total cost of stay for the patient and her family is approximately rs . however , poor socio - economic status was a major problem to travel for brachytherapy ( 69.23% ) rather than coming for ebrt at first ( 52.78% cases ) . surprisingly it was found that 64.29% of cases who had completed the entire treatment ( group c ) had a poor socio - economic status , i.e. hence , subgroup analysis of group c was performed to critically further analyze all the six evaluable socio - demographic factors respectively in subgroup c1 ( those who completed treatment within 8 weeks ) and c2 ( < 8 weeks to complete ) . only 28.57% cases of group c could complete the treatment within 8 weeks and 87.5% of cases out of these group c1 were not of bpl category . 71.42% cases were in group c2 and 45% cases of group c2 were of bpl category , which reflected in the broad group c as the majority of cases . hence , the critical analysis of group c did showed that a high association of good treatment compliance with good socio - economic status in a select subgroup of patients . nearly 68.75% cases of group c1 lived near nbmch compared to 55% cases of group c2 who lived far away when the factor of distance was compared . this also suggested that people living near had to travel less and hence could complete the treatment in time inspite of having a poor socio - economic status . women were of middle age ( 40 - 50 years ) mostly and did not have more than two children at home in both groups c1 and c2 . this indirectly reflected that good treatment in young or middle aged women who at the same time did not have a great burden of children . literacy was not a very strong decisive factor as reflected from this study as most of the cases in any group were either illiterate or not better than just having primary education and there was no major difference between the number of illiterate ( 41.66% ) women and those who had merely primary education ( 43.05% cases only ) . factors like advanced age related poor treatment compliance and outcome have been addressed in the study by brun et al . had shown that in the us many women with cervical cancer are not treated properly and the factors associated included lack of access to the health - care system , access to a medical specialist , lack of third - party coverage and lack of the necessary social support to help patients be compliant with their medical treatment . the authors emphasized that we must implement research to identify the obstacles that may prevent adherence to treatment recommendations . similar such studies were performed by authors like formenti et al . at los angeles , carpenter and cowell at texas , and suggested several possible reasons like poor understanding of the disease process and the importance of treatment , as well as concerns about transportation , childcare , employment for high rates of non - compliance among inner - city minority patients treated with radiation for cervical cancer . there has been a lot of emphasis on socio - demographic issues regarding cervical cancer screening program and the current study is our initial attempt in india to address these issues and find out the possible suggestions to ensure proper treatment compliance . however , larger more detailed studies will be needed to clarify the reasons for these failures and to dene any solution for better treatment outcome . the limitations of this study this study was an initial attempt and probably the first reported study of the issue of non - compliance to rt treatment of cervical cancer in a peripheral rural medical college hospital . however , this study needs to be carried out for a more prolonged time and include more number of patients so that a proper re - evaluation of more number of important factors leading to non - compliance can be analyzed statistically . this study strongly suggests that a family burden of more than three children is a major determinant of poor treatment compliance . the best outcome is directly linked with a good socio - economic status though a bpl category does not necessarily mean that she will not comply to treatment . this will ensure lesser number of unwanted travel and expensesevery effort should be made to increase the facilities of arranging the patient 's hostel near to the rt department at a cheap and subsidized rate from the government so that they can stay with their family during the entire treatment and avoid undue excess expenditure in a limited source of incomeeven though , the bpl category patients get free treatment facilities for radiation and chemotherapy ; this should be extended in an uncomplicated manner to all patients irrespective of their financial status with regards not only to treatment , but to travel both by rail and specially by road as people hear have to travel long distances by road alonesmall incentives like giving a small cash token amount of money after completing ebrt and brachytherapy can be given as a reward so as to encourage more treatment compliance and goal directed attitude among the patient and the caregivers quite like the concept of mid - day meal for ensuring good formal education among children and also like getting cash money after getting tubal ligation carried out in camps and hospitallast but not the least every effort made to increase the number of rt centers with basic external beam and brachytherapy facilities in this part of the country so that people have to travel less from their homes . this will ensure lesser number of unwanted travel and expenses every effort should be made to increase the facilities of arranging the patient 's hostel near to the rt department at a cheap and subsidized rate from the government so that they can stay with their family during the entire treatment and avoid undue excess expenditure in a limited source of income even though , the bpl category patients get free treatment facilities for radiation and chemotherapy ; this should be extended in an uncomplicated manner to all patients irrespective of their financial status with regards not only to treatment , but to travel both by rail and specially by road as people hear have to travel long distances by road alone small incentives like giving a small cash token amount of money after completing ebrt and brachytherapy can be given as a reward so as to encourage more treatment compliance and goal directed attitude among the patient and the caregivers quite like the concept of mid - day meal for ensuring good formal education among children and also like getting cash money after getting tubal ligation carried out in camps and hospital last but not the least every effort made to increase the number of rt centers with basic external beam and brachytherapy facilities in this part of the country so that people have to travel less from their homes .

uranium is an environmental contaminant that arises as a result of authorized and accidental releases at various stages in the nuclear fuel cycle , including from uranium ore mining activities and post - reactor operations . additionally , in many countries , uranium - containing radioactive wastes , including spent nuclear fuel and intermediate - level waste , are likely to be disposed in deep geological disposal facilities ( gdf ) . here , uranium will typically be the most significant radionuclide by mass in the waste inventory . after deep disposal has been implemented , it is inevitable that , on geological time scales , uranium ( and other radionuclides ) will be released from within the waste containers and , importantly , due to its long half - life ( 4.5 ga ) , the behavior of uranium and of its resultant decay chain will be important to any safety case for geological disposal over extended time frames . it is , therefore , crucial that we understand the fate of uranium in these natural and engineered environments to be able to both predict and constrain its environmental impact . iron ( oxyhydr)oxides ( e.g. , hematite -fe2o3 ) are ubiquitous and are known to be effective at reducing the mobility of u(vi ) through either their high sorption capacity ( e.g. , surface adsorption ) or , where fe(ii ) is present , via reductive precipitation to poorly soluble u(iv ) phases . studies of uranium retardation mechanisms in the environment have tended to focus on adsorption of u(vi ) to various mineral phases or reduction of u(vi ) to u(iv ) either directly or indirectly as a result of microbial or abiotic pathways . however , a change in the geochemical conditions may reverse these processes ( e.g. , reduction in ph leading to desorption or reoxidation of u(iv ) ) and cause remobilization of the contaminant . incorporation of uranium into stable mineral phases , such as iron ( oxyhydr)oxides , offers a pathway for sequestration with the potential for long - term immobilization . it has been shown that goethite and hematite are able to accommodate various impurities ( e.g. , si , ti , mn , ni ) into their structure . specifically , u(vi ) and reportedly even u(v ) may be incorporated into goethite ( -feooh ) during fe(ii)-catalyzed crystallization of ferrihydrite , and evidence for u(vi ) incorporation into hematite during coprecipitation has been reported . precipitated ferrihydrite from a solution containing u(vi ) and fe(iii ) and induced hematite formation by aging at ph 11 and 60 c . here , they reported incorporation of u(vi ) into hematite in a uranate - like coordination environment with the resultant loss of the short uranyl bonds . followed the method of duff et al . and reported a similar structure for incorporated u(vi ) . atomistic simulations of u(iv ) , u(v ) , and u(vi ) incorporation into hematite using various different charge compensation mechanisms , based on the duff et al . incorporation model , indicated that u(vi ) maintained octahedral coordination in most cases but that the predicted interatomic distances differed from the experimental data . furthermore , in a similar study , chemical extractions on u(vi ) associated with ferrihydrite showed a decrease in leachable uranium as the solid phase aged and the formation of u(vi)-labeled crystalline goethite and hematite occurred , suggesting a change in speciation during crystallization . the lack of agreement between the spectroscopic and atomistic modeling approaches in the literature to date indicates that the mechanism of uranium incorporation , and the details of the molecular - level bonding environment within the hematite structure warrant further investigation . in addition to forming in soil and sediments , predominantly as a weathering product of iron - bearing minerals , iron ( oxyhydr)oxides form as corrosion products of steel and are present in intermediate level radioactive wastes . they are also reported to form in deep geological systems on tunnel walls due to biological oxidation of fe(ii ) . many geological disposal concepts utilize cementitious materials ( often within the wasteform itself or in the engineered barrier system ) and many contaminated soils at nuclear facilities will be in contact with cements and concrete construction materials . leaching of the cementitious materials will buffer the ph to hyperalkaline conditions , creating a chemically disturbed zone ( cdz ) in the host rock or local environment . thus , understanding the changes in speciation ( i.e. , adsorbed versus incorporated ) of actinides during crystallization of iron ( oxyhydr)oxides under these geochemical conditions is key to predicting their long - term stability and mobility in natural and engineered environments . ferrihydrite crystallizes to hematite or goethite depending upon solution conditions , with ph , ionic strength , and temperature all having an influence . hematite formation is favored under near - neutral conditions and higher temperature and ionic strength , whereas goethite forms under extremes of ph ( less than 4 , greater than 10 ) and at lower temperature and ionic strength . the hematite formation process begins with ferrihydrite particle aggregation , followed by recrystallization within the aggregate via dissolution and reprecipitation processes that occur at the nanoscale . this crystallization involves a variety of processes including dehydration of the ferrihydrite particles , deprotonation of hydroxyl groups , creation of oxy - linkages , and redistribution of cation vacancies . during this process , adsorbed uranium has the potential to become incorporated into the structure of the hematite . however , the mechanism of this reaction is poorly constrained , and how much of the adsorbed uranium is incorporated , at which stage in the crystallization process uranium is incorporated , and what the final site of uranium is within the hematite structure are all worthy of attention . in this contribution , we provide a detailed insight into the mechanism(s ) of uranium incorporation during hematite formation under conditions relevant to both geological disposal and contaminated land to determine whether significant amounts of uranium could be sequestered into this phase in the long term . we have combined aqueous chemical data with x - ray diffraction ( xrd ) , transmission electron microscopy ( tem ) , and x - ray absorption spectroscopy ( xas ) to characterize the solid phase crystallization at elevated ph ( 10.5 ) . throughout , we have focused on the fate of uranium during ferrihydrite transformation to hematite to determine the mechanism(s ) of uranium incorporation , and our aim was to define the atomic scale bonding environment of uranium within this environmentally important phase . batch experiments were used to follow the crystallization of u(vi)-adsorbed ferrihydrite in a synthetic cement leachate ( 0.015 g l ca(oh)2 ; ph 10.5 ) system . full experimental setup and sampling and analysis details g l and spiked with u(vi ) to give an initial u(aq ) concentration of 1 ppm ( 4.2 10 mol l ) , which was thermodynamically modeled ( phreeqc ) to be below the solubility of any u(vi ) phase in the synthetic leachate . the experiments were placed in an oven at 60 c for up to 70 days . some experiments were also placed into an oven at 105 c for up to 45 days to suppress the formation of goethite and favor hematite formation . all experiments were maintained between ph 10.310.7 and were purged with co2-free air throughout . partitioning of uranium between the solid and the solution was determined by analysis of uranium in solution ( u(aq ) ) . chemical extractions were performed to assess the partitioning of uranium to the solid phase . the surface - bound uranium ( u(ads ) ) was determined by titration of the iron ( oxyhydr)oxide suspension to ph 2.5 , below the u adsorption edge , using hcl . the resulting supernatant was analyzed for uranium , and u(ads ) was calculated by subtracting u(aq ) . the nonleachable uranium ( u(s ) ) was then calculated from the mass balance according to aqueous samples were analyzed for u by icp - ms , and solids were characterized by powder xrd and surface area using the bet method . uranium liii - edge xas spectra were collected on beamline b18 , diamond light source , at room temperature in fluorescence mode using a nine - element ge detector . reference spectra from u(vi ) and u(iv ) standards ( schoepite ( ( uo2)8o2(oh)1212(h2o ) ) and uraninite ( uo2 ) , respectively ) were collected in transmission mode . in - line yttrium foil reference spectra were also collected for each sample for energy calibration . background subtraction , data normalization , and fitting to the exafs spectra were performed using the software packages athena and artemis . xrd patterns for the products from the 60 c crystallization experiment show hematite formed rapidly from 2-line ferrihydrite over the first 2448 h of aging ( figure 1 ) . quantitative analysis of the xrd patterns ( qxrd ) ( supporting information table si-1 ) reveals that greater than 80% of the 24 h sample was hematite , decreasing to 55% hematite with the remaining phase 45% goethite after 30 days . hematite formation is favored over goethite with increasing temperature , therefore , to obtain a high purity end - member hematite sample , a 105 c crystallization at ph 10.5 was performed . quantitative analysis of the xrd patterns from the high temperature experiment confirmed that the sample was greater than 90% hematite after 45 days aging . xrd pattern time series for the 60 c crystallization and the end - point of the 105 c crystallization . observable peaks are indexed with f , h , or g signifying ferrihydrite , hematite , and goethite , respectively . tem photomicrographs of the solid samples ( supporting information figure si-1 ) illustrate the crystallization pathway of hematite and goethite from ferrihydrite at 60 c . at 0 h , the ferrihydrite nanoparticles were 35 nm and had no visible structure within the particles . after 24 h at 60 c , the ferrihydrite had aggregated and clumps of nanocrystalline hematite and acicular goethite became evident ( supporting information figure si-1 ) . thereafter , the amount of ferrihydrite rapidly decreased and the size of the hematite / goethite crystals gradually increased . over the first 48 h of crystallization , there was a rapid decrease in surface area from 164 3 to 21 1 m g , followed by a slow continued decrease to 17 1 m g after 30 days ( figure 2 ) . the xrd and qxrd , tem , and bet data indicated that during the first 2448 h , there was rapid aggregation of ferrihydrite and crystallization of hematite / goethite , causing a large and rapid decrease in surface area . this was followed by a stage of crystal ripening , with some transformation of hematite to goethite ( supporting information table si-1 ) . no evidence for discrete uranium phases was detected using xrd or tem , as expected from phreeqc modeling of the system . partitioning of uranium ( % ) and bet surface area ( m g ) during the crystallization of ferrihydrite at 60 c . green triangles = uranium in solution ( u(aq ) ) ; red diamonds = surface bound uranium ( u(ads ) ) ; blue diamonds = nonleachable uranium ( u(s ) ) ; purple crosses = bet surface area . the majority of the uranium ( 91.9 0.2% ) was instantaneously adsorbed to the solid phase on its addition to the ferrihydrite slurry ( figure 2 ) . during the aggregation and initial crystallization phase , u(ads ) rapidly decreased to 79.6 3.2% at 1 h and 51.3 2.1% at 48 h , with a continued decrease to 23.2 0.9% after 70 days of aging . on the basis of the uranium mass balance , this shows an increase in u(s ) from 20.2 2.6% at 1 h to 75.0 9.6% after 70 days ( figure 2 ) . thus , the chemical extraction data strongly suggest that a significant proportion of the uranium is becoming increasingly strongly associated with , and possibly structurally incorporated into , hematite / goethite during crystallization . reflecting this , we suggest that uranium adsorbed to the surface of the ferrihydrite particles is trapped within the solid phase during the aggregation process at the early stages of the crystallization process , consistent with the incorporation mechanism of pb into hematite during hydrothermal crystallization of ferrihydrite . the gradual increase in u(s ) during crystallization and ripening indicates that u(vi ) then continues to be incorporated as the iron ( oxyhdr)oxide crystals form and grow . this is in contrast to the behavior observed for pb , where the contaminant was slowly released from the hematite structure during ripening because of its incompatibility ( i.e. , located within defect sites ) with the mineral structure . this does not occur with uranium , suggesting that it may become located within a stable crystallographic site within the newly formed mineral , in agreement with modeling simulations . xanes spectra from a time series of samples spanning the crystallization of ferrihydrite at 60 and 105 c , along with u(vi ) and u(iv ) reference spectra are shown in figure 3 . reference spectra for u(vi ) ( schoepite ( ( uo2)8o2(oh)1212(h2o ) ) ; purple ) and u(iv ) ( uraninite ( uo2 ) ; green ) are shown for comparison . the edge positions of all xanes spectra from the four iron ( oxyhydr)oxide samples aligned to the u(vi ) schoepite standard ( 17 172 ev ) indicating that uranium remained as u(vi ) during crystallization . two prominent resonance features are visible in the xanes spectra of uranyl - containing compounds ( a and b , figure 3 ) and are due to resonance from the different u o bonds . feature a ( 17 190 ev ) is attributable to the short axial u = o bonds in the dioxygenyl species , and feature b ( 17 21017 215 ev ) is attributable to the longer u o equatorial bonds . feature a is absent from the xanes spectra of compounds that do not have the axial u = o bonds , such as uraninite , but is clearly present in all our u - bearing fe sample spectra ( figure 3 ) . the xanes spectrum of the 0 h solid associated sample is very similar to that of schoepite , indicating uranyl coordination . however , during the experiment the resonance features migrate in energy with time indicating a change in the local bonding environment of uranium throughout crystallization . here , feature a migrates to a lower energy ( 17 188 to 17 182 ev ) , whereas feature b migrates to a higher energy ( 17 211 to 17 228 ev ) over time ( figure 3 ) . changes in the energy of these resonance features in the xanes region are reportedly inversely proportional to the changes in the corresponding bond length . the time series xanes data , therefore , suggest that during reaction , the axial uranyl oxygen bond elongates while the average bond length in the equatorial plane shortens . the xanes spectra of the 60 c , 30 day sample and the 105 c , 45 day sample are very similar to reported xanes for alkali metal uranate compounds . this indicates that the u(vi ) associated with the crystalline iron ( oxyhydr)oxide is likely in a uranate - like coordination and presumably relates to the uranium becoming structurally incorporated . it is important to note that feature a remains during incorporation , indicating retention of the uranyl bonds , albeit with an increase in the bond distance inferred from its migration to lower energy . the xanes spectra from previous studies of u(vi ) incorporation into hematite are also very similar to our data , suggesting the same u(vi ) local environment is favored in several experimental systems . ( a ) uranium liii - edge exafs spectra and ( b ) corresponding fourier transforms of the exafs data from u(vi ) during hematite crystallization from ferrihydrite . black lines are k - weighted data , and red lines are model fits to the data . further information on the bonding environment of the uranium can be determined from analysis of the exafs spectra and their fourier transform ( figure 4a and b respectively ) . the model of waite et al . for u(vi ) adsorption to ferrihydrite in a mononuclear bidentate complex was applied to the 0 h data and provided a good fit ( figure 4 , table 1 ) . to test our hypothesis of uranium incorporation into hematite during crystallization , fitting was performed using the hematite structure with u(vi ) substituted for fe(iii ) in the mineral structure . the model was then applied to the 105 c , 45 day data first because this was 90.8 0.8% hematite and had only limited potential contributions from goethite ( figure 1 ) . the refined fit model from the 105 c system was then applied to the 60 c , 30 day data ( 55% hematite ) to assess the goodness of fit to hematite - incorporated uranium in a more environmentally relevant system . in the hematite structure ( table 1 ) , each fe is octahedrally coordinated by oxygen and is surrounded by fe o octahedra which are face , edge , or corner sharing ( table 1 , figure 5 ) . we assumed an octahedral u o coordination for our exafs fits based on our xanes , previous modeling , and the working hypothesis that the fe(iii ) that was replaced by u(vi ) was octahedrally coordinated . the best fit to the exafs data showed that the optimal coordination was a fit with three separate u o shells , each with a coordination of 2 at u these u o distances are similar to those in barium uranate ( bauo4 ) , in which uranium is also octahedrally coordinated with oxygen , with 2 at u o distances of 1.89 and 4 at 2.20 . we were then able to fit all of the four closest neighboring fe shells expected from the hematite structure with a good level of statistical significance ( supporting information table si-7 ) . fe bond distances for face sharing fe ( fef ) and the nearer corner sharing fe ( fec1 ) are at approximately the same distance as the fe fe distance in hematite , whereas an increased atomic distance to the other two fe shells ( fee and fec2 ) is observed suggesting some strain in the structure ( table 1 ) . u(vi ) has a larger crystal radius than fe(iii ) ( 0.870 versus 0.785 ) , and thus , upon incorporation , it would be expected to cause expansion and distortion to the host octahedral site . waller factor ; e0 denotes the shift in energy from the calculated fermi level ; s0 denotes the amplitude factor which was constrained to between 0.85 and 1.05 ; xv denotes the reduced square value ; r denotes the goodness of fit factor ; the subscript ms denotes multiple scattering paths in the axial o u o unit . the multiple scattering paths considered were linear paths and their r and parameters were evaluated as multiples of the corresponding single scattering path parameter . the usual shell - by - shell approach to exafs fitting was not possible here because the fe scatterers mostly contribute to the exafs spectrum in the low to middle k range ( 410 ) ( supporting information figure si-2 ) ; hence , any model that excludes these contributions will be unsatisfactory . additionally , we found that having data with good signal - to - noise ratio in the high k range ( > 10 ) was essential to adequately fit the u o shells ( supporting information figure si-2 ) . therefore , with our incorporation hypothesis in mind , we iteratively refined the u o shells and u fe shells simultaneously . once the u o shells had been satisfactorily resolved , we then constructed a model from a single fe shell to the full model including four fe shells refining the model each time and assessing the statistical relevance of each additional shell by way of an f - test ( supporting information table si-7 ) . the f - test results confirmed that the addition of each subsequent fe shell significantly improved the fit of the model to the data and was statistically valid . o distance , the axial oxygens , is the highest of the three oxygen shells , when normally it would be expected that they would be the tightest bound and , thus , have the lowest debye this may be due to static disorder , possibly related to the averaged nature of the exafs spectrum and the complexity of the structure with u o octahedra potentially in several different orientations , resulting in a range of u o axial oxygen distances that are averaged in the fit . additionally , the outermost fe shell has a comparatively large debye again , this is likely to be due to the relative increase in static disorder in the spectrum as the distance from the central uranium atom increases . overall , these data , coupled to tem and qxrd show that the u(vi ) within the 105 c , 45 day aged samples was incorporated into the hematite structure by replacing fe(iii ) . the fit model from the 105 c data can be fitted to the 60 c data but requires the removal of the outer two fe shells from the model . this may be due to the reduced useable k range of the 60 c data or to the heterogeneity of the uranium location ( e.g. , an adsorbed and incorporated component ) in this sample compared to the high temperature experiment . the fit parameters for the two closest fe shells ( table 1 ) were statistically valid and were essentially the same as the 105 c fit . quantitative analysis of the xrd revealed up to 45% goethite present in this sample ( supporting information table si-1 ) . the fe fe distances in goethite are similar to those in hematite , although the hematite face - sharing octahedra at 2.90 is absent in goethite . however , amplitude at this distinctive distance was clearly present in our data , meaning that the majority of the u(vi ) must reside within the hematite , although it was not possible to eliminate some fraction of uranium residing within the goethite . the fit parameters for the o shells in the 60 c fit did not remain the same as in the 105 c fit . oax bond distance ( 1.84 ) in this sample was close to that of the adsorbed model ( 1.81 ) , and both the equatorial shells have longer u o octahedra in goethite and hematite are nearly identical , u(vi ) present in goethite was unlikely to be the cause for the changed u approximately 30% of the uranium in the 60 c sample was acid leachable , indicating a significant proportion remains surface bound after 30 days aging . hence , it seems the bulk exafs data contained a significant component of signal from u(vi ) in a surface adsorption site , which caused the average u o bond length in the exafs signal to be closer to those of surface bound u(vi ) . waller factors for each u o shell were > 0.008 , indicating significant disorder : our model did not account for the adsorbed component , and trying to fit two similar u o environments simultaneously to the 60 c data set resulted in large disorder in the u o shells and , thus , was unjustifiable . linear combination fitting of the 60 c , 30 day data , using the 0 h and 105 c data as end members , indicated a contribution from the adsorbed u(vi ) species of approximately 20% ( see supporting information ) , which is in agreement with the chemical extraction data ( figure 2 ) . applying the same linear combination fitting to data taken after 24 h aging at 60 c indicated approximately 40% u was adsorbed , whereas the chemical extraction suggested closer to 50% was adsorbed . this modest discrepancy may be partially due to an overestimate of the adsorbed fraction by the operationally defined chemical extraction . this is not uncommon for indirect techniques and suggests a small proportion of the partially crystalline iron oxyhydroxide was dissolved at ph 2.5 . for our end - member 105 c experiment , the exafs analysis showed that during hematite crystallization and u(vi ) incorporation , the uranyl axial bonds lengthen by 0.06 and the average equatorial bonds shorten by 0.17 . o bond lengths are in agreement with our interpretation of the changes in energy of the resonance features in the xanes data . the exafs analysis is consistent with 6-fold coordinated u(vi ) residing in a distorted uranate - like octahedral site within the hematite structure ( figure 5 ) , although we accept there may be a contribution from minor amounts of u(vi ) in goethite . ( a ) hematite structure showing fe fe distances of blake et al . ( b ) uranium incorporated hematite showing u fe distances obtained from exafs fitting of the 105 c data . subscript notation indicates the polygon sharing relationship : f = face ; e = edge ; c = corner . redrawn after cornell and schwertmann . in earlier work , duff et al . formed u(vi)-containing hematite via a coprecipitation method at ph 11 and interpreted their exafs as showing incorporation of uranium into the crystal structure , with an oxygen coordination of approximately 4 at radial distances of 2.19 ( n = 1.4 15% ) and 2.36 ( n = 2.1 20% ) , with a single fe atom ( n = 1.12 25% ) at a distance of 3.19 . the implication is that uranium was incorporated into hematite with the loss of the axial u o bonds . latterly , it has been suggested that the duff model had an unexpectedly low u o coordination , suggesting that not all of the u o bond distances were fully resolved from the exafs . the same approach to uranium incorporation into hematite was followed by ilton et al . who reported a similar uranium environment to duff et al . our exafs data analysis showed that these models are incorrect and that u(vi ) is fully coordinated by 6 oxygens within a distorted octahedral site in the hematite structure . our interpretation is supported by recent work on atomic simulations of uranium incorporation into hematite , which shows that incorporation of octahedrally coordinated u(vi ) , with reduction of fe(iii ) as the charge compensation mechanism , maintains an average u o bond distance of 2.06 . this is identical to the average u o bond distance obtained from the fit to our data ( 2.06 0.02 ) . fe atomic distances returned by the simulations were in excess of those obtained from our exafs fitting , in particular the calculated single u o bond length ; this does not take into account the shape of the distorted uo6 octahedron that we propose . the corresponding simulation that considers incorporation of the u(vi ) into an unoccupied interstitial site within the hematite structure returns similar average u o and u fe bond distances to those of our exafs fit . however , the calculated fe shells in the simulation are doubly overcoordinated compared to our fit and we were unable to reconcile this model with our data , leading us to discount u(vi ) incorporation into a vacancy site . we can see no viable mechanism to achieve charge compensation by reduction of fe(iii ) to fe(ii ) in our fully oxidized system . similarly , reduction of u(vi ) to u(v ) again seems to be improbable in the absence of a suitable reducing agent . although distinction of u(v ) from u(iv ) and u(vi ) has been shown to be possible with high resolution xas techniques , it is not possible to do so for our samples at their low u - loadings . furthermore , in crystalline materials , reportedly the u(v ) cation may occur in octahedral or pentagonal bipyramidal coordination with a near linear o u o unit , but the u o bond length is typically around 2 , which is in vast excess to the 1.87 we observed , giving confidence that u(v ) was not present in our samples . another potential charge compensation mechanism postulated by kerisit et al . for u(vi ) substituting for fe(iii ) is via creation of an fe vacancy in its vicinity . to test for this we refitted the model described above , but with the coordination number of each fe shell reduced by one , sequentially ( supporting information tables si-3 and si-4 ) . none of these fits gave a statistical improvement on that presented here , and in fact , the omission of the face - sharing fe significantly worsened the fit ( supporting information table si-7 ) . this suggests that if the charge compensation is via creation of a fe(iii ) vacancy , then the vacancy is ( a ) located in the edge - sharing or corner - sharing shells and ( b ) is randomly distributed relative to u(vi ) or is undetectable within the constraints of the exafs measurements we made . overall , in this study , we present clear evidence for u(vi ) incorporation into hematite in an octahedrally coordinated environment and via direct substitution for fe(iii ) . our model requires retention of the uranyl bonds as evidenced by the xanes and exafs analyses , albeit elongated within the structure , which is in direct contrast to previous studies . our data also evidence the importance of high quality spectroscopic data out to high k when attempting to model actinide incorporation into iron oxides . our work highlights that under conditions relevant to both geological disposal and contaminated land , a significant proportion of u(vi ) adsorbed to ferrihydrite is incorporated into the hematite crystal structure during crystallization . in our experiments , hematite showed the ability to incorporate approximately 3000 ppm u(vi ) ( 0.3 wt % ) in the solid . this is relevant to a wide range of nuclear decommissioning and waste management scenarios where iron oxides are ubiquitous . indeed , the incorporation of uranium into iron oxides , specifically hematite , has implications for reducing the long - term environmental mobility of u(vi ) , especially given the long - term stability of hematite , which is found in geological settings older than 1 ga . it is also worth noting that elevated temperatures associated with disposal of heat - yielding radioactive wastes may enhance hematite formation and , thereby , u(vi ) immobilization . in addition , under conditions where biogeochemical processes can occur , it is interesting to note that hematite is recalcitrant to microbial reduction due to its crystallinity , with only a thin surface layer of bioavailable fe(iii ) present , again suggesting its stability may be significant in , for example , oxic - contaminated land environments . fe(ii)aq has been shown to enhance the release of iron oxide incorporated trace metals , although interestingly , natural iron oxides substituted with , for example , al are less susceptible to fe(ii)-activated recrystallization , and as such , trace metal release may be inhibited in these phases . in particular , the alkaline conditions used in this study show that these processes are directly relevant to the conditions expected around a cementitious disposal facility for radioactive watse as well as alkaline waste management scenarios ( e.g. , hanford tanks ) . thus , our results show that substantial incorporation of u(vi ) into hematite can occur , which is potentially a significant new pathway to immobilize u(vi ) and has clear implications for the environmental mobility of this important radionuclide , especially in high ph conditions relevant to engineered waste environments .

ferrihydrite was exposed to u(vi)-containing cement leachate ( ph 10.5 ) and aged to induce crystallization of hematite . a combination of chemical extractions , tem , and xas techniques provided the first evidence that adsorbed u(vi ) ( 3000 ppm ) was incorporated into hematite during ferrihydrite aggregation and the early stages of crystallization , with continued uptake occurring during hematite ripening . analysis of exafs and xanes data indicated that the u(vi ) was incorporated into a distorted , octahedrally coordinated site replacing fe(iii ) . fitting of the exafs showed the uranyl bonds lengthened from 1.81 to 1.87 , in contrast to previous studies that have suggested that the uranyl bond is lost altogether upon incorporation into hematite . the results of this study both provide a new mechanistic understanding of uranium incorporation into hematite and define the nature of the bonding environment of uranium within the mineral structure . immobilization of u(vi ) by incorporation into hematite has clear and important implications for limiting uranium migration in natural and engineered environments .

Introduction Results and Discussion

it is , therefore , crucial that we understand the fate of uranium in these natural and engineered environments to be able to both predict and constrain its environmental impact . , hematite -fe2o3 ) are ubiquitous and are known to be effective at reducing the mobility of u(vi ) through either their high sorption capacity ( e.g. studies of uranium retardation mechanisms in the environment have tended to focus on adsorption of u(vi ) to various mineral phases or reduction of u(vi ) to u(iv ) either directly or indirectly as a result of microbial or abiotic pathways . , reduction in ph leading to desorption or reoxidation of u(iv ) ) and cause remobilization of the contaminant . specifically , u(vi ) and reportedly even u(v ) may be incorporated into goethite ( -feooh ) during fe(ii)-catalyzed crystallization of ferrihydrite , and evidence for u(vi ) incorporation into hematite during coprecipitation has been reported . precipitated ferrihydrite from a solution containing u(vi ) and fe(iii ) and induced hematite formation by aging at ph 11 and 60 c . here , they reported incorporation of u(vi ) into hematite in a uranate - like coordination environment with the resultant loss of the short uranyl bonds . atomistic simulations of u(iv ) , u(v ) , and u(vi ) incorporation into hematite using various different charge compensation mechanisms , based on the duff et al . incorporation model , indicated that u(vi ) maintained octahedral coordination in most cases but that the predicted interatomic distances differed from the experimental data . furthermore , in a similar study , chemical extractions on u(vi ) associated with ferrihydrite showed a decrease in leachable uranium as the solid phase aged and the formation of u(vi)-labeled crystalline goethite and hematite occurred , suggesting a change in speciation during crystallization . the lack of agreement between the spectroscopic and atomistic modeling approaches in the literature to date indicates that the mechanism of uranium incorporation , and the details of the molecular - level bonding environment within the hematite structure warrant further investigation . many geological disposal concepts utilize cementitious materials ( often within the wasteform itself or in the engineered barrier system ) and many contaminated soils at nuclear facilities will be in contact with cements and concrete construction materials . , adsorbed versus incorporated ) of actinides during crystallization of iron ( oxyhydr)oxides under these geochemical conditions is key to predicting their long - term stability and mobility in natural and engineered environments . this crystallization involves a variety of processes including dehydration of the ferrihydrite particles , deprotonation of hydroxyl groups , creation of oxy - linkages , and redistribution of cation vacancies . during this process , adsorbed uranium has the potential to become incorporated into the structure of the hematite . however , the mechanism of this reaction is poorly constrained , and how much of the adsorbed uranium is incorporated , at which stage in the crystallization process uranium is incorporated , and what the final site of uranium is within the hematite structure are all worthy of attention . in this contribution , we provide a detailed insight into the mechanism(s ) of uranium incorporation during hematite formation under conditions relevant to both geological disposal and contaminated land to determine whether significant amounts of uranium could be sequestered into this phase in the long term . we have combined aqueous chemical data with x - ray diffraction ( xrd ) , transmission electron microscopy ( tem ) , and x - ray absorption spectroscopy ( xas ) to characterize the solid phase crystallization at elevated ph ( 10.5 ) . throughout , we have focused on the fate of uranium during ferrihydrite transformation to hematite to determine the mechanism(s ) of uranium incorporation , and our aim was to define the atomic scale bonding environment of uranium within this environmentally important phase . batch experiments were used to follow the crystallization of u(vi)-adsorbed ferrihydrite in a synthetic cement leachate ( 0.015 g l ca(oh)2 ; ph 10.5 ) system . partitioning of uranium between the solid and the solution was determined by analysis of uranium in solution ( u(aq ) ) . the nonleachable uranium ( u(s ) ) was then calculated from the mass balance according to aqueous samples were analyzed for u by icp - ms , and solids were characterized by powder xrd and surface area using the bet method . reference spectra from u(vi ) and u(iv ) standards ( schoepite ( ( uo2)8o2(oh)1212(h2o ) ) and uraninite ( uo2 ) , respectively ) were collected in transmission mode . background subtraction , data normalization , and fitting to the exafs spectra were performed using the software packages athena and artemis . quantitative analysis of the xrd patterns ( qxrd ) ( supporting information table si-1 ) reveals that greater than 80% of the 24 h sample was hematite , decreasing to 55% hematite with the remaining phase 45% goethite after 30 days . quantitative analysis of the xrd patterns from the high temperature experiment confirmed that the sample was greater than 90% hematite after 45 days aging . tem photomicrographs of the solid samples ( supporting information figure si-1 ) illustrate the crystallization pathway of hematite and goethite from ferrihydrite at 60 c . thereafter , the amount of ferrihydrite rapidly decreased and the size of the hematite / goethite crystals gradually increased . over the first 48 h of crystallization , there was a rapid decrease in surface area from 164 3 to 21 1 m g , followed by a slow continued decrease to 17 1 m g after 30 days ( figure 2 ) . the xrd and qxrd , tem , and bet data indicated that during the first 2448 h , there was rapid aggregation of ferrihydrite and crystallization of hematite / goethite , causing a large and rapid decrease in surface area . this was followed by a stage of crystal ripening , with some transformation of hematite to goethite ( supporting information table si-1 ) . partitioning of uranium ( % ) and bet surface area ( m g ) during the crystallization of ferrihydrite at 60 c . thus , the chemical extraction data strongly suggest that a significant proportion of the uranium is becoming increasingly strongly associated with , and possibly structurally incorporated into , hematite / goethite during crystallization . reflecting this , we suggest that uranium adsorbed to the surface of the ferrihydrite particles is trapped within the solid phase during the aggregation process at the early stages of the crystallization process , consistent with the incorporation mechanism of pb into hematite during hydrothermal crystallization of ferrihydrite . , located within defect sites ) with the mineral structure . xanes spectra from a time series of samples spanning the crystallization of ferrihydrite at 60 and 105 c , along with u(vi ) and u(iv ) reference spectra are shown in figure 3 . reference spectra for u(vi ) ( schoepite ( ( uo2)8o2(oh)1212(h2o ) ) ; purple ) and u(iv ) ( uraninite ( uo2 ) ; green ) are shown for comparison . however , during the experiment the resonance features migrate in energy with time indicating a change in the local bonding environment of uranium throughout crystallization . this indicates that the u(vi ) associated with the crystalline iron ( oxyhydr)oxide is likely in a uranate - like coordination and presumably relates to the uranium becoming structurally incorporated . it is important to note that feature a remains during incorporation , indicating retention of the uranyl bonds , albeit with an increase in the bond distance inferred from its migration to lower energy . the xanes spectra from previous studies of u(vi ) incorporation into hematite are also very similar to our data , suggesting the same u(vi ) local environment is favored in several experimental systems . ( a ) uranium liii - edge exafs spectra and ( b ) corresponding fourier transforms of the exafs data from u(vi ) during hematite crystallization from ferrihydrite . further information on the bonding environment of the uranium can be determined from analysis of the exafs spectra and their fourier transform ( figure 4a and b respectively ) . to test our hypothesis of uranium incorporation into hematite during crystallization , fitting was performed using the hematite structure with u(vi ) substituted for fe(iii ) in the mineral structure . in the hematite structure ( table 1 ) , each fe is octahedrally coordinated by oxygen and is surrounded by fe o octahedra which are face , edge , or corner sharing ( table 1 , figure 5 ) . we assumed an octahedral u o coordination for our exafs fits based on our xanes , previous modeling , and the working hypothesis that the fe(iii ) that was replaced by u(vi ) was octahedrally coordinated . the best fit to the exafs data showed that the optimal coordination was a fit with three separate u o shells , each with a coordination of 2 at u these u o distances are similar to those in barium uranate ( bauo4 ) , in which uranium is also octahedrally coordinated with oxygen , with 2 at u o distances of 1.89 and 4 at 2.20 . u(vi ) has a larger crystal radius than fe(iii ) ( 0.870 versus 0.785 ) , and thus , upon incorporation , it would be expected to cause expansion and distortion to the host octahedral site . the usual shell - by - shell approach to exafs fitting was not possible here because the fe scatterers mostly contribute to the exafs spectrum in the low to middle k range ( 410 ) ( supporting information figure si-2 ) ; hence , any model that excludes these contributions will be unsatisfactory . the f - test results confirmed that the addition of each subsequent fe shell significantly improved the fit of the model to the data and was statistically valid . o distance , the axial oxygens , is the highest of the three oxygen shells , when normally it would be expected that they would be the tightest bound and , thus , have the lowest debye this may be due to static disorder , possibly related to the averaged nature of the exafs spectrum and the complexity of the structure with u o octahedra potentially in several different orientations , resulting in a range of u o axial oxygen distances that are averaged in the fit . overall , these data , coupled to tem and qxrd show that the u(vi ) within the 105 c , 45 day aged samples was incorporated into the hematite structure by replacing fe(iii ) . quantitative analysis of the xrd revealed up to 45% goethite present in this sample ( supporting information table si-1 ) . however , amplitude at this distinctive distance was clearly present in our data , meaning that the majority of the u(vi ) must reside within the hematite , although it was not possible to eliminate some fraction of uranium residing within the goethite . oax bond distance ( 1.84 ) in this sample was close to that of the adsorbed model ( 1.81 ) , and both the equatorial shells have longer u o octahedra in goethite and hematite are nearly identical , u(vi ) present in goethite was unlikely to be the cause for the changed u approximately 30% of the uranium in the 60 c sample was acid leachable , indicating a significant proportion remains surface bound after 30 days aging . hence , it seems the bulk exafs data contained a significant component of signal from u(vi ) in a surface adsorption site , which caused the average u o bond length in the exafs signal to be closer to those of surface bound u(vi ) . linear combination fitting of the 60 c , 30 day data , using the 0 h and 105 c data as end members , indicated a contribution from the adsorbed u(vi ) species of approximately 20% ( see supporting information ) , which is in agreement with the chemical extraction data ( figure 2 ) . for our end - member 105 c experiment , the exafs analysis showed that during hematite crystallization and u(vi ) incorporation , the uranyl axial bonds lengthen by 0.06 and the average equatorial bonds shorten by 0.17 . o bond lengths are in agreement with our interpretation of the changes in energy of the resonance features in the xanes data . the exafs analysis is consistent with 6-fold coordinated u(vi ) residing in a distorted uranate - like octahedral site within the hematite structure ( figure 5 ) , although we accept there may be a contribution from minor amounts of u(vi ) in goethite . formed u(vi)-containing hematite via a coprecipitation method at ph 11 and interpreted their exafs as showing incorporation of uranium into the crystal structure , with an oxygen coordination of approximately 4 at radial distances of 2.19 ( n = 1.4 15% ) and 2.36 ( n = 2.1 20% ) , with a single fe atom ( n = 1.12 25% ) at a distance of 3.19 . the implication is that uranium was incorporated into hematite with the loss of the axial u o bonds . latterly , it has been suggested that the duff model had an unexpectedly low u o coordination , suggesting that not all of the u o bond distances were fully resolved from the exafs . the same approach to uranium incorporation into hematite was followed by ilton et al . our exafs data analysis showed that these models are incorrect and that u(vi ) is fully coordinated by 6 oxygens within a distorted octahedral site in the hematite structure . our interpretation is supported by recent work on atomic simulations of uranium incorporation into hematite , which shows that incorporation of octahedrally coordinated u(vi ) , with reduction of fe(iii ) as the charge compensation mechanism , maintains an average u o bond distance of 2.06 . the corresponding simulation that considers incorporation of the u(vi ) into an unoccupied interstitial site within the hematite structure returns similar average u o and u fe bond distances to those of our exafs fit . however , the calculated fe shells in the simulation are doubly overcoordinated compared to our fit and we were unable to reconcile this model with our data , leading us to discount u(vi ) incorporation into a vacancy site . similarly , reduction of u(vi ) to u(v ) again seems to be improbable in the absence of a suitable reducing agent . although distinction of u(v ) from u(iv ) and u(vi ) has been shown to be possible with high resolution xas techniques , it is not possible to do so for our samples at their low u - loadings . furthermore , in crystalline materials , reportedly the u(v ) cation may occur in octahedral or pentagonal bipyramidal coordination with a near linear o u o unit , but the u o bond length is typically around 2 , which is in vast excess to the 1.87 we observed , giving confidence that u(v ) was not present in our samples . for u(vi ) substituting for fe(iii ) is via creation of an fe vacancy in its vicinity . this suggests that if the charge compensation is via creation of a fe(iii ) vacancy , then the vacancy is ( a ) located in the edge - sharing or corner - sharing shells and ( b ) is randomly distributed relative to u(vi ) or is undetectable within the constraints of the exafs measurements we made . overall , in this study , we present clear evidence for u(vi ) incorporation into hematite in an octahedrally coordinated environment and via direct substitution for fe(iii ) . our model requires retention of the uranyl bonds as evidenced by the xanes and exafs analyses , albeit elongated within the structure , which is in direct contrast to previous studies . our work highlights that under conditions relevant to both geological disposal and contaminated land , a significant proportion of u(vi ) adsorbed to ferrihydrite is incorporated into the hematite crystal structure during crystallization . in our experiments , hematite showed the ability to incorporate approximately 3000 ppm u(vi ) ( 0.3 wt % ) in the solid . indeed , the incorporation of uranium into iron oxides , specifically hematite , has implications for reducing the long - term environmental mobility of u(vi ) , especially given the long - term stability of hematite , which is found in geological settings older than 1 ga . thus , our results show that substantial incorporation of u(vi ) into hematite can occur , which is potentially a significant new pathway to immobilize u(vi ) and has clear implications for the environmental mobility of this important radionuclide , especially in high ph conditions relevant to engineered waste environments .

trypanosomatids are parasitic protozoa responsible for several tropical diseases of which african sleeping sickness ( trypanosoma brucei gambiense and t. b. rhodesiense ) , chagas disease ( t. cruzi ) , and the different forms of leishmaniasis ( leishmania donovani and l. infantum ) are considered the most lethal.(1 ) at present , the chemotherapies associated with all forms of trypanosomiasis have many deficiencies including poor efficacy , toxicity to humans , high cost , and the emergence of drug - resistant parasitic strains.(2 ) unfortunately , because of the low profit margins associated with developing improved therapies for tropical diseases , there is little motivation for the pharmaceutical industry to develop better tropical disease drugs . accordingly , many agencies , including the special program for research and training in tropical diseases at the world health organization ( who / tdr ) , numerous academic research groups , various international / national bodies , and philanthropic foundations , have been supporting the discovery of new drug candidates for a number of tropical diseases.(3 ) one of the most promising strategies to develop potent and specific antiparasitic drugs relies on the exploitation of metabolic differences between the pathogen and the host . the mammalian redox defense system , based on the glutathione / glutathione reductase ( graaabbreviations : tryr , trypanothione reductase ; gr , glutathione reductase ; sbdd , structure - based drug design ; shop , scaffold hopping ; mcs , maximum common substructure ; spd , spermidine ; cs , consensus scoring ; ad , autodock ; xs , x - score ; z , screening window coefficient . ) couple , is replaced in trypanosomatids by an analogous , but distinct , system based on the thiolpolyamine conjugate trypanothione and the flavoenzyme trypanothione reductase(4 ) ( figure 1 ) . the almost total mutual substrate exclusivity of these two enzymes and the essential role of tryr in the survival of all trypanosomatids studied so far have served as the basis for rational design of many types of selective inhibitors of tryr in the presence of host gr . abbreviations : tryr , trypanothione reductase ; gr , glutathione reductase ; sbdd , structure - based drug design ; shop , scaffold hopping ; mcs , maximum common substructure ; spd , spermidine ; cs , consensus scoring ; ad , autodock ; xs , x - score ; z , screening window coefficient . structure of trypanothione ( t(s)2 ) and the reaction catalyzed by trypanothione that reduces t(s)2 to dihydrotrypanothione , t(sh)2 . structure - based drug design(7 ) ( sbdd ) has emerged as a very effective and low - cost strategy to improve the rate of success at any stage of the drug discovery pipeline . there are two broad categories of sbdd computational techniques : ( 1 ) proteinligand docking and ( 2 ) ligand similarity methods . proteinligand docking attempts to use the 3d protein structure of the protein target to predict binding modes and affinities of ligands to biologically relevant targets , while ligand - similarity methods capitalize on the fact that ligands similar to an active ligand are more likely to be active than random ligands . the latter method considers two- or three - dimensional chemistry , shape , electrostatic , and interaction points ( e.g. , pharmacophore points ) to assess similarity . the discovery and optimization of antiparasitic compounds has also profited from the use of computational docking techniques.(8 ) the elucidation of the 3d structure of tryr in free form and complexed with nadph,(11 ) glutathionylspermidine,(12 ) trypanothione,(13 ) the competitive inhibitor quinacrine(14 ) and its alkylating derivative quinacrine mustard,(15 ) have all provided detailed information about the interactions involved in enzyme recognition and substrate binding . despite this , tryr has been the target of just a few computational approaches and only modest results have been obtained so far . for instance horvath s early work employed a rigid - body docking algorithm(16 ) to predict binding affinity of ligands to tryr . the validity of this docking model is particularly noteworthy in relation to similar computational approaches because it predicted an interaction between the alkyl amino moiety of the acridine ring and the side chain of glu 19 as observed in the crystal structure of the tryrquinacrine complex.(14 ) the discovery of the macrocyclic alkaloid lunarine(13 ) and the recent identification of chlorhexidine and its derivatives(17 ) via high - throughput virtual screening are successful examples of the employment of structure - based methodologies to identify novel tryr inhibitors . at present . the active site of tryr is extremely wide and it allows for multiple binding orientations and/or the simultaneous accommodation of more than one inhibitor . in addition , proteinligand interactions of even very similar compounds can be unpredictable . for these reasons , the search for novel structure - based strategies with the ability to identify more potent and selective tryr inhibitors is desirable . in this context , we have decided that the combination of ligand similarity and protein docking strategies in a single workflow could be useful for this purpose . the fact that both methods , when used individually , focus only on one part of the structural information available has recently prompted the development of hybrid similarity - based docking methods . these hybrid methods aim at fully exploiting all the structural information present in ligand - bound protein structures , both from the protein and ligand perspective . in principle , a strategy combining 3d ligand similarity and protein docking should provide more accurate prediction about ligand binding modes.(25 ) although the majority of ligand similarity - based methods for molecular comparisons , compound classifications , or database searching utilize molecular descriptors for the definition of chemical reference spaces,(26 ) it has been demonstrated that molecular fragment profiles could also be successfully employed as queries for database searching and that significant recovery rates could be achieved.(27 ) fragment profiles differ from molecular fingerprints(28 ) because , on the one hand , they do not organize predefined or catalogued descriptors in a specific manner , and on the other , they are based on the randomization of structural information rather than canonical bit string representations . stemming from our interest in the development of new antiprotozoal drugs,(29 ) we report herein the first implementation of a novel virtual screening cascade protocol that can be used effectively for virtual screening and identification of prospective ligands that bind to trypanothione reductase . to validate this strategy , we used tryr inhibition assays to measure the activity of 19 of these prospective ligands and we found that many of them exhibit sufficiently promising characteristics to warrant further ligand optimization . the novelty of this approach lies in the sequential combination of a similarity virtual screening based on fragment profile comparisons facilitated through the calculation of compound class - specific fragment frequencies using naive bayesian statistics , with a docking protocol ( autodock and x - score ) for screening against the tryr target . another important feature of this computational approach is the enhancement of its predictive power by incorporating an exhaustive conformational consensus scoring process using a rank - by - rank strategy in order to provide a list of suitable compounds to be considered for bioscreening . the computational approach we developed for screening molecules binding to tryr combines both ligand similarity and protein docking methods within an integrated framework . a flowchart describing various phases of the virtual screening cascade protocol is shown in figure 2 . miscreen(30 ) was selected as the base methodology to perform the ligand - based virtual screening . briefly , the miscreen toolkit first analyses a training set of active structures to build a usable ligand model and then compares it with inactive molecules by using sophisticated bayesian statistics . on the basis of this analysis , a bioactivity model is developed , where for each substructure fragment a bioactivity contribution is calculated . once a model is built , the bioactivity of the screened molecules may be then calculated as a sum of the activity contributions of the fragments in these molecules . this approach provides an activity score for each compound , which represents the probability that the particular molecule will have the desired biological activity . the higher this score , the higher the probability that the compound will be active . molecules that bind reversibly to trypanothione reductase constitute the largest group of inhibitors studied so far and are among the most effective with inhibition constant ( ki ) values , usually in the low micromolar range . to date , the only reports about the structural determination of tryr complexed with a non - natural substrate are related to the competitive inhibitor quinacrine(14 ) and its chloro derivative , quinacrine mustard.(15 ) the availability of these structures certainly provides some useful insights into how these type of ligands fit into the active site of the enzyme and the interactions involved . in principle , a single active molecule is sufficient to build a usable model , but larger fragment populations have higher information content , leading to similarity analysis at higher resolution and increasingly accurate compound ranking . the initial training set was assembled using 135 molecules extracted from different bibliographic sources ( see supporting information ) and selected according to their inhibition mode and high binding affinity profiles . only compounds with ki values ranging between 0.150 m the majority of the chosen compounds fell into one of the three known general categories of reversible tryr inhibitors : hydrophobic polyamines , tricyclic compounds , and diphenylsulfane derivatives ( figure 3 ) . compounds included in the training set were transformed to smiles coordinates to generate a bioactivity model . the distribution of antiprotozoal activity for 100000 molecules with druglike properties extracted from pubchem by diversity selection to represent the average druglike chemical space and the training set of active structures is shown in figure 4 . according to this diagram , the bioactivity model provides a good separation between active compounds and the average background . subsequently , in silico screening of the zinc database ( http://zinc.docking.org/ ) , containing over eight million purchasable compounds , was performed by calculating the bioactivity of each molecule as a sum of activity contributions of fragments . a data set of 1312 compounds , listed according to their activity score , typically between 4.3 ( higher score ) and 1 ( lower score ) , was retrieved . distribution of antiprotozoal activity for average background molecules and the training set based on the bioactivity model . 100000 molecules may be screened in less than an hour ) permitting the processing of very large molecular libraries . filtering of the output data set derived from the similarity - based virtual screening ( 1312 compounds ) was performed by faf - drugs(31 ) ( http://bioserv.rpbs.jussieu.fr/help/faf drugs.html ) . this online service , based on frowns ( a chemoinformatics toolkit ) , allows users to process their own compound collections via simple adme / tox filtering rules such as molecular weight , polar surface area , logp , or number of rotatable bonds . notably , faf - drugs turned out to be very convenient in our case because its customizable protocol allowed us to input filtering criteria not only related with bioavailability issues but also with substrate specificity . in contrast to gr , the active site of tryr shows an overall negative charge and is much wider and more hydrophobic.(32 ) therefore , special attention was given to the input values of total charge ( 02 ) , logp ( 15 ) , and molecular mass ( 200500 ) . the rest of the parameters were either in compliance with lipinski s rule of five or set as default . compounds matching the filtering criteria ( 603 molecules ) comprised the enriched library to be used as input data for docking studies . one of the advantages of using a virtual screening protocol based on bayesian statistics is that it can generalize , i.e. , it is able to learn general structure requirements that are necessary for bioactivity . as a result , the newly identified bioactive molecules not only contain building blocks found in the training set . furthermore , the protocol is also able to identify new chemotypes via a process called scaffold hopping ( shop ) . to shed some light about this point , clustering analysis of the initial training set and the enriched library was carried out through libmcs,(33 ) a stand - alone application program , part of the jklustor package . librarymcs clusters a set of chemical structures in a hierarchical manner based on the concept of maximum common substructure ( mcs ) . structures that share a large common substructure ( a core or scaffold structure ) are grouped together . in the next step , these cores are clustered to form the next level in the hierarchy . two hierarchical clusterings , corresponding to the initial training set and the enriched library , respectively , are depicted in figure 5 . initial structures are found at the bottom of the hierarchy , and the next level contains the mcs of clusters of initial molecules . the highest clustering values observed for the enriched library ( cluster level : 4 , total cluster count : 257 ) , figure 5b , with respect to the initial training set ( cluster level : 3 , total cluster count : 101 ) , figure 5a , confirms the effectiveness of the adopted similarity - based strategy to generate new chemotypes by using the information of already known bioactive molecules . virtual docking was performed using the lamarckian genetic algorithm implemented in autodock4.(34 ) this is commonly regarded as best of the three docking methods in autodock in terms of its ability to find the lowest energy and its structure prediction accuracy.(35 ) the protein structure for the unbound trypanosoma cruzi trypanothione reductase was chosen as the target , and coordinates were downloaded from the protein data bank ( pdb code 1aog).(36 ) prior to docking , fad cofactors and water molecules were removed manually from the pdb file . grid maps were prepared using the autogrid utility with 126 126 102 points and grid spacing set to 0.204 . docking parameters modified from the defaults were : number of individuals in the population ( set to 300 ) , maximum number of energy evaluations ( set to 2500000 ) , maximum number of generations ( set to 2700 ) , and number of hybrid ga - ls runs ( set to 100 ) . initial conformations of the ligands were placed next to the aromatic ring of tyr111 , a central residue in the active site having a hydrophobic interaction with trypanothione . all rotatable bonds in the ligands were allowed to rotate during the docking trials . because amino acid positions in the enzyme were found , experimentally , to change insignificantly upon binding the native substrate , the active site was assumed to be rigid and therefore nonflexible docking was carried out . ligand structures ( 603 ) were taken from version 8.0 of the zinc database.(37 ) for some compounds that were not available in the zinc database , sets of coordinates were generated by submitting their smile strings to zinc s user upload utility . in - house perl scripts were used to extract the 100 conformations generated by autodock from the docking output file for each of the compounds in order to allow them to be evaluated by the program x - score.(38 ) all ligand files that had the same logp values calculated by x - score were inspected visually to verify their structures . compounds whose files were found to be incorrect had their smile strings submitted to zinc s user upload utility to regenerate their three - dimensional structures and were rescreened . to minimize the computing time , docking jobs as well as x - score evaluations were carried out on a westgrid computer cluster designed for serial processing and parallel jobs ( http://www.westgrid.ca ) . jobs were submitted using portable batch system ( pbs ) files invoking either autodock or x - score . in - house perl scripts were used to extract the top conformations for each compound docked to the protein . pymol v0.99(39 ) was used to visualize the compounds docked to the active site of the protein , and schematic diagrams of proteinligand interactions were obtained by the ligplot program.(40 ) combining multiple scoring functions has been shown in various virtual database screening studies to be an effective way for improving hit rates.(41 ) while autodock is one of the most cited docking programs(42 ) and has recorded many successes in virtual screening approaches,(43 ) the stand - alone scoring function x - score has shown good correlations between theoretical and experimentally determined proteinligand binding affinities.(44 ) consequently , x - score was combined in this study with the function implemented in the autodock program to provide a list of ligands sorted by consensus scoring adopting the rank - by - rank strategy.(45 ) autodock and x - score were applied to rank all the conformations generated by the 603 compounds ( 60300 conformations in total ) . for each conformation , the final rank was calculated as the average rank from the two scoring functions . only the best scored conformation of each compound a representation of the 20 top ranked ligands according to consensus scoring is shown in figure 6 . these compounds exhibit structural characteristics typical of several known tryr inhibitors , including extended hydrophobic moieties tethered to positive charge fragments mostly represented by bulky quaternary cyclic amines . the latter feature agrees with the previous finding that positive charges play a crucial role for ligand binding in the active site of tryr . phenothiazines and diphenylsulfanes , included in the initial training set , were successfully recovered among the top scored ligands , highlighting the importance of these building blocks for binding affinity . the presence of a chloro substituent is another common structural motif found in the retrieved molecules . in the tryrquinacrine complex , obviously the halogen is stabilized by interacting with the positively polarized hydrogen of the indolyl nitrogen . the same type of interaction is also very likely to occur for some of the newly identified chloro derivatives . calculated tanimoto similarity coefficient with respect to quinacrine mustard ( in parenthesis ) . to test the efficacy of our computational protocol , 19 compounds were screened for inhibition using a high - throughput microplate assay previously developed for this purpose.(47 ) the tested set included 10 of the best rank structures according consensus scoring ( compounds 17 , 10 , 12 , 16 , figure 6 ) . additionally , some of the best hits by autodock ( 21 ) and x - score ( 22 , 23 , 24 ) and several high - ranked new chemotypes ( 2529 ) depicted in figure 7 , were also considered for screening . the mean z value(48 ) over the total 19 testing plates was 0.88 ( range 0.560.96 ) , indicating a well performing assay . the triplicate ic50 values were used to calculate a mean weighted to the standard error , and these values are given in table 1 . gratifyingly some of the best hits by consensus scoring ( 1 , 5 , 6 , 10 ) displayed good inhibitory activity against tryr from both species of parasite , with ic50 values ranging between 2156 m . to shed some light about the probable binding modes of these ligands , a computational model of the complex between tryr and the 2-(3-(2-chloro-10h - phenothiazin-0-yl)-3-oxopropyl ) decahydropyrido[1,2-a]pyrazine-2,5-diium ( 1 ) and 1-(10,11-dihydrodibenzo[b , f]thiepin-10-yl)-4-ethylpiperazine-1,4-diium ( 5 ) as well as of particular importance for substrate specificity are five residues in the disulfide substrate binding site that are not conserved when comparing tryr and human gr : glu19 , trp22 , ser110 , met114 , and ala343 . as shown in figure 8 , the low - energy model for both ligands exhibited contacts with several of these key residues in accordance with a pure competitive type of inhibition . view of possible binding modes of protonated compounds 1 ( a ) and 5 ( c ) in the active site of tryr predicted by consensus scoring ( pink ) , autodock ( green ) , and x - score ( blue ) . residues of the active site are shown in atomic colors . predicted hydrophobic and electrostatic interactions of compounds 1 ( b ) and 5 ( d ) at the active site according to ligplot . three possible binding modes were calculated for compound 1 at the active site , figure 8a . consensus scoring predicted a very specific hydrogen bond interaction with the oxygen atom of glu19 side chain and placed the pyridopyrazine-2,5-diium moiety near trp22 , tyr111 , and leu18 . this is the region in tryr that also fixes the spermidine moiety ( spd site ) and is thus responsible for the mutually exclusive substrate specificities when comparing the parasite tryr and human gr.(13 ) in turn . asn340 . these residues form a pocket that has been reported to accommodate the glui component of t[s]2,(13 ) figure 8b . in contrast to the binding mode predicted by consensus scoring , the other two calculated conformations positioned the phenothiazine ring near the spd site and , according to x - score , the positively charged fragment is at hydrogen bonding distance from tyr111 . on the other hand , two very similar orientations were found for compound 5 , figure 8c . in both cases , trp22 , met114 , leu18 , and tyr11 , meanwhile the heavily charged methylpiperazine-1,4-diium moiety displays strong electrostatic interaction with glu19 , figure 8d . a similar docking pattern was found for 3-((10h - phenothiazin-10-yl)methyl)-1-azoniabicyclo[2.2.2]octane ( 6 ) , and n - methyl - n-(2-oxo-2-(2-(phenylthio)phenylamino ) ethyl)cyclohexanaminium ( 10 ) . on the basis of these preliminary docking results , we could assume that the presence of bulky quaternary amines fragments in the analyzed molecules has an important role in enhancing tryr binding affinity . a possible explanation to this fact could be the effective combination of strong electrostatic and hydrophobic interactions between these positively charge moieties and several key residues at the active site that are intimately associated with substrate specificity . compounds 2 and 3 , table 1 , showed poor correlation between their theoretical binding affinity and the experimental inhibitory potency . a likely explanation for this fact could be the lack of rigidity in the diphenylsulfide class and the consequent difficulty to accurately predict the energy associated with the ligandreceptor complex . compounds 12 , 14 , 16 , and 20 are the only neutral molecules among the 20 top ranked ligands . in contrast to positively charged aromatic compounds , it has been found that neutral species are less likely to induce host toxicity upon dna binding and they also possess a better availability to cross the bloodbrain barrier.(5 ) the latter property is particularly desirable in the treatment of late - stage parasitic diseases when the parasite have established themselves in the central nervous system.(5 ) according to the results in table 1 , neither 12 nor 16 displayed significant inhibitory activity against tryr . in the same manner , some of the best ranked ligands by autodock ( 21 ) and x - score ( 22 - 24 ) failed to show significant inhibition at concentrations below 100 m . the low values of the tanimoto similarity coefficient , with respect to the acridine quinacrine mustard , calculated for some of the newly identified chemotypes depicted in figure 6 and 7 ( 7 , 0.21 ; 5 , 0.18 ; 11 , 0.23 ; 25 , 0.19 ; 26 , 0.15 ; 27 , 0.21 ; 28 , 0.18 ; 29 , 0.26 ) , also confirm the good shop performance of the implemented similarity strategy . in this context , the possibility of identifying novel tricyclic - related inhibitors would be of great value because most of the synthetic approaches in this area have been focused toward the decoration of known templates rather than the search for new tricyclic scaffolds . gratifyingly , we found that molecules containing dibenzothiepine 5 , dibenzooxathiepine 25 , and dibenzodithiepine 26 heterocycles as a central core could be considered as suitable candidates to increase the limited repertoire of existing tricyclic inhibitors . these heterocyclic systems are present in several of the top - ranked molecules , and they have been used as inhibitors of tumor necrosis factor-,(49 ) dna helicases,(50 ) as well as neurotropic and psychotropic agents.(51 ) moreover , these tricyclic derivatives displayed a good inhibitory activity compound , 25 being the most potent among the tested ligands with ic50 values of 11.5 and 14.6 m for t. cruzi and t. brucei tryr , respectively , table 1 . the preferred conformation found for 3-((11h - dibenzo[b , e]oxathiepin-11-yl)methyl)-1-methylpiperidinium ( 25 ) and 3-(11h - dibenzo[b , e]dithiepin-11-yl)-n , n - dimethylpropan-1-aminium ( 26 ) at the active cleft , and the ligandprotein interactions involved are depicted in figure 9 . as expected , these compounds display a binding pattern similar to that proposed for the tricyclic inhibitors acridine and phenothiazine . three modes of binding were found for compound 25 , figure 9a . even though all the conformations anchored the tricyclic dibenzooxathiepine in the proximity of the hydrophobic patch ( trp22 , met114 , tyr111 , leu18 ) , only autodock and consensus scoring positioned the 1-methylpiperidinium moiety at hydrogen bond distance from glu19 , figure 9b . the bulky dibenzodithiepine moiety is anchored at the spd site ( trp22 , met114 , ser110 ) , and the alkyl amino side chain exhibits electrostatic interactions with glu19 via hydrogen bonding , figure 9d . view of possible binding modes of compounds 25 ( a ) and 26 ( c ) in the active site of tryr predicted by consensus scoring ( pink ) , autodock ( green ) and x - score ( blue ) . residues of the active site are shown in atomic colors . predicted hydrophobic and electrostatic interactions of compounds 25 ( b ) and 26 ( d ) at the active site according to ligplot . the thia-3-azatetracyclo 27 and the 2,4,6-trioxohexahydropyrimidine 28 also constitutes novel chemotypes found among the top rank molecules . compound 27 possesses moderate inhibitory activity ( 5875 m ) , meanwhile 28 shows an unexpected selectivity for inhibiting t. cruzi tryr ( 45 m ) because little inhibition was detected under 100 m for t. brucei tryr . recently , the importance of terminally substituted guanidines and biguanides for antitrypanosomal activity has been highlighted . the biguanide clorhexidine,(18 ) and several alkylpolyaminoguanidines and alkylpolyaminobiguanides,(52 ) have shown potent inhibitory activity against tyrr . assuming that these derivatives would produce an entropy penalty upon binding , the employment of 4-oxo-1,4,5,5-tetrahydropyrimidin-2-yl - piperazin-1-ium 29 , as a conformationally constrained bioisosters of guanidine , seems to be a viable alternative . the development of a novel virtual screening cascade protocol to identify potential inhibitors of the parasitic enzyme trypanothione reductase has been described . on the basis of molecular fragment profiles , a similarity search on the zinc database was performed by the miscreen program using known reversible inhibitors of tryr as reference structures . after adme / tox filtering , the resulting enriched library was docked into the binding site of tryr to provide a list of putative ligands ranked according to consensus scoring . clustering analysis and tanimoto values of selected compounds confirms a notable improvement of the molecular diversity of the enriched library with respect to the initial training set . the study of electrostatic and hydrophobic interactions of the best hits at the active cleft highlighted the importance of conformationally constrained quaternary amine moieties in enhancing tryr binding affinity . enzymatic inhibition assays on t. cruzi and t. brucei tryr confirmed the inhibitory effect of some of the top ranked ligands . in addition , the scaffold hopping ( shop ) process derived from the developed computational approach allowed the identification of several chemotypes not previously tested as antiprotozoal agents . to our knowledge , this is the first report about the inhibitory effect of molecules - containing dibenzothiepine , dibenzooxathiepine , dibenzodithiepine , thiaazatetracyclo , and 2,4,6-trioxohexahydro pyrimidine on trypanothione reductase . further rational optimization of these polycyclic ring systems and the bioscreening of the rest of the structures contained in the database are the theme of future research . compounds 5 ( salt , ch3so3h ) , 24 , 25 ( salt , hooccooh ) , 26 ( salt , hooccooh ) , and 27 ( salt , cl h2o ) were from specs ( http : www.specs.net ) ; 3 , 4 ( salt , hcl ) , and 22 were from chembridge ( http : www.chembridge.com ) ; 6 , 7 ( salt , cl ) , and 28 ( salt , i ) were from ibscreen ( http : www.ibscreen.com ) ; 2 , 10 , 12 , 16 , 23 , and 29 were from enamine ( http : www.enamine.net ) ; 1 ( salt 2 hoocchchcooh ) was from timtec ( http : www.timtec.net ) , and 21 ( salt co3 ) was from bachem . the purity of the tested compounds was determined by liquid chromatography - ms ( phenomenex gemini c18 column , 50 mm 3.0 mm , 5 m particle size ; mobile phase , water / acetonitrile + 0.1% hcooh 80:20 to 5:95 over 3.5 min and then held for 1.5 min ; flow rate 0.5 ml / min ) . samples were diluted with acetonitrile / water ( 1:1 ) and analyzed using uv detection at 254 nm . according to the analytical data the purity of the tested compounds is higher than 95% except for compound 10 ( 43% ) . inhibition of tryr was carried out in a microplate format as described previously.(45 ) assays were set up in 96-well plates using a biotek precision 2000 automated liquid handler and initiated using nadph . the final assay mixtures ( 0.18 ml ) contained tr ( 20 mu / ml ) in the presence of 40 mm hepes ( ph 7.5 ) , 1 mm edta , 0.15 mm nadph , 50 m 5,5-dithio - bis(2-nitrobenzoic acid ) , 6 m t[s]2 , and inhibitor ( 100 m to 10 nm in 3-fold serial dilutions ) . the linear rate of thionitrobenzoate ion formation was monitored over 5 min in a spectramax 340pc plate reader ( molecular devices ) at 412 nm . grafit 5.0 ( erithacus software ) was used to fit the data to a three - parameter equation to determine ic50 values . ic50 determinations were carried out in triplicate for each compound and the mean weighted to standard error calculated .

the implementation of a novel sequential computational approach that can be used effectively for virtual screening and identification of prospective ligands that bind to trypanothione reductase ( tryr ) is reported . the multistep strategy combines a ligand - based virtual screening for building an enriched library of small molecules with a docking protocol ( autodock , x - score ) for screening against the tryr target . compounds were ranked by an exhaustive conformational consensus scoring approach that employs a rank - by - rank strategy by combining both scoring functions . analysis of the predicted ligandprotein interactions highlights the role of bulky quaternary amine moieties for binding affinity . the scaffold hopping ( shop ) process derived from this computational approach allowed the identification of several chemotypes , not previously reported as antiprotozoal agents , which includes dibenzothiepine , dibenzooxathiepine , dibenzodithiepine , and polycyclic cationic structures like thiaazatetracyclo - nonadeca - hexaen-3-ium . assays measuring the inhibiting effect of these compounds on t. cruzi and t. brucei tryr confirm their potential for further rational optimization .

Introduction Computational Methods Results and Discussion Conclusions Experimental Section

trypanosomatids are parasitic protozoa responsible for several tropical diseases of which african sleeping sickness ( trypanosoma brucei gambiense and t. b. rhodesiense ) , chagas disease ( t. cruzi ) , and the different forms of leishmaniasis ( leishmania donovani and l. infantum ) are considered the most lethal. (2 ) unfortunately , because of the low profit margins associated with developing improved therapies for tropical diseases , there is little motivation for the pharmaceutical industry to develop better tropical disease drugs . accordingly , many agencies , including the special program for research and training in tropical diseases at the world health organization ( who / tdr ) , numerous academic research groups , various international / national bodies , and philanthropic foundations , have been supporting the discovery of new drug candidates for a number of tropical diseases. the mammalian redox defense system , based on the glutathione / glutathione reductase ( graaabbreviations : tryr , trypanothione reductase ; gr , glutathione reductase ; sbdd , structure - based drug design ; shop , scaffold hopping ; mcs , maximum common substructure ; spd , spermidine ; cs , consensus scoring ; ad , autodock ; xs , x - score ; z , screening window coefficient . ) the almost total mutual substrate exclusivity of these two enzymes and the essential role of tryr in the survival of all trypanosomatids studied so far have served as the basis for rational design of many types of selective inhibitors of tryr in the presence of host gr . abbreviations : tryr , trypanothione reductase ; gr , glutathione reductase ; sbdd , structure - based drug design ; shop , scaffold hopping ; mcs , maximum common substructure ; spd , spermidine ; cs , consensus scoring ; ad , autodock ; xs , x - score ; z , screening window coefficient . structure - based drug design(7 ) ( sbdd ) has emerged as a very effective and low - cost strategy to improve the rate of success at any stage of the drug discovery pipeline . proteinligand docking attempts to use the 3d protein structure of the protein target to predict binding modes and affinities of ligands to biologically relevant targets , while ligand - similarity methods capitalize on the fact that ligands similar to an active ligand are more likely to be active than random ligands . (8 ) the elucidation of the 3d structure of tryr in free form and complexed with nadph,(11 ) glutathionylspermidine,(12 ) trypanothione,(13 ) the competitive inhibitor quinacrine(14 ) and its alkylating derivative quinacrine mustard,(15 ) have all provided detailed information about the interactions involved in enzyme recognition and substrate binding . the validity of this docking model is particularly noteworthy in relation to similar computational approaches because it predicted an interaction between the alkyl amino moiety of the acridine ring and the side chain of glu 19 as observed in the crystal structure of the tryrquinacrine complex. (14 ) the discovery of the macrocyclic alkaloid lunarine(13 ) and the recent identification of chlorhexidine and its derivatives(17 ) via high - throughput virtual screening are successful examples of the employment of structure - based methodologies to identify novel tryr inhibitors . in addition , proteinligand interactions of even very similar compounds can be unpredictable . the fact that both methods , when used individually , focus only on one part of the structural information available has recently prompted the development of hybrid similarity - based docking methods . these hybrid methods aim at fully exploiting all the structural information present in ligand - bound protein structures , both from the protein and ligand perspective . (27 ) fragment profiles differ from molecular fingerprints(28 ) because , on the one hand , they do not organize predefined or catalogued descriptors in a specific manner , and on the other , they are based on the randomization of structural information rather than canonical bit string representations . stemming from our interest in the development of new antiprotozoal drugs,(29 ) we report herein the first implementation of a novel virtual screening cascade protocol that can be used effectively for virtual screening and identification of prospective ligands that bind to trypanothione reductase . to validate this strategy , we used tryr inhibition assays to measure the activity of 19 of these prospective ligands and we found that many of them exhibit sufficiently promising characteristics to warrant further ligand optimization . the novelty of this approach lies in the sequential combination of a similarity virtual screening based on fragment profile comparisons facilitated through the calculation of compound class - specific fragment frequencies using naive bayesian statistics , with a docking protocol ( autodock and x - score ) for screening against the tryr target . another important feature of this computational approach is the enhancement of its predictive power by incorporating an exhaustive conformational consensus scoring process using a rank - by - rank strategy in order to provide a list of suitable compounds to be considered for bioscreening . the computational approach we developed for screening molecules binding to tryr combines both ligand similarity and protein docking methods within an integrated framework . a flowchart describing various phases of the virtual screening cascade protocol is shown in figure 2 . miscreen(30 ) was selected as the base methodology to perform the ligand - based virtual screening . molecules that bind reversibly to trypanothione reductase constitute the largest group of inhibitors studied so far and are among the most effective with inhibition constant ( ki ) values , usually in the low micromolar range . to date , the only reports about the structural determination of tryr complexed with a non - natural substrate are related to the competitive inhibitor quinacrine(14 ) and its chloro derivative , quinacrine mustard. (15 ) the availability of these structures certainly provides some useful insights into how these type of ligands fit into the active site of the enzyme and the interactions involved . only compounds with ki values ranging between 0.150 m the majority of the chosen compounds fell into one of the three known general categories of reversible tryr inhibitors : hydrophobic polyamines , tricyclic compounds , and diphenylsulfane derivatives ( figure 3 ) . the distribution of antiprotozoal activity for 100000 molecules with druglike properties extracted from pubchem by diversity selection to represent the average druglike chemical space and the training set of active structures is shown in figure 4 . subsequently , in silico screening of the zinc database ( http://zinc.docking.org/ ) , containing over eight million purchasable compounds , was performed by calculating the bioactivity of each molecule as a sum of activity contributions of fragments . filtering of the output data set derived from the similarity - based virtual screening ( 1312 compounds ) was performed by faf - drugs(31 ) ( http://bioserv.rpbs.jussieu.fr/help/faf drugs.html ) . (32 ) therefore , special attention was given to the input values of total charge ( 02 ) , logp ( 15 ) , and molecular mass ( 200500 ) . the rest of the parameters were either in compliance with lipinski s rule of five or set as default . compounds matching the filtering criteria ( 603 molecules ) comprised the enriched library to be used as input data for docking studies . one of the advantages of using a virtual screening protocol based on bayesian statistics is that it can generalize , i.e. furthermore , the protocol is also able to identify new chemotypes via a process called scaffold hopping ( shop ) . to shed some light about this point , clustering analysis of the initial training set and the enriched library was carried out through libmcs,(33 ) a stand - alone application program , part of the jklustor package . two hierarchical clusterings , corresponding to the initial training set and the enriched library , respectively , are depicted in figure 5 . initial structures are found at the bottom of the hierarchy , and the next level contains the mcs of clusters of initial molecules . the highest clustering values observed for the enriched library ( cluster level : 4 , total cluster count : 257 ) , figure 5b , with respect to the initial training set ( cluster level : 3 , total cluster count : 101 ) , figure 5a , confirms the effectiveness of the adopted similarity - based strategy to generate new chemotypes by using the information of already known bioactive molecules . (35 ) the protein structure for the unbound trypanosoma cruzi trypanothione reductase was chosen as the target , and coordinates were downloaded from the protein data bank ( pdb code 1aog). initial conformations of the ligands were placed next to the aromatic ring of tyr111 , a central residue in the active site having a hydrophobic interaction with trypanothione . in - house perl scripts were used to extract the 100 conformations generated by autodock from the docking output file for each of the compounds in order to allow them to be evaluated by the program x - score. (38 ) all ligand files that had the same logp values calculated by x - score were inspected visually to verify their structures . to minimize the computing time , docking jobs as well as x - score evaluations were carried out on a westgrid computer cluster designed for serial processing and parallel jobs ( http://www.westgrid.ca ) . jobs were submitted using portable batch system ( pbs ) files invoking either autodock or x - score . pymol v0.99(39 ) was used to visualize the compounds docked to the active site of the protein , and schematic diagrams of proteinligand interactions were obtained by the ligplot program. (40 ) combining multiple scoring functions has been shown in various virtual database screening studies to be an effective way for improving hit rates. (41 ) while autodock is one of the most cited docking programs(42 ) and has recorded many successes in virtual screening approaches,(43 ) the stand - alone scoring function x - score has shown good correlations between theoretical and experimentally determined proteinligand binding affinities. (44 ) consequently , x - score was combined in this study with the function implemented in the autodock program to provide a list of ligands sorted by consensus scoring adopting the rank - by - rank strategy. (45 ) autodock and x - score were applied to rank all the conformations generated by the 603 compounds ( 60300 conformations in total ) . only the best scored conformation of each compound a representation of the 20 top ranked ligands according to consensus scoring is shown in figure 6 . these compounds exhibit structural characteristics typical of several known tryr inhibitors , including extended hydrophobic moieties tethered to positive charge fragments mostly represented by bulky quaternary cyclic amines . phenothiazines and diphenylsulfanes , included in the initial training set , were successfully recovered among the top scored ligands , highlighting the importance of these building blocks for binding affinity . the same type of interaction is also very likely to occur for some of the newly identified chloro derivatives . (47 ) the tested set included 10 of the best rank structures according consensus scoring ( compounds 17 , 10 , 12 , 16 , figure 6 ) . additionally , some of the best hits by autodock ( 21 ) and x - score ( 22 , 23 , 24 ) and several high - ranked new chemotypes ( 2529 ) depicted in figure 7 , were also considered for screening . the triplicate ic50 values were used to calculate a mean weighted to the standard error , and these values are given in table 1 . gratifyingly some of the best hits by consensus scoring ( 1 , 5 , 6 , 10 ) displayed good inhibitory activity against tryr from both species of parasite , with ic50 values ranging between 2156 m . to shed some light about the probable binding modes of these ligands , a computational model of the complex between tryr and the 2-(3-(2-chloro-10h - phenothiazin-0-yl)-3-oxopropyl ) decahydropyrido[1,2-a]pyrazine-2,5-diium ( 1 ) and 1-(10,11-dihydrodibenzo[b , f]thiepin-10-yl)-4-ethylpiperazine-1,4-diium ( 5 ) as well as of particular importance for substrate specificity are five residues in the disulfide substrate binding site that are not conserved when comparing tryr and human gr : glu19 , trp22 , ser110 , met114 , and ala343 . as shown in figure 8 , the low - energy model for both ligands exhibited contacts with several of these key residues in accordance with a pure competitive type of inhibition . view of possible binding modes of protonated compounds 1 ( a ) and 5 ( c ) in the active site of tryr predicted by consensus scoring ( pink ) , autodock ( green ) , and x - score ( blue ) . residues of the active site are shown in atomic colors . consensus scoring predicted a very specific hydrogen bond interaction with the oxygen atom of glu19 side chain and placed the pyridopyrazine-2,5-diium moiety near trp22 , tyr111 , and leu18 . in contrast to the binding mode predicted by consensus scoring , the other two calculated conformations positioned the phenothiazine ring near the spd site and , according to x - score , the positively charged fragment is at hydrogen bonding distance from tyr111 . on the basis of these preliminary docking results , we could assume that the presence of bulky quaternary amines fragments in the analyzed molecules has an important role in enhancing tryr binding affinity . compounds 2 and 3 , table 1 , showed poor correlation between their theoretical binding affinity and the experimental inhibitory potency . compounds 12 , 14 , 16 , and 20 are the only neutral molecules among the 20 top ranked ligands . in the same manner , some of the best ranked ligands by autodock ( 21 ) and x - score ( 22 - 24 ) failed to show significant inhibition at concentrations below 100 m . the low values of the tanimoto similarity coefficient , with respect to the acridine quinacrine mustard , calculated for some of the newly identified chemotypes depicted in figure 6 and 7 ( 7 , 0.21 ; 5 , 0.18 ; 11 , 0.23 ; 25 , 0.19 ; 26 , 0.15 ; 27 , 0.21 ; 28 , 0.18 ; 29 , 0.26 ) , also confirm the good shop performance of the implemented similarity strategy . gratifyingly , we found that molecules containing dibenzothiepine 5 , dibenzooxathiepine 25 , and dibenzodithiepine 26 heterocycles as a central core could be considered as suitable candidates to increase the limited repertoire of existing tricyclic inhibitors . these heterocyclic systems are present in several of the top - ranked molecules , and they have been used as inhibitors of tumor necrosis factor-,(49 ) dna helicases,(50 ) as well as neurotropic and psychotropic agents. (51 ) moreover , these tricyclic derivatives displayed a good inhibitory activity compound , 25 being the most potent among the tested ligands with ic50 values of 11.5 and 14.6 m for t. cruzi and t. brucei tryr , respectively , table 1 . the preferred conformation found for 3-((11h - dibenzo[b , e]oxathiepin-11-yl)methyl)-1-methylpiperidinium ( 25 ) and 3-(11h - dibenzo[b , e]dithiepin-11-yl)-n , n - dimethylpropan-1-aminium ( 26 ) at the active cleft , and the ligandprotein interactions involved are depicted in figure 9 . as expected , these compounds display a binding pattern similar to that proposed for the tricyclic inhibitors acridine and phenothiazine . even though all the conformations anchored the tricyclic dibenzooxathiepine in the proximity of the hydrophobic patch ( trp22 , met114 , tyr111 , leu18 ) , only autodock and consensus scoring positioned the 1-methylpiperidinium moiety at hydrogen bond distance from glu19 , figure 9b . the bulky dibenzodithiepine moiety is anchored at the spd site ( trp22 , met114 , ser110 ) , and the alkyl amino side chain exhibits electrostatic interactions with glu19 via hydrogen bonding , figure 9d . view of possible binding modes of compounds 25 ( a ) and 26 ( c ) in the active site of tryr predicted by consensus scoring ( pink ) , autodock ( green ) and x - score ( blue ) . compound 27 possesses moderate inhibitory activity ( 5875 m ) , meanwhile 28 shows an unexpected selectivity for inhibiting t. cruzi tryr ( 45 m ) because little inhibition was detected under 100 m for t. brucei tryr . the development of a novel virtual screening cascade protocol to identify potential inhibitors of the parasitic enzyme trypanothione reductase has been described . after adme / tox filtering , the resulting enriched library was docked into the binding site of tryr to provide a list of putative ligands ranked according to consensus scoring . clustering analysis and tanimoto values of selected compounds confirms a notable improvement of the molecular diversity of the enriched library with respect to the initial training set . the study of electrostatic and hydrophobic interactions of the best hits at the active cleft highlighted the importance of conformationally constrained quaternary amine moieties in enhancing tryr binding affinity . enzymatic inhibition assays on t. cruzi and t. brucei tryr confirmed the inhibitory effect of some of the top ranked ligands . in addition , the scaffold hopping ( shop ) process derived from the developed computational approach allowed the identification of several chemotypes not previously tested as antiprotozoal agents . to our knowledge , this is the first report about the inhibitory effect of molecules - containing dibenzothiepine , dibenzooxathiepine , dibenzodithiepine , thiaazatetracyclo , and 2,4,6-trioxohexahydro pyrimidine on trypanothione reductase . further rational optimization of these polycyclic ring systems and the bioscreening of the rest of the structures contained in the database are the theme of future research . the purity of the tested compounds was determined by liquid chromatography - ms ( phenomenex gemini c18 column , 50 mm 3.0 mm , 5 m particle size ; mobile phase , water / acetonitrile + 0.1% hcooh 80:20 to 5:95 over 3.5 min and then held for 1.5 min ; flow rate 0.5 ml / min ) . the final assay mixtures ( 0.18 ml ) contained tr ( 20 mu / ml ) in the presence of 40 mm hepes ( ph 7.5 ) , 1 mm edta , 0.15 mm nadph , 50 m 5,5-dithio - bis(2-nitrobenzoic acid ) , 6 m t[s]2 , and inhibitor ( 100 m to 10 nm in 3-fold serial dilutions ) .

spinal stability is the vertebral ability to maintain their relationship and limit their relative displacements during physiologic postures and loads.1 instability develops when the spinal stabilizing system fails to maintain the physiological limit of spinal neutral zone , which may result in progressive deterioration of the structural components of the spine leading to incapacitating symptoms [ e.g. , low back pain ( lbp ) with or without sciatica , increasing disability and progressive deterioration of quality of life ] . the concept of lumbar segmental instability ( lsi ) is not new and degenerative and lytic spondylolisthesis comprises the principal aetiology . the clinical symptoms and proposed clinical tests had limited diagnostic significance,2 hence the radiological criteria has been emphasized.3 although the initial treatment is conservative ( e.g. , patient education , exercise , bracing , physical therapy ) , surgery is the last resort for symptomatic instability.4 spinal fusion procedures are indicated with severe disabling symptoms and radiographic evidence of increased segmental motion that fails to respond to adequate conservative trial.5 segmental fusion provides solid fixation , restores the spinal stability , and maintains loadbearing capacity of spine.6 considering all these advantages , posterior lumbar interbody fusion ( plif ) has long been the gold standard surgical technique for lsi,78 but since transforaminal lumbar interbody fusion ( tlif ) ( a modification of plif by harms9 ) has been introduced , it has been found to be better technique for different other spinal disorders.101112 to the best of our knowledge , there is no study comparing these two techniques in spinal instability in english language literature . our aim was to assess and compare the early clinical and radiological outcome of plif and tlif in the two most common causes of symptomatic lumbar instability . a retrospective review of the records of 52 patients , 11 men and 41 women aged 40 to 59 years ( mean 46.73 , sd 05.88 ) who underwent plif ( group - i ) , and 50 patients , 14 men and 36 women aged 40 to 59 years ( mean 49.04 , sd 06.88 ) who underwent tlif ( group - ii ) were reviewed between january 2005 and december 2011 . patients with lumbar instability ( degenerative and lytic ) with uni / bilateral radiculopathy who failed adequate conservative therapy ( e.g. , bracing , physiotherapy , and exercises ) for 6 - 15 months ( mean 9 months , sd 2.75 ) were included , but patients with ( i ) symptomatic radiological instability in > 1 segment , ( ii ) previous history of spondylodiscitis ; and ( iii ) pathological condition of lumbar spine ( e.g. , trauma , tumor ) were excluded . higher grades ( > grade - ii ) of spondylolisthesis were also excluded because of unavailability of reduction pedicle screws and in situ reduction devices . preoperative x - ray lumbosacral ( l / s ) spine antero / posterior ( a / p ) , lateral [ figure 1 ] and dynamic views were done to assess the instability by the radiographic criteria of posner3 and meyerding's13 grading was done to assess forward slip . magnetic resonance imaging ( mri ) of the l / s spine the operative time , intraoperative blood loss , postoperative hospital stay , improvement of neurological status was recorded . preoperative and postoperative pain status was recorded by self - evaluated visual analog score ( vas)14 and disability by oswestry disability index ( odi).15 followup was done at 6 weeks , 3 months , 6 months , and then every yearly16 for radiological fusion,17 structural restoration ( disc height , foraminal height , angle of total lumbar lordosis and slip reduction ) in standing lateral films18 [ figure 2 ] , and maintenance of stability.3 computed tomography ( ct ) scan had been reserved for cases where radiological fusion was doubtful or delayed or suspected of pseudarthrosis , as recommended.19 the overall functional outcome was assessed by macnab's20 criteria . x - ray lumbosacral spine lateral view showing radiological assessment of structural restoration ( disc height , foraminal height and angle of total lumbar lordosis ) . disc height has been measured as ( da+dp ) / 2 ; the foraminal height has been measured as the distance between the midpoint of the superior and inferior neural arch ( f1-f2 ) ; the angle of total lumbar lordosis has been measured by the angle formed by the perpendicular lines from the two lines drawn along the superior end plate of l1 and superior end plate of s1 ( angle abc ) x - ray lumbosacral spine lateral view showing ( a ) measurement of instability according to posner;3 dynamic lateral film ( flexion ) in standing posture showing > 10 sagittal rotation ( angular displacement ) ; measurement is done by the angle formed between the two adjacent end - plates . ( c ) the dynamic films producing anterior translation measured as the distance between a and b= d ( in flexion ) . ( d ) the distance of a1 and b1 = d1 ( in extension ) . the distance difference ( d ) in flexion ( d ) and extension ( d1 ) calculated as the percentage ( d = d d1 above vertebral width 100 ) of the width of the above vertebra after proper explanation of the management options and surgical technique , an informed written consent was taken , the patients were operated under general anaesthesia . patients of both groups i.e. plif and tlif , were placed in prone position and a routine posterior midline approach was used . the pedicle screws were inserted using a standard free hand targeting technique under fluoroscopic control . in plif [ figure 3 ] , decompression was commenced by laminectomy and removal of interspinous ligaments and ligamentum flavum with sufficient decompressive laminotomy superiorly and inferiorly . but in tlif [ figure 4 ] , unilateral laminotomy and partial facetectomy were performed on the side consistent with the patient 's symptoms or anatomical abnormalities . unlike most other literature , we used to excise the spinous process to collect adequate autografts for the prepared disc space intended to achieve good fusion . discectomy was done bilaterally in plif but unilaterally in tlif . degenerative instability managed by plif ; ( a ) x - ray lumbosacral ( l / s ) spine anteroposterior view . ( b ) x - ray l / s spine lateral view showing degenerative spondylolisthesis at l4 over l5 . ( e ) t2w mri scan of that patient with degeneration and spondylolisthesis at l4/5 level . ( a ) postoperative x - ray lumbosacral spine anteroposterior view and ( b ) lateral view showing good implant position . ( c ) 1 year followup x - ray anteroposterior view and ( d ) lateral view showing listhesis reduction and radiological fusion ( arrow ) spondylolytic instability managed by tlif : x - ray lumbosacral spine anteroposterior view ( a ) and lateral view ( b ) showing grade - i spondylolytic spondylolisthesis instability at l5/s1 level . ( c ) dynamic lateral view ( flexion ) showing spondylolysis ( arrow ) and anterior translation . ( d ) dynamic lateral view ( extension ) showing spondylolysis ( arrow ) and angular motion at l5/s1 level . ( e ) t2w mri scan of that patient showing involvement of l5/s1 level ( a ) postoperative x - ray lumbosacral spine anteroposterior view and ( b ) lateral view showing good implant position and the transforaminal approach ( arrow ) . ( c ) 1 year followup x - ray anteroposterior view and ( d ) lateral view showing listhesis reduction and radiological fusion ( arrow ) disc space preparation was done by unilateral distractor instrumentation with bilateral curettage in plif ; however , in tlif we used unilateral curettage by using special curved curettes to remove the end plates . the morcelized bone grafts taken from the excised spinous process and/or laminar bone were introduced to the anterior part of the disc space and impacted with an l - shaped impactor . in both techniques , we used single banana cage ( titanium , size 9 mm , 10 mm , or 11 mm ) which was packed with cortico - cancellous autografts and inserted in the prepared space from the surgeon 's side . the final position of the cage was confirmed fluoroscopically or by check x - ray . patients were mobilized on the 2 or 3 postoperative day with a brace support which was continued for minimum 6 weeks postoperaively . during the followup period , the clinical and radiological parameters were measured by the same assessor and the statistical analysis was performed using the spss statistical software where results were achieved from the chi square test and z test where applicable . after proper explanation of the management options and surgical technique , an informed written consent was taken , the patients were operated under general anaesthesia . patients of both groups i.e. plif and tlif , were placed in prone position and a routine posterior midline approach was used . the pedicle screws were inserted using a standard free hand targeting technique under fluoroscopic control . in plif [ figure 3 ] , decompression was commenced by laminectomy and removal of interspinous ligaments and ligamentum flavum with sufficient decompressive laminotomy superiorly and inferiorly . but in tlif [ figure 4 ] , unilateral laminotomy and partial facetectomy were performed on the side consistent with the patient 's symptoms or anatomical abnormalities . unlike most other literature , we used to excise the spinous process to collect adequate autografts for the prepared disc space intended to achieve good fusion . discectomy was done bilaterally in plif but unilaterally in tlif . degenerative instability managed by plif ; ( a ) ( b ) x - ray l / s spine lateral view showing degenerative spondylolisthesis at l4 over l5 . ( e ) t2w mri scan of that patient with degeneration and spondylolisthesis at l4/5 level . ( a ) postoperative x - ray lumbosacral spine anteroposterior view and ( b ) lateral view showing good implant position . ( c ) 1 year followup x - ray anteroposterior view and ( d ) lateral view showing listhesis reduction and radiological fusion ( arrow ) spondylolytic instability managed by tlif : x - ray lumbosacral spine anteroposterior view ( a ) and lateral view ( b ) showing grade - i spondylolytic spondylolisthesis instability at l5/s1 level . ( c ) dynamic lateral view ( flexion ) showing spondylolysis ( arrow ) and anterior translation . ( d ) dynamic lateral view ( extension ) showing spondylolysis ( arrow ) and angular motion at l5/s1 level . ( e ) t2w mri scan of that patient showing involvement of l5/s1 level ( a ) postoperative x - ray lumbosacral spine anteroposterior view and ( b ) lateral view showing good implant position and the transforaminal approach ( arrow ) . ( c ) 1 year followup x - ray anteroposterior view and ( d ) lateral view showing listhesis reduction and radiological fusion ( arrow ) disc space preparation was done by unilateral distractor instrumentation with bilateral curettage in plif ; however , in tlif we used unilateral curettage by using special curved curettes to remove the end plates . the morcelized bone grafts taken from the excised spinous process and/or laminar bone were introduced to the anterior part of the disc space and impacted with an l - shaped impactor . in both techniques , we used single banana cage ( titanium , size 9 mm , 10 mm , or 11 mm ) which was packed with cortico - cancellous autografts and inserted in the prepared space from the surgeon 's side . the final position of the cage was confirmed fluoroscopically or by check x - ray . patients were mobilized on the 2 or 3 postoperative day with a brace support which was continued for minimum 6 weeks postoperaively . during the followup period , the clinical and radiological parameters were measured by the same assessor and the statistical analysis was performed using the spss statistical software where results were achieved from the chi square test and z test where applicable . in both the groups , female patients of the 40 - 49 years age group were significant ( chi - square test , p < 0.05 ) . maximum cases had spondylolytic instability [ plif = 27 ( 51.92% ) , tlif = 24 ( 48.00% ) ] and l4/5 level involved most commonly [ plif = 27 ( 51.92% ) , tlif = 28 ( 56.00% ) ] in both groups . grade - ii slip [ plif = 41 ( 78.85% ) , tlif = 40 ( 80.00% ) ] had been significant ( chi - square test , p < 0.05 ) in both groups [ table 1 ] . preoperative lbp and radiculopathy ( leg pain ) was significantly present in both groups ( chi - square test , p < 0.05 ) having a highly significant improvement of their vas scores ( z test , p < 0.001 ) at 1 year . lbp score improved from 07.25 01.04 to 02.25 00.55 in the plif group and from 06.64 01.24 to 01.92 00.63 in the tlif group . similar significant improvement of the leg pain status was achieved from 05.25 00.52 to 01.75 00.79 in the plif group and from 05.24 00.43 to 01.74 00.63 in the tlif group . 0.001 ) improvement from 60.75 11.37 to 11.25 02.91 in the plif group and 56.71 11.10 to 07.46 02.09 in the tlif group . the neurological status ( e.g. , motor and sensory ) of both groups also had highly significant recovery ( z test , none of the pain score , disability score , and neurological recovery had significant difference between the groups ( z test , p > 0.05 ) . although overall satisfactory outcome [ plif = 49 ( 94.23% ) , tlif = 48 ( 96.00% ) ] had no significant difference , there had been more excellent results with tlif [ 35 ( 70.00% ) ] than that of plif [ 29 ( 55.77% ) ] [ table 2 ] . demographics of the patients ( n=102 ) clinical , functional , and overall outcome of the patients ( n=102 ) assessment of radiological restoration of structural components revealed a significant ( z test , p < 0.05 ) rise of mean disc height ( mdh ) from 07.50 02.73 to 12.00 01.97 mm in the plif group and from 07.76 02.77 to 12.24 01.89 mm in the tlif group . the mean foraminal height ( mfh ) increase was recorded from 13.25 01.61 to 17.50 01.85 mm in plif and from 13.30 1.55 to 17.50 01.87 mm in the tlif group , which was also significant ( z test , p < 0.05 ) . the preoperative value of mean total lumbar lordosis ( mtll ) increased to 24.60 00.85 ( from 18.30 03.75 ) in cases the plif group and from 18.60 03.16 to 24.00 02.00 in the tlif group . the mean anterior slip ( mas ) in the plif group was 30.25 05.75% and in the tlif group it was 34.80 03.25% . there had been a significant slip reduction in both the groups ( plif = 90 03.85% for grade - i and 81 05.24% for grade - ii , tlif = 92 03.63% for grade - i and 84 01.50% for grade - ii ) , which had been maintained even at their last followup . the preoperative mean anterior translation ( mat ) was 09.50 0.87% at l3/4 , 09.21 1.03% at l4/5 , and 07.75 1.93% at l5/s1 in the plif group and 09.22 1.07% at l3/4 , 09.00 1.12% at l4/5 , and 08.26 1.07% at l5/s1 in the tlif group . the preoperative mean angular displacement ( mad ) was 10.75 01.58 at l3/4 , 12.40 00.70 at l4/5 , and 04.50 01.98 at l5/s1 in the plif group and 11.12 01.70 at l3/4 , 12.14 00.95 at l4/5 , and 04.20 01.10 at l5/s1 in the tlif group [ table 3 ] . but all these variables of structural restoration ( e.g. , mdh , mfh , mtll , mas , mat , mad ) had no statistical significant differences ( z test , p > 0.05 ) between the groups . postoperative dynamic films did not reveal abnormal range of movement in patients of either of the group , hence achieving radiological stability . time required for achieving signs of radiological fusion [ plif ( 17.23 02.95 weeks ) , tlif ( 16.80 03.94 weeks ) ] also had no significant difference ( z test , p > 0.05 ) [ table 4 ] . radiological outcome of the patients ( n=102 ) operative outcome and complications in both groups ( n=102 ) plif surgery took longer time ( 182 10.91 minutes ) and higher intraoperative bleeding ( 245 19 ml ) than tlif ( 165 06.63 minutes and 215 12 ml ) . the postoperative hospital stay ( 07.50 02.30 days for plif versus 05.76 01.60 days for tlif ) and the intraoperative complication [ plif ( 09.61% ) , tlif ( 02.00% ) ] and postoperative complication [ plif ( 13.47% ) , tlif ( 04.00% ) ] had no significant difference between the groups . accidental durotomy had occurred in four ( 07.69% ) cases - all of which could be repaired primarily and none of them had any postoperative csf leakage . three ( 05.77% ) cases had intraoperative root injury ( during the insertion of interbody cage ) , among which one case developed foot drop . five ( 09.62% ) cases in plif and two ( 04.00% ) cases in tlif developed pseud - arthrosis ( chi - square test , p > 0.05 ) . these cases were managed conservatively and had their self - assessed vas pain score ranged within 02 to 04 , odi disability score from 19.80% to 28.20% ( moderate disability ) and none of them had any signs of neurological compromise , hence did not require revision surgery . there were no cases of superficial wound infection in the tlif group but two ( 03.85% ) cases with plif developed superficial wound infection , which were managed conservatively [ table 4 ] . the concept of lsi has received an increased attention from the clinicians and researchers as a potential cause of chronic lbp , which has been commonly associated with spondylolisthesis and spondylolysis.2122 restoration of the segmental stability by adequate neural decompression , fusion , and stabilization helps to improve clinical symptoms and achieve normal spinal anatomy.23 failure of restoration can result in inadequate clinical improvement potentially leading to poor long term results.24 significant clinical improvement was observed in both plif and tlif techniques in different spinal disorders2526 and found to be superior due to proper neural decompression , structural restoration , and segmental stabilization that ultimately lead to improved pain , disability , and functional capability . in our study , both the techniques resulted in significant clinical and functional improvement , structural restoration , fusion , and stability but plif had been associated with higher rates of intraoperative neural complications . firstly , the diagnosis was solely depended upon the radiological findings which may have misinterpretations.27 secondly , t2-weighted kinetic mri and three - dimensional ct reconstruction had been recommended for a precise diagnosis of lsi,2728 but we could not perform these due to unavailability of expertise . thirdly , foraminal widening and fusion assessment needs ct evaluation,29 but was ignored due to patients financial constraints . the cases with multi - segmental instability were not included with an assumption of bias . fourthly , we did not intend to assess whether there is any biomechanical relationship between pseudarthrosis and postoperative instability . lastly , the study population was not large enough and followup period was short , as a result we could not evaluate the long term complications , pseudarthrosis requiring revision , adjacent segment degeneration , implant failure , or even the cases with failed back syndrome . in literatures , comparison of plif and tlif in spondylolisthesis and degenerative lumbar spine had been done . the improvement of vas score of the initial series [ plif ( 07.08 01.13 to 02.84 0.89 ) and tlif ( 07.18 01.09 to 02.84 0.91 ) ] was comparable to ours . according to audat30 excellent outcome had been observed around 60% cases in plif and around 70% cases in tlif , which was also comparable to ours . the overall satisfactory clinical outcome was not measured by the same criteria in different literatures2630 but even then , the overall outcome had also been similar . interbody cages are used to restore the disc height , foraminal height and stabilize the affected segment.31 these parameters have significant correlation regarding structural restoration and maintenance of stability.32 the cage was inserted from the patients left side irrespective of neurological involvement but decompression was done by changing the side with contralateral involvements . we observed a significant increase of disc and foraminal height as well as neurological improvement comparable to other studies.3334 the increased foraminal height effectively decompresses the nerve roots32 and restores lumbar lordosis which ultimately maintains the lumbar sagittal profile.33 restoration of local and regional lordosis ultimately achieves clinical and biomechanical stability.35 kim36 recommended to place the graft anterior to the cage and hsieh31 recommended to apply compression using the graft and cage as a fulcrum to achieve the desired lordosis . in our study , we were able to achieve 06.500.55 and 05.700.75 improvement of lordotic angles in plif and tlif , that were comparable to other studies.3334 despite of no statistical difference of postoperative lordotic angles between the groups , we observed lesser degree of correction in tlif which assumed to be due to intact posterior structures which had also been observed by hsieh.31 the correction of forward slip restores sagittal alignment and physiological transmission of weight . inadequate restoration and abnormal lordosis is the primary predisposing factor for adjacent segment degeneration.37 moreover , it shifts the spinal vertical axis anteriorly to induce further degeneration and results in chronic lbp.38 the percentage of correction of slip in our study had been significant by both techniques which was comparable to yan26 [ plif ( 72.60 05.20% ) , tlif ( 72.40 05.40% ) ] . but the reports of increased chance of postoperative instability with plif due to loss of integrity of posterior structures should not be abandoned.32 interbody fusion with cage has been well accepted for its superior fusion results,3940 and has been reported to be significant by both these techniques.2526 theoretically , tlif is advantageous to plif , as it provides a full 360 fusion because of intact contralateral laminar surface that increases the surface area for new bone to grow and bridge the gap.41 we had observed signs of radiological union at 17.23 02.95 weeks in plif and 16.90 03.94 weeks in tlif . but these signs might not conclude regarding the achievement of fusion as because , kim et al.42 showed around 35% patients achieved solid fusion at 1 year despite of radiological union signs and it even took minimum 4 years to achieve solid fusion in 82% cases . but brantigan16 compared radiological fusion with exploratory fusion and stated > 97% sensitivity , > 94% positive predictive value and > 93% accuracy of the radiological parameters . autografts had been the gold standard for achieving fusion , but there are recent reports of graft substitutes for fusion enhancement and has become a new arena of research . we placed autografts anteriorly and impacted before the introduction of cage in all the cases of plif and tlif with a theoretical background of anterior column load transmission ( 80%)39 and enhancement of fusion.43 the radiological fusion in both the groups had no statistical difference as like other studies.111230 the biomechanical concept of fusion stability is assessed postoperatively by dynamic films to determine the achievement of stability even after fusion . although posterior instrumentations enhances the stability and fusion,8 biomechanically stable spine is achieved only when solid fusion is achieved.44 we achieved the target motion of stability in all the cases of plif and tlif at 1 year according to posner et al.,3 but according to kumar et al.,45 postoperative segmental stability is achieved only when radiological motion is < 2 in dynamic films . development of pseudarthrosis is one of the most common ( range , 05 - 45% ) complications of interbody fusion.46 it can be managed conservatively and does not necessarily need revision surgery.47 pseud arthrosis was present in two ( 2.60% ) and two ( 4.60% ) patients with plif or tlif in a series of mehta10 which is comparable to our results . in different literatures , plif had been reported to be associated with more neural complications.10111226 the excess medial retraction of the dura has been blamed to be the cause of such injuries during the placement of the cage.11 on the contrary , tlif approaches the disc space through far lateral portion of the vertebral foramen , which ultimately reduces the thecal manipulation and the chances of complications . in this series , we had iatrogenic durotomy in four ( 7.69% ) cases and root injury in three ( 5.77% ) cases . there were no such complications in the tlif group . intraoperative dural repair were sufficient to control the leaks but one case with root injury ultimately developed foot drop , whereas other two cases were transient and were under the process of gradual recovery . there was a case each in both groups with hardware misplacement which we identified as an instrumental default . two ( 3.85% ) cases in plif had superficial wound infection that had been managed with intravenous antibiotics following culture sensitivity ( staphylococcus aureus ) and regular dressing , and the wound was later healed with secondary intention . to conclude , both methods are effective in relieving symptoms , achieving stability and fusion , but tlif can be preferred for its shorter operative time , less blood loss , and lesser complication rates in surgical management of symptomatic lumbar instability .

background : transforaminal lumbar interbody fusion ( tlif ) has been preferred to posterior lumbar interbody fusion ( plif ) for different spinal disorders but there had been no study comparing their outcome in lumbar instability . a comparative retrospective analysis of the early results of tlif and plif in symptomatic lumbar instability was conducted between 2005 and 2011.materials and methods : review of the records of 102 operated cases of lumbar instability with minimum 1 year followup was done . a total of 52 cases ( 11 men and 41 women , mean age 46 years sd 05.88 , range 40 - 59 years ) underwent plif and 50 cases ( 14 men and 36 women , mean age 49 years sd 06.88 , range 40 - 59 years ) underwent tlif . the surgical time , duration of hospital stay , intraoperative blood loss were compared . self - evaluated low back pain and leg pain status ( using visual analog score ) , disability outcome ( using oswestry disability questionnaire ) was analyzed . radiological structural restoration ( e.g. , disc height , foraminal height , lordotic angle , and slip reduction ) , stability ( using posner criteria ) , fusion ( using hackenberg criteria ) , and overall functional outcome ( using macnab 's criteria ) were compared.results:pain , disability , neurology , and overall functional status were significantly improved in both groups but plif required more operative time and caused more blood loss . postoperative hospital stay , structural restoration , stability , and fusion had no significant difference but neural complications were relatively more with plif.conclusions:both methods were effective in relieving symptoms , achieving structural restoration , stability , and fusion , but tlif had been associated with shorter operative time , less blood loss , and lesser complication rates for which it can be preferred for symptomatic lumbar instability .

I M Operative procedure R D

spinal stability is the vertebral ability to maintain their relationship and limit their relative displacements during physiologic postures and loads.1 instability develops when the spinal stabilizing system fails to maintain the physiological limit of spinal neutral zone , which may result in progressive deterioration of the structural components of the spine leading to incapacitating symptoms [ e.g. , low back pain ( lbp ) with or without sciatica , increasing disability and progressive deterioration of quality of life ] . the concept of lumbar segmental instability ( lsi ) is not new and degenerative and lytic spondylolisthesis comprises the principal aetiology . the clinical symptoms and proposed clinical tests had limited diagnostic significance,2 hence the radiological criteria has been emphasized.3 although the initial treatment is conservative ( e.g. , patient education , exercise , bracing , physical therapy ) , surgery is the last resort for symptomatic instability.4 spinal fusion procedures are indicated with severe disabling symptoms and radiographic evidence of increased segmental motion that fails to respond to adequate conservative trial.5 segmental fusion provides solid fixation , restores the spinal stability , and maintains loadbearing capacity of spine.6 considering all these advantages , posterior lumbar interbody fusion ( plif ) has long been the gold standard surgical technique for lsi,78 but since transforaminal lumbar interbody fusion ( tlif ) ( a modification of plif by harms9 ) has been introduced , it has been found to be better technique for different other spinal disorders.101112 to the best of our knowledge , there is no study comparing these two techniques in spinal instability in english language literature . our aim was to assess and compare the early clinical and radiological outcome of plif and tlif in the two most common causes of symptomatic lumbar instability . a retrospective review of the records of 52 patients , 11 men and 41 women aged 40 to 59 years ( mean 46.73 , sd 05.88 ) who underwent plif ( group - i ) , and 50 patients , 14 men and 36 women aged 40 to 59 years ( mean 49.04 , sd 06.88 ) who underwent tlif ( group - ii ) were reviewed between january 2005 and december 2011 . patients with lumbar instability ( degenerative and lytic ) with uni / bilateral radiculopathy who failed adequate conservative therapy ( e.g. , bracing , physiotherapy , and exercises ) for 6 - 15 months ( mean 9 months , sd 2.75 ) were included , but patients with ( i ) symptomatic radiological instability in > 1 segment , ( ii ) previous history of spondylodiscitis ; and ( iii ) pathological condition of lumbar spine ( e.g. , trauma , tumor ) were excluded . preoperative x - ray lumbosacral ( l / s ) spine antero / posterior ( a / p ) , lateral [ figure 1 ] and dynamic views were done to assess the instability by the radiographic criteria of posner3 and meyerding's13 grading was done to assess forward slip . magnetic resonance imaging ( mri ) of the l / s spine the operative time , intraoperative blood loss , postoperative hospital stay , improvement of neurological status was recorded . preoperative and postoperative pain status was recorded by self - evaluated visual analog score ( vas)14 and disability by oswestry disability index ( odi).15 followup was done at 6 weeks , 3 months , 6 months , and then every yearly16 for radiological fusion,17 structural restoration ( disc height , foraminal height , angle of total lumbar lordosis and slip reduction ) in standing lateral films18 [ figure 2 ] , and maintenance of stability.3 computed tomography ( ct ) scan had been reserved for cases where radiological fusion was doubtful or delayed or suspected of pseudarthrosis , as recommended.19 the overall functional outcome was assessed by macnab's20 criteria . x - ray lumbosacral spine lateral view showing radiological assessment of structural restoration ( disc height , foraminal height and angle of total lumbar lordosis ) . disc height has been measured as ( da+dp ) / 2 ; the foraminal height has been measured as the distance between the midpoint of the superior and inferior neural arch ( f1-f2 ) ; the angle of total lumbar lordosis has been measured by the angle formed by the perpendicular lines from the two lines drawn along the superior end plate of l1 and superior end plate of s1 ( angle abc ) x - ray lumbosacral spine lateral view showing ( a ) measurement of instability according to posner;3 dynamic lateral film ( flexion ) in standing posture showing > 10 sagittal rotation ( angular displacement ) ; measurement is done by the angle formed between the two adjacent end - plates . patients of both groups i.e. plif and tlif , were placed in prone position and a routine posterior midline approach was used . discectomy was done bilaterally in plif but unilaterally in tlif . ( e ) t2w mri scan of that patient with degeneration and spondylolisthesis at l4/5 level . ( c ) 1 year followup x - ray anteroposterior view and ( d ) lateral view showing listhesis reduction and radiological fusion ( arrow ) spondylolytic instability managed by tlif : x - ray lumbosacral spine anteroposterior view ( a ) and lateral view ( b ) showing grade - i spondylolytic spondylolisthesis instability at l5/s1 level . ( c ) 1 year followup x - ray anteroposterior view and ( d ) lateral view showing listhesis reduction and radiological fusion ( arrow ) disc space preparation was done by unilateral distractor instrumentation with bilateral curettage in plif ; however , in tlif we used unilateral curettage by using special curved curettes to remove the end plates . the morcelized bone grafts taken from the excised spinous process and/or laminar bone were introduced to the anterior part of the disc space and impacted with an l - shaped impactor . the final position of the cage was confirmed fluoroscopically or by check x - ray . after proper explanation of the management options and surgical technique , an informed written consent was taken , the patients were operated under general anaesthesia . patients of both groups i.e. plif and tlif , were placed in prone position and a routine posterior midline approach was used . discectomy was done bilaterally in plif but unilaterally in tlif . ( e ) t2w mri scan of that patient with degeneration and spondylolisthesis at l4/5 level . ( c ) 1 year followup x - ray anteroposterior view and ( d ) lateral view showing listhesis reduction and radiological fusion ( arrow ) spondylolytic instability managed by tlif : x - ray lumbosacral spine anteroposterior view ( a ) and lateral view ( b ) showing grade - i spondylolytic spondylolisthesis instability at l5/s1 level . ( c ) 1 year followup x - ray anteroposterior view and ( d ) lateral view showing listhesis reduction and radiological fusion ( arrow ) disc space preparation was done by unilateral distractor instrumentation with bilateral curettage in plif ; however , in tlif we used unilateral curettage by using special curved curettes to remove the end plates . in both techniques , we used single banana cage ( titanium , size 9 mm , 10 mm , or 11 mm ) which was packed with cortico - cancellous autografts and inserted in the prepared space from the surgeon 's side . the final position of the cage was confirmed fluoroscopically or by check x - ray . in both the groups , female patients of the 40 - 49 years age group were significant ( chi - square test , p < 0.05 ) . maximum cases had spondylolytic instability [ plif = 27 ( 51.92% ) , tlif = 24 ( 48.00% ) ] and l4/5 level involved most commonly [ plif = 27 ( 51.92% ) , tlif = 28 ( 56.00% ) ] in both groups . grade - ii slip [ plif = 41 ( 78.85% ) , tlif = 40 ( 80.00% ) ] had been significant ( chi - square test , p < 0.05 ) in both groups [ table 1 ] . preoperative lbp and radiculopathy ( leg pain ) was significantly present in both groups ( chi - square test , p < 0.05 ) having a highly significant improvement of their vas scores ( z test , p < 0.001 ) at 1 year . similar significant improvement of the leg pain status was achieved from 05.25 00.52 to 01.75 00.79 in the plif group and from 05.24 00.43 to 01.74 00.63 in the tlif group . the neurological status ( e.g. , motor and sensory ) of both groups also had highly significant recovery ( z test , none of the pain score , disability score , and neurological recovery had significant difference between the groups ( z test , p > 0.05 ) . although overall satisfactory outcome [ plif = 49 ( 94.23% ) , tlif = 48 ( 96.00% ) ] had no significant difference , there had been more excellent results with tlif [ 35 ( 70.00% ) ] than that of plif [ 29 ( 55.77% ) ] [ table 2 ] . demographics of the patients ( n=102 ) clinical , functional , and overall outcome of the patients ( n=102 ) assessment of radiological restoration of structural components revealed a significant ( z test , p < 0.05 ) rise of mean disc height ( mdh ) from 07.50 02.73 to 12.00 01.97 mm in the plif group and from 07.76 02.77 to 12.24 01.89 mm in the tlif group . the mean foraminal height ( mfh ) increase was recorded from 13.25 01.61 to 17.50 01.85 mm in plif and from 13.30 1.55 to 17.50 01.87 mm in the tlif group , which was also significant ( z test , p < 0.05 ) . there had been a significant slip reduction in both the groups ( plif = 90 03.85% for grade - i and 81 05.24% for grade - ii , tlif = 92 03.63% for grade - i and 84 01.50% for grade - ii ) , which had been maintained even at their last followup . the preoperative mean anterior translation ( mat ) was 09.50 0.87% at l3/4 , 09.21 1.03% at l4/5 , and 07.75 1.93% at l5/s1 in the plif group and 09.22 1.07% at l3/4 , 09.00 1.12% at l4/5 , and 08.26 1.07% at l5/s1 in the tlif group . the preoperative mean angular displacement ( mad ) was 10.75 01.58 at l3/4 , 12.40 00.70 at l4/5 , and 04.50 01.98 at l5/s1 in the plif group and 11.12 01.70 at l3/4 , 12.14 00.95 at l4/5 , and 04.20 01.10 at l5/s1 in the tlif group [ table 3 ] . but all these variables of structural restoration ( e.g. , mdh , mfh , mtll , mas , mat , mad ) had no statistical significant differences ( z test , p > 0.05 ) between the groups . postoperative dynamic films did not reveal abnormal range of movement in patients of either of the group , hence achieving radiological stability . time required for achieving signs of radiological fusion [ plif ( 17.23 02.95 weeks ) , tlif ( 16.80 03.94 weeks ) ] also had no significant difference ( z test , p > 0.05 ) [ table 4 ] . radiological outcome of the patients ( n=102 ) operative outcome and complications in both groups ( n=102 ) plif surgery took longer time ( 182 10.91 minutes ) and higher intraoperative bleeding ( 245 19 ml ) than tlif ( 165 06.63 minutes and 215 12 ml ) . the postoperative hospital stay ( 07.50 02.30 days for plif versus 05.76 01.60 days for tlif ) and the intraoperative complication [ plif ( 09.61% ) , tlif ( 02.00% ) ] and postoperative complication [ plif ( 13.47% ) , tlif ( 04.00% ) ] had no significant difference between the groups . five ( 09.62% ) cases in plif and two ( 04.00% ) cases in tlif developed pseud - arthrosis ( chi - square test , p > 0.05 ) . there were no cases of superficial wound infection in the tlif group but two ( 03.85% ) cases with plif developed superficial wound infection , which were managed conservatively [ table 4 ] . the concept of lsi has received an increased attention from the clinicians and researchers as a potential cause of chronic lbp , which has been commonly associated with spondylolisthesis and spondylolysis.2122 restoration of the segmental stability by adequate neural decompression , fusion , and stabilization helps to improve clinical symptoms and achieve normal spinal anatomy.23 failure of restoration can result in inadequate clinical improvement potentially leading to poor long term results.24 significant clinical improvement was observed in both plif and tlif techniques in different spinal disorders2526 and found to be superior due to proper neural decompression , structural restoration , and segmental stabilization that ultimately lead to improved pain , disability , and functional capability . in our study , both the techniques resulted in significant clinical and functional improvement , structural restoration , fusion , and stability but plif had been associated with higher rates of intraoperative neural complications . firstly , the diagnosis was solely depended upon the radiological findings which may have misinterpretations.27 secondly , t2-weighted kinetic mri and three - dimensional ct reconstruction had been recommended for a precise diagnosis of lsi,2728 but we could not perform these due to unavailability of expertise . thirdly , foraminal widening and fusion assessment needs ct evaluation,29 but was ignored due to patients financial constraints . in literatures , comparison of plif and tlif in spondylolisthesis and degenerative lumbar spine had been done . according to audat30 excellent outcome had been observed around 60% cases in plif and around 70% cases in tlif , which was also comparable to ours . interbody cages are used to restore the disc height , foraminal height and stabilize the affected segment.31 these parameters have significant correlation regarding structural restoration and maintenance of stability.32 the cage was inserted from the patients left side irrespective of neurological involvement but decompression was done by changing the side with contralateral involvements . in our study , we were able to achieve 06.500.55 and 05.700.75 improvement of lordotic angles in plif and tlif , that were comparable to other studies.3334 despite of no statistical difference of postoperative lordotic angles between the groups , we observed lesser degree of correction in tlif which assumed to be due to intact posterior structures which had also been observed by hsieh.31 the correction of forward slip restores sagittal alignment and physiological transmission of weight . inadequate restoration and abnormal lordosis is the primary predisposing factor for adjacent segment degeneration.37 moreover , it shifts the spinal vertical axis anteriorly to induce further degeneration and results in chronic lbp.38 the percentage of correction of slip in our study had been significant by both techniques which was comparable to yan26 [ plif ( 72.60 05.20% ) , tlif ( 72.40 05.40% ) ] . but the reports of increased chance of postoperative instability with plif due to loss of integrity of posterior structures should not be abandoned.32 interbody fusion with cage has been well accepted for its superior fusion results,3940 and has been reported to be significant by both these techniques.2526 theoretically , tlif is advantageous to plif , as it provides a full 360 fusion because of intact contralateral laminar surface that increases the surface area for new bone to grow and bridge the gap.41 we had observed signs of radiological union at 17.23 02.95 weeks in plif and 16.90 03.94 weeks in tlif . but brantigan16 compared radiological fusion with exploratory fusion and stated > 97% sensitivity , > 94% positive predictive value and > 93% accuracy of the radiological parameters . autografts had been the gold standard for achieving fusion , but there are recent reports of graft substitutes for fusion enhancement and has become a new arena of research . we placed autografts anteriorly and impacted before the introduction of cage in all the cases of plif and tlif with a theoretical background of anterior column load transmission ( 80%)39 and enhancement of fusion.43 the radiological fusion in both the groups had no statistical difference as like other studies.111230 the biomechanical concept of fusion stability is assessed postoperatively by dynamic films to determine the achievement of stability even after fusion . although posterior instrumentations enhances the stability and fusion,8 biomechanically stable spine is achieved only when solid fusion is achieved.44 we achieved the target motion of stability in all the cases of plif and tlif at 1 year according to posner et al.,3 but according to kumar et al.,45 postoperative segmental stability is achieved only when radiological motion is < 2 in dynamic films . development of pseudarthrosis is one of the most common ( range , 05 - 45% ) complications of interbody fusion.46 it can be managed conservatively and does not necessarily need revision surgery.47 pseud arthrosis was present in two ( 2.60% ) and two ( 4.60% ) patients with plif or tlif in a series of mehta10 which is comparable to our results . in different literatures , plif had been reported to be associated with more neural complications.10111226 the excess medial retraction of the dura has been blamed to be the cause of such injuries during the placement of the cage.11 on the contrary , tlif approaches the disc space through far lateral portion of the vertebral foramen , which ultimately reduces the thecal manipulation and the chances of complications . there was a case each in both groups with hardware misplacement which we identified as an instrumental default . two ( 3.85% ) cases in plif had superficial wound infection that had been managed with intravenous antibiotics following culture sensitivity ( staphylococcus aureus ) and regular dressing , and the wound was later healed with secondary intention . to conclude , both methods are effective in relieving symptoms , achieving stability and fusion , but tlif can be preferred for its shorter operative time , less blood loss , and lesser complication rates in surgical management of symptomatic lumbar instability .

to reduce an inequitable and often catastrophic reliance on out - of - pocket ( oop ) payments to finance health care,1 various forms of social health insurance ( shi ) are being introduced by a number of developing countries in response to the call of the world health organization ( who ) to move towards universal health care.2 shi schemes in developing countries commonly take the form of contributions from employees and employers linked to earnings for the formal sector , and various forms of private or community - based health insurance are potentially available for the informal sector and members of rural communities.3,4 developing countries that have recently introduced or expanded shi include china , vietnam , colombia , mexico , nigeria , ghana , south africa , and tanzania.4,5 although there is evidence that some of these schemes are having a positive impact in reducing oop payments and improving equity in access to health care,6,7 gaps in provision and coverage are common and will need to be addressed.8 such gaps can only be met by one or a combination of the following : ( 1 ) oop payments , ( 2 ) general taxation , or ( 3 ) private health insurance ( phi ) . while progressive taxation and shi systems potentially offer the most equitable means of financing health care,9 phi is a feature of almost all organization for economic co - operation and development ( oecd ) countries10 and is recognized as an important option for developing countries.11 phi can be classified under three general categories : ( 1 ) free market health insurance , ( 2 ) controlled market health insurance , and ( 3 ) medical savings accounts ( msas ) . in applications where phi is optional , it is referred to as voluntary health insurance ( vhi).12 phi can also play several different roles with regard to health care systems . in some applications , it acts as the primary source of financing , while in other countries it operates in a supplementary role to cover gaps in health care coverage . in still other countries , phi is complementary to publicly financed health care through cost - sharing arrangements . phi ( vhi ) may also be substitutive to a public system whereby subpopulations are allowed , or encouraged , to opt out of the statutory system and use comprehensive phi as an alternative.10,13 in free - market phi , although mostly regulated through legislation , actuarially fair premiums are applied whereby high - risk individuals pay higher premiums than those at low risk . in controlled - market phi , the government imposes regulations and mechanisms to equalize risk across the community by applying community ratings such that premiums are set according to criteria such as age of entry or membership of a defined community.14 msas , alternatively known as health savings accounts ( hsas ) , offer a means of providing phi cover for emergencies and a separate fund for elective treatments and oop expenditure through regular savings.12 insurance companies providing phi take various measures to manage risk associated with two phenomena : moral hazard and adverse selection.12 moral hazard refers to the behavior of the insured and potentially results in an additional quantity of potentially unnecessary health care being demanded . measures to control moral hazard include co - payments and deductibles , which vary according to the nature of premiums and cover provided . adverse selection ( of high risks ) arises from asymmetric information whereby individuals are able to purchase insurance at premiums below their actuarially fare rate . insurance companies typically address this by screening potential clients regarding preexisting conditions , age , and lifestyle characteristics.12 with this background in mind , the aim of this study is to provide an overview of the application of phi in an informative sample of different health care systems . the factors that affect the uptake of phi are also discussed with the objective of informing policymakers in developing countries regarding how phi can be part of the mix of funding arrangements that can be applied in moving towards universal health care . after reviewing health care systems according to their typology ( tax - based , shi , phi , and mixed public / phi),12 and the application of phi within them,10 the countries selected as representative case studies were : the us , the uk , the netherlands , france , australia , and latvia . latvia was included as it is a former eastern bloc country and offers some important insights regarding its use of phi since this transition . descriptions of countries health care systems were obtained from the european observatory on health systems and policies health systems in transition series,15 the latest commonwealth fund s study of international profiles of health care systems,16 and other relevant literature . pubmed was searched to locate additional studies that reported the factors that affect the uptake of phi . for each country , summary statistics on health care spending from the oecd health database17 and world bank18 were included for the following variables : total health expenditure as a percentage of gross domestic product ( gdp ) , per capita health expenditure , public health expenditure , private oop expenditure , and phi spending as a percentage of total expenditure . the summary data for each country selected are provided in table 1 and are used to augment case study descriptions in the following sections . the us is categorized as a mixed system12 as it has a large free - market phi ( vhi ) system as a primary source of financing , which operates in conjunction with tax - funded public programs for specified groups . although phi plays a major role , table 1 shows it accounts for less than half ( 35% ) of total health expenditure , while public expenditure accounts for 44.9% . although the us has the highest health expenditure in the world,16 public insurance and phi coverage is highly fragmented , with multiple private and public sources and gaps in coverage rates across the us population.16 msas were initially authorized in the us using a combination of consumer - driven health plans usually low - cost insurance for catastrophic events with high deductibles and msa accounts to fund oop expenditure and elective care . this phi arrangement postulates the prudent use of health care and thus potentially controls for moral hazard . however , users may avoid seeking treatment or utilizing preventive health care measures in preserving their savings . a modified employee - only version , the hsa , is being adopted in the us since the passing of hsa legislation in 2003 as part of the medicaid prescription drug act.12 publicly funded health care is principally delivered via the medicare and medicaid programs , although a number of other programs are available for specific sectors of the population.19 medicare is a national program that covers approximately 95% of those aged 65 years and older in addition to many disabled people receiving social security . medicaid is a state - run program , with matching federal funding , that provides health care coverage to certain vulnerable individuals or groups on low incomes . each state establishes its own eligibility criteria , range of services covered , and tariffs , within broad criteria specified by the federal government.12 however , the poorest individuals are not covered unless they belong to specified groups , such as children less than 6 years of age or pregnant women from families with incomes below 133% of the federal poverty level.12 despite these arrangements , a significant proportion of the us population have no , or inadequate , health insurance.20 in 2010 , 81 million people ( 44% of us adults ) were uninsured or under - insured ( having high medical expenditures relative to their incomes ) : this figure represents an increase of almost one - third from 2003.20 in an attempt to reduce the number of uninsured americans , the affordable care act ( aca ) was introduced in 2010 by the obama administration.20,21 the aim is to expand medicaid coverage to include individuals with incomes below 133% of the federal poverty level , and provide assistance to limit premiums for those with incomes of up to 400% of the federal poverty level.20,21 a recent study suggests that full implementation of the aca could reduce the number of underinsured by up to 70%.20 this case study indicates that the use of nonstatutory and free - market phi as a primary source of funding , with public provision for vulnerable groups , is expensive , complex , and associated with gaps in coverage . achieving universal health care the uk has a tax - based health care system that provides universal health care coverage through its national health service ( nhs ) with supplementary free - market phi ( vhi ) . heath services in the uk are largely free at the point of use within the nhs and cover qualifying residents from the cradle to the grave . it is the only option for many long - term chronic conditions as well as accident and emergency care.22 funding sources include direct and indirect taxes plus national insurance contributions . however , some services that are not covered are funded by user charges , direct oop payments , or through phi.22table 1 shows that total health expenditure is 9.6% of gdp , and public funding accounts for 81.3% of total health care spending . oop payments constitute the largest single element of private expenditure , accounting for 11.1% of total health care expenditure.22 phi in the uk is taken out by about 12% of the population16 and accounts for only 2.9% of total health expenditure ( table 1 ) . these include relatively long hospital waiting lists , limited choice of specialist provider , and potentially lower standards of hospital facilities compared with phi provision.12 it is also utilized to cover care no longer provided by the nhs such as optician services and cost sharing within dental care . in terms of those who purchase phi , data suggest that individual and corporate purchasers account for approximately equal proportions of expenditure.22 the underlying stimulus behind supplementary phi in the uk , therefore , is dissatisfaction with the public nhs , cost sharing , or in meeting gaps in provision . france is a statutory shi country that provides an example of complementary phi in order to meet gaps in social security reimbursements . health care coverage in france is now virtually universal at 99.9% as a result of the introduction of couverture maladie universelle ( cmu ) in 2000.23 while shi seeks to cover the entire population , it does not cover 100% of expenditures that are paid using fee - for - service arrangements . to cover this gap , the vast majority ( 92% ) of the population has phi ( vhi ) either through their employers or via means - tested vouchers.16,23 only 6.8% of phi is purchased by individuals.23 as can be seen in table 1 , france spends about 12% of its gdp on health care , which is one of highest percentages in the european union ( eu).19,24 most of this ( 73.2% ) is classified as social security funding , and the remainder is general taxation . oop expenditure is low in france ( 7.2% ) , and per capita spending is relatively high ( us$4691 ) . in addition to cost - sharing through coinsurance , which can be fully reimbursed by phi ( vhi ) , some co - payments associated with doctors visits , prescription drugs , and hospital treatment are not reimbursed up to an annual ceiling of 50 ( us$70 ) to avoid catastrophic payments for health care and control for moral hazard.16 to assist the poorest in society , people with a taxable income of less than 9020 per year are exempt from paying shi contributions , while those with incomes above this threshold pay 8% of their taxable income . in addition , free phi coverage is offered to people with incomes below a defined ceiling ( 7521 as of january 2010 ) ; this covers 8% of the population.23 to facilitate access to phi by people at the margin of the cmu income threshold , a voucher scheme , aide pour une complmentaire sant , was established in 2004 for people whose income is below 120% of the cmu threshold . in 2010 , the amounts available under this scheme ranged from 100 per year for people aged less than 25 years to 400 for people aged over 60 years . this scheme covers about 5.3% of the overall population.23,25 the case of france illustrates the added complexity of shi , compared with tax - based universal systems , in that individuals and the government take many additional steps to achieve financial protection in health care and the near - universal coverage that is provided . the netherlands was previously a statutory shi country that utilized substitutive phi for high income earners . since being reformed in 2006 , however , substitutive health insurance has been abolished.16 the reformed system now offers a good example of complementary and supplementary phi ( vhi ) to statutory and controlled phi operating in conjunction with an shi component to provide comprehensive coverage . as shown in table 1 , the netherlands spends about 12% of its gdp on health care and has the second highest per capita spending behind the us . seventy - five percent of health expenditure is public , with a very low percentage of oop contributions compartmentalized system with two principal components.26 the first is long - term and disability care , which is funded by statutory shi , regulated through the exceptional medical expenses act ( algemene wet bijzondere ziektekosten ; awbz ) . the second component consists of basic health insurance for short - term medical care , which is funded by compulsory and controlled phi . individuals pay a community - rated nominal premium and an income - dependent employer contribution , which is deducted directly from their wages ; a health care allowance is available for lower income groups . there is competition between private health insurers , who can negotiate to a certain extent with health care providers on the price , volume , and quality of care delivered . they are also allowed to pay dividends to shareholders , unlike traditional shi schemes which are not - for - profit.12 most of the population purchases phi ( vhi ) from the same health insurers who provide statutory coverage . this form of phi covers , for example , dentistry and extra visits to a physiotherapist . in 2009 , 91% of people who were insured under the statutory schemes also had phi ( vhi ) . it should be noted that those with vhi do not receive faster access to any type of care , nor do they have more choice of specialist or hospital.16 although health insurance is statutory , about 1% of the population ( approximately 171,000 persons ) is uninsured , mainly due to objections to insurance on religious grounds or for other reasons . such individuals can purchase an insurance policy when they require care , but risk a penalty of 130% of total nominal premiums for the time that they were uninsured.26 the system of the netherlands therefore illustrates a minor role for phi ( vhi ) with a dramatic shift from its former shi system to a reliance on compulsory and highly controlled phi for short - term care . like france , a complex set of arrangements is used to provide near - universal coverage of the population . latvia offers a particularly useful example of phi ( vhi ) in a complementary and supplementary role alongside a tax - funded statutory shi system within a former eastern bloc country . a number of fairly radical changes have occurred since achieving membership of the eu in 2004 . the central government is responsible for financing the shi system through tax revenue , and additional funding comes directly from oop payments and from phi ( vhi ) . as shown in table 1 , latvia spends the lowest among the sample countries as a share of gdp ( 6.7% ) , but this has shown a marked increase from only 3.4% of gdp in 2005.27 latvia also has the lowest per capita spending of us$718 , which reflects the relative size of its economy and population compared with other countries in the sample . the percentage of total health expenditure on phi is estimated to be 5% ( table 1 ) . the proportion of total health care expenditure accounted for by phi increased to this figure from 1.8% in 1997 and appears to have stabilized since the 2001 estimate.27 interest in phi ( vhi ) was first shown in 1996 , following the introduction of user charges , and the market has increased rapidly since then as shown in figure 1 ; using latvian currency data and 1997 prices it can be seen that premiums rose from about ls 3 million in 1997 to about ls 18 million in 2008 , while claims rose from about ls 2 million to ls 11 million over the same period . although comparative data for oop expenditure as a share of total expenditure are not available for 2007 , data for oop spending as a percentage of private expenditure show a very high figure ( 97.3% ) using 2010 figures . this compares less favorably with the uk ( 62% ) , france ( 33.1% ) , the netherlands ( 38% ) , australia ( 64% ) , and the us ( 25.2%).18 supplementary phi is used to cover health care services and/or prescription drugs which are not financed by the statutory system , such as dentistry for adults , routine health checkups needed for specific job security requirements , physiotherapy and massage , rehabilitation , and some vaccines . complementary phi ( vhi ) schemes cover only oop charges such as optician services , hearing aids , and prostheses . actuarially fair premiums are calculated according to companies tariffs , based on the purchaser s age and health status.27 the observed growth in supplementary and complementary phi ( vhi ) and oop expenditure in latvia reflects the inability of public funding to meet an increasing demand for health care services.27 australia offers an example of how incentives are utilized to encourage the uptake of controlled phi for some aspects of care ( hospital ) as an alternative to a universal tax - based system . this is a form of complementary phi,16 as users are not allowed to opt out of their tax commitments to fund the public system . as shown in table 1 , australia spends a comparatively low percentage of gdp on health at under 9% , but achieves per capita spending above other countries that spend less as a share of gdp . the health care system is mainly financed through general taxation as public spending on health is 67% , while oop expenditure is the highest in the sample at almost 20% . funding also includes a small statutory insurance levy and phi accounts for a relatively low figure of 8.2% . the public , tax - funded national health insurance scheme , medicare , provides universal access to subsidized medical services , subsidized pharmaceuticals , and free hospital treatment as a public patient . people can also take out controlled phi to cover or partially cover the financial costs of hospital treatment as private patients to facilitate quicker access to elective surgery with private patient status , or to cover or partially cover dental and other allied health services.16,28 in 2000 , the government introduced lifetime health cover ( lhc ) , an initiative designed to encourage people to take up phi at an early age and maintain their cover throughout their lives.29 under lhc , early uptake of phi is rewarded with lower premiums ( in recognition of the fact that younger people are less likely to require hospital treatment than older people ) , and premiums are eligible for age - related tax rebates of 30%40% , which increase with age.29,30 insurers can not refuse coverage , and premiums are community - rated to equalize risk.29 evidence suggests that these initiatives have increased uptake of phi by 50% , and decreased the average age of purchasers.31 some commentators suggest , however , that the penalty of 2% per year for delaying insurance , introduced as part of the lhc plan , is too low to be effective.32 findings suggest that some insurers flouted community rating systems by screening older consumers into more expensive plans . the lhc may not , therefore , have increased health care access via phi as much as intended . in spite of these mixed messages , figure 2 provides an informative representation of estimated hospital coverage in australia , expressed as a percentage of the population , assuming that the current policy of lhc and a 30% tax rebate on private health insurance premiums is either maintained or withdrawn . as can be seen , the projected percentage if lhc is maintained is an approximately 20% higher uptake , which is sustained over the period 20042042 . the case study of australia is informative as it illustrates that some governments seek to shift health care financing away from general taxation . the study used for figure 2 has estimated that if lhc and the tax rebate were withdrawn , the decreased use of phi would increase projected public spending on health care by 0.5% of gdp by 2042.30 phi with sufficient controls and risk equalization , therefore , provides a potentially more equitable form of private health care in comparison with free - market phi . mixed system12 as it has a large free - market phi ( vhi ) system as a primary source of financing , which operates in conjunction with tax - funded public programs for specified groups . although phi plays a major role , table 1 shows it accounts for less than half ( 35% ) of total health expenditure , while public expenditure accounts for 44.9% . although the us has the highest health expenditure in the world,16 public insurance and phi coverage is highly fragmented , with multiple private and public sources and gaps in coverage rates across the us population.16 msas were initially authorized in the us using a combination of consumer - driven health plans usually low - cost insurance for catastrophic events with high deductibles and msa accounts to fund oop expenditure and elective care . this phi arrangement postulates the prudent use of health care and thus potentially controls for moral hazard . however , users may avoid seeking treatment or utilizing preventive health care measures in preserving their savings . a modified employee - only version , the hsa , is being adopted in the us since the passing of hsa legislation in 2003 as part of the medicaid prescription drug act.12 publicly funded health care is principally delivered via the medicare and medicaid programs , although a number of other programs are available for specific sectors of the population.19 medicare is a national program that covers approximately 95% of those aged 65 years and older in addition to many disabled people receiving social security . medicaid is a state - run program , with matching federal funding , that provides health care coverage to certain vulnerable individuals or groups on low incomes . each state establishes its own eligibility criteria , range of services covered , and tariffs , within broad criteria specified by the federal government.12 however , the poorest individuals are not covered unless they belong to specified groups , such as children less than 6 years of age or pregnant women from families with incomes below 133% of the federal poverty level.12 despite these arrangements , a significant proportion of the us population have no , or inadequate , health insurance.20 in 2010 , 81 million people ( 44% of us adults ) were uninsured or under - insured ( having high medical expenditures relative to their incomes ) : this figure represents an increase of almost one - third from 2003.20 in an attempt to reduce the number of uninsured americans , the affordable care act ( aca ) was introduced in 2010 by the obama administration.20,21 the aim is to expand medicaid coverage to include individuals with incomes below 133% of the federal poverty level , and provide assistance to limit premiums for those with incomes of up to 400% of the federal poverty level.20,21 a recent study suggests that full implementation of the aca could reduce the number of underinsured by up to 70%.20 this case study indicates that the use of nonstatutory and free - market phi as a primary source of funding , with public provision for vulnerable groups , is expensive , complex , and associated with gaps in coverage . achieving universal health care is being addressed through the aca in the us , but substantial challenges remain . the uk has a tax - based health care system that provides universal health care coverage through its national health service ( nhs ) with supplementary free - market phi ( vhi ) . heath services in the uk are largely free at the point of use within the nhs and cover qualifying residents from the cradle to the grave . it is the only option for many long - term chronic conditions as well as accident and emergency care.22 funding sources include direct and indirect taxes plus national insurance contributions . however , some services that are not covered are funded by user charges , direct oop payments , or through phi.22table 1 shows that total health expenditure is 9.6% of gdp , and public funding accounts for 81.3% of total health care spending . oop payments constitute the largest single element of private expenditure , accounting for 11.1% of total health care expenditure.22 phi in the uk is taken out by about 12% of the population16 and accounts for only 2.9% of total health expenditure ( table 1 ) . these include relatively long hospital waiting lists , limited choice of specialist provider , and potentially lower standards of hospital facilities compared with phi provision.12 it is also utilized to cover care no longer provided by the nhs such as optician services and cost sharing within dental care . in terms of those who purchase phi , data suggest that individual and corporate purchasers account for approximately equal proportions of expenditure.22 the underlying stimulus behind supplementary phi in the uk , therefore , is dissatisfaction with the public nhs , cost sharing , or in meeting gaps in provision . france is a statutory shi country that provides an example of complementary phi in order to meet gaps in social security reimbursements . health care coverage in france is now virtually universal at 99.9% as a result of the introduction of couverture maladie universelle ( cmu ) in 2000.23 while shi seeks to cover the entire population , it does not cover 100% of expenditures that are paid using fee - for - service arrangements . to cover this gap , the vast majority ( 92% ) of the population has phi ( vhi ) either through their employers or via means - tested vouchers.16,23 only 6.8% of phi is purchased by individuals.23 as can be seen in table 1 , france spends about 12% of its gdp on health care , which is one of highest percentages in the european union ( eu).19,24 most of this ( 73.2% ) is classified as social security funding , and oop expenditure is low in france ( 7.2% ) , and per capita spending is relatively high ( us$4691 ) . in addition to cost - sharing through coinsurance , which can be fully reimbursed by phi ( vhi ) , some co - payments associated with doctors visits , prescription drugs , and hospital treatment are not reimbursed up to an annual ceiling of 50 ( us$70 ) to avoid catastrophic payments for health care and control for moral hazard.16 to assist the poorest in society , people with a taxable income of less than 9020 per year are exempt from paying shi contributions , while those with incomes above this threshold pay 8% of their taxable income . in addition , free phi coverage is offered to people with incomes below a defined ceiling ( 7521 as of january 2010 ) ; this covers 8% of the population.23 to facilitate access to phi by people at the margin of the cmu income threshold , a voucher scheme , aide pour une complmentaire sant , was established in 2004 for people whose income is below 120% of the cmu threshold . in 2010 , the amounts available under this scheme ranged from 100 per year for people aged less than 25 years to 400 for people aged over 60 years . this scheme covers about 5.3% of the overall population.23,25 the case of france illustrates the added complexity of shi , compared with tax - based universal systems , in that individuals and the government take many additional steps to achieve financial protection in health care and the near - universal coverage that is provided . the netherlands was previously a statutory shi country that utilized substitutive phi for high income earners . since being reformed in 2006 , however , substitutive health insurance has been abolished.16 the reformed system now offers a good example of complementary and supplementary phi ( vhi ) to statutory and controlled phi operating in conjunction with an shi component to provide comprehensive coverage . as shown in table 1 , the netherlands spends about 12% of its gdp on health care and has the second highest per capita spending behind the us . seventy - five percent of health expenditure is public , with a very low percentage of oop contributions compartmentalized system with two principal components.26 the first is long - term and disability care , which is funded by statutory shi , regulated through the exceptional medical expenses act ( algemene wet bijzondere ziektekosten ; awbz ) . the second component consists of basic health insurance for short - term medical care , which is funded by compulsory and controlled phi . individuals pay a community - rated nominal premium and an income - dependent employer contribution , which is deducted directly from their wages ; a health care allowance is available for lower income groups . there is competition between private health insurers , who can negotiate to a certain extent with health care providers on the price , volume , and quality of care delivered . they are also allowed to pay dividends to shareholders , unlike traditional shi schemes which are not - for - profit.12 most of the population purchases phi ( vhi ) from the same health insurers who provide statutory coverage . this form of phi covers , for example , dentistry and extra visits to a physiotherapist . in 2009 , 91% of people who were insured under the statutory schemes also had phi ( vhi ) . it should be noted that those with vhi do not receive faster access to any type of care , nor do they have more choice of specialist or hospital.16 although health insurance is statutory , about 1% of the population ( approximately 171,000 persons ) is uninsured , mainly due to objections to insurance on religious grounds or for other reasons . such individuals can purchase an insurance policy when they require care , but risk a penalty of 130% of total nominal premiums for the time that they were uninsured.26 the system of the netherlands therefore illustrates a minor role for phi ( vhi ) with a dramatic shift from its former shi system to a reliance on compulsory and highly controlled phi for short - term care . like france , a complex set of arrangements is used to provide near - universal coverage of the population . latvia offers a particularly useful example of phi ( vhi ) in a complementary and supplementary role alongside a tax - funded statutory shi system within a former eastern bloc country . a number of fairly radical changes have occurred since achieving membership of the eu in 2004 . the central government is responsible for financing the shi system through tax revenue , and additional funding comes directly from oop payments and from phi ( vhi ) . as shown in table 1 , latvia spends the lowest among the sample countries as a share of gdp ( 6.7% ) , but this has shown a marked increase from only 3.4% of gdp in 2005.27 latvia also has the lowest per capita spending of us$718 , which reflects the relative size of its economy and population compared with other countries in the sample . the percentage of total health expenditure on phi is estimated to be 5% ( table 1 ) . the proportion of total health care expenditure accounted for by phi increased to this figure from 1.8% in 1997 and appears to have stabilized since the 2001 estimate.27 interest in phi ( vhi ) was first shown in 1996 , following the introduction of user charges , and the market has increased rapidly since then as shown in figure 1 ; using latvian currency data and 1997 prices it can be seen that premiums rose from about ls 3 million in 1997 to about ls 18 million in 2008 , while claims rose from about ls 2 million to ls 11 million over the same period . although comparative data for oop expenditure as a share of total expenditure are not available for 2007 , data for oop spending as a percentage of private expenditure show a very high figure ( 97.3% ) using 2010 figures . this compares less favorably with the uk ( 62% ) , france ( 33.1% ) , the netherlands ( 38% ) , australia ( 64% ) , and the us ( 25.2%).18 supplementary phi is used to cover health care services and/or prescription drugs which are not financed by the statutory system , such as dentistry for adults , routine health checkups needed for specific job security requirements , physiotherapy and massage , rehabilitation , and some vaccines . complementary phi ( vhi ) schemes cover only oop charges such as optician services , hearing aids , and prostheses . actuarially fair premiums are calculated according to companies tariffs , based on the purchaser s age and health status.27 the observed growth in supplementary and complementary phi ( vhi ) and oop expenditure in latvia reflects the inability of public funding to meet an increasing demand for health care services.27 australia offers an example of how incentives are utilized to encourage the uptake of controlled phi for some aspects of care ( hospital ) as an alternative to a universal tax - based system . this is a form of complementary phi,16 as users are not allowed to opt out of their tax commitments to fund the public system . as shown in table 1 , australia spends a comparatively low percentage of gdp on health at under 9% , but achieves per capita spending above other countries that spend less as a share of gdp . the health care system is mainly financed through general taxation as public spending on health is 67% , while oop expenditure is the highest in the sample at almost 20% . funding also includes a small statutory insurance levy and phi accounts for a relatively low figure of 8.2% . the public , tax - funded national health insurance scheme , medicare , provides universal access to subsidized medical services , subsidized pharmaceuticals , and free hospital treatment as a public patient . people can also take out controlled phi to cover or partially cover the financial costs of hospital treatment as private patients to facilitate quicker access to elective surgery with private patient status , or to cover or partially cover dental and other allied health services.16,28 in 2000 , the government introduced lifetime health cover ( lhc ) , an initiative designed to encourage people to take up phi at an early age and maintain their cover throughout their lives.29 under lhc , early uptake of phi is rewarded with lower premiums ( in recognition of the fact that younger people are less likely to require hospital treatment than older people ) , and premiums are eligible for age - related tax rebates of 30%40% , which increase with age.29,30 insurers can not refuse coverage , and premiums are community - rated to equalize risk.29 evidence suggests that these initiatives have increased uptake of phi by 50% , and decreased the average age of purchasers.31 some commentators suggest , however , that the penalty of 2% per year for delaying insurance , introduced as part of the lhc plan , is too low to be effective.32 findings suggest that some insurers flouted community rating systems by screening older consumers into more expensive plans . the lhc may not , therefore , have increased health care access via phi as much as intended . in spite of these mixed messages , figure 2 provides an informative representation of estimated hospital coverage in australia , expressed as a percentage of the population , assuming that the current policy of lhc and a 30% tax rebate on private health insurance premiums is either maintained or withdrawn . as can be seen , the projected percentage if lhc is maintained is an approximately 20% higher uptake , which is sustained over the period 20042042 . the case study of australia is informative as it illustrates that some governments seek to shift health care financing away from general taxation . the study used for figure 2 has estimated that if lhc and the tax rebate were withdrawn , the decreased use of phi would increase projected public spending on health care by 0.5% of gdp by 2042.30 phi with sufficient controls and risk equalization , therefore , provides a potentially more equitable form of private health care in comparison with free - market phi . the case studies presented in this paper have illustrated the wide range of applications and roles that phi plays within a representative sample of countries that utilize , between them , the principal categories of health care systems according to their methods of financing health care . for countries considering the potential of phi in improving equity in financing and access to health care , and addressing gaps in provision , it would be beneficial to consider the factors that affect its uptake and impact on equity , as outlined in the following . the first points of relevance are whether or not phi is statutory or voluntary , and whether it is free market or controlled with risk equalization . in statutory phi with risk equalization as found in the netherlands , uptake is high but not 100% , and defaulters face a penalty if they purchase phi only when needed or if they abstain . in australia , positive and negative incentives exist to encourage uptake of controlled but voluntary phi , with a focus on equity through measures such as community rating with risk equalization . in the case of the us , however , phi ( vhi ) plays a primary role alongside medicare or medicaid , with a large proportion of the population uninsured or underinsured in the face of actuarially fair premium assessments . achieving universal coverage under these conditions is a challenge which the obama administration is seeking to address.19,20 the uptake of phi is clearly also influenced by health care system typology . the case study of the uk illustrates that long waiting times to access secondary care has been a significant cause of user dissatisfaction with its nhs.33,34 similarly , the increase in phi in latvia during the last decade can largely be attributed to the inability of public funding to meet a rising demand for services coupled with the introduction of regressive oop payments.27 in australia , evidence also confirms that long waiting times for elective procedures has increased the probability of phi uptake.35 in statutory shi and phi systems the issue is not usually long waiting lists or a perceived need to use alternative providers , but rather with gaps in reimbursement or services , as illustrated by france and the netherlands . analyses of these countries indicate the complexity of responses using either taxation and/or phi in order to achieve universal or near - universal coverage of the population . phi ( vhi ) has been predominantly purchased by high - income groups,10,13,36 which can lead to inequalities in access.10,13,36 in the us , evidence confirms that low income is an important factor contributing to under - insurance,20 and conversely , higher incomes are associated with greater uptake of phi.37 in a uk study covering the period from 1997 to 2000 , income was a principal determinant of uptake of phi,36 and there is evidence to suggest this remains the case.22 evidence regarding access to phi in france also indicates a correlation with income , particularly affecting the elderly.23 socioeconomic status has also been shown to be an important factor determining phi uptake in latvia.27 similarly , an australian study found a strong association between high rates of phi cover and high socioeconomic status.38 proxies of lower income , such as lower educational achievement or presence of mental health problems , have also found to be associated with lower uptake of phi.37,39,40 there is some evidence , however , that phi uptake in lower income groups may increase in the future if initiatives , as exemplified in the case of australia , are actively adopted.41 age has also been shown to be a factor affecting uptake of phi . in the uk , for example , age was identified as a major determinant of phi uptake and may reflect both increasing disposable income and greater concern about acquiring timely access to health care among older working people.36 as a result , older patients in the uk often pay full user charges to obtain access to health care , with one study finding that 30% of people aged over 75 years pay oop user charges for private operations in england and wales.42 in contrast , the australian 45 and up study reported higher uptake of private insurance among younger individuals incentivized by tax and lifetime cover incentives.43 in the us , one study found a graying of the phi market as older , high - income users increasingly predominate.44 however , older people may find free - marked phi difficult to obtain because actuarially fair premiums are higher than in younger people . msas , as a form of incentivized phi , have been particularly popular in asian countries with a strong savings ethos , such as singapore , but their replication in other settings with very different health care systems and socioeconomic characteristics has been shown to be problematic.45 although an in - depth analysis of the impact of phi on equity is beyond the scope of the present study , it is clearly an important consideration for policymakers . concerning equity in financing , the seminal work of wagstaff et al9,46 offers some useful insights . their analysis , although relating to the 1980s and 1990s , showed the uk to have , overall , a progressive system of financing , with phi having a progressive effect ; the us exhibited an overall regressive effect , and free - market phi was shown to be regressive ; france was progressive overall , with phi having a regressive effect ; the shi systems with substitutive phi ( such as the netherlands at that stage ) were shown to be regressive overall , but substitutive phi itself was progressive . although no similar data are available for australia , the use of controlled phi with community - rating , life - time cover premiums mean the application of phi is likely to be progressive , but the financial cost to the government of non - means tested rebates may well dampen this effect estimated in 2011 to be a$4.5 billion ( us$4.7 billion ) annually.16 in terms of equity in access to health care , it should be noted that the benefits provided by some forms of supplementary and complementary phi accrue exclusively to those who are covered by it . furthermore , recent mortality data in the usa suggest that a lack of access to quality medical care appears to be responsible for widening the mortality gap between groups of higher education ( a proxy for income ) and lower education.47 if access to quality care , in terms of early diagnosis and appropriate treatment , is generally available only through phi coverage , then this would suggest expansion of phi to improve the overall quality of health care may be a productive development . it is therefore possible to conclude that phi can have either a positive or negative impact on equity depending on its application . however , the principal benefit of any form of phi is protection against financial barriers to health care . well run schemes may thus reduce oop expenditures , which place a disproportionate burden on low - income individuals and therefore especially applicable to developing countries.11 reducing a reliance on oop expenditures is the major hurdle to be overcome in developing countries as many have oop expenditures three or four times that of the highest country in the sample used in the present study . the who has quantified this impact in its 2010 report ; oop expenditures of 60%70% of total health expenditure result in 2% of the population becoming impoverished and 3.5% facing financial catastrophe.2 moreover , informal economies , unemployment , self - employment , and aging populations with diminishing workforces may limit the available revenue from social insurance contributions and tax - based funding.13 most of these factors and also the cost of premiums have a major limiting impact in developing countries , which often have a majority of the population living in abject poverty and are striving under many pressures to achieve universal health care.48 in terms of the limitations of our study , we acknowledge that forming strong conclusions from case studies has some shortfalls and we have intentionally selected specific countries with the aim of illustrating the common applications of phi . as a financing mechanism , phi also has a number of other components that we have not addressed . these include premium costs and collection methods , the impact on equity of variations in benefits packages , the payment mechanism used to reimburse providers , and the impact of different financing methods on health outcomes . in developing countries , the impact on equity regarding external donor funding to support weak health care financing may also be substantial as it is reported to be up to 80% in some countries.2 too strong a reliance on donor funding is known to have adverse effects such as dampening the supply - side efforts of governments in financing health care.2 they should seek to increase internal and progressive methods of funding through general taxation and shi where feasible , but also consider the role of phi for specific groups to relieve pressure on public systems , as illustrated in the present case studies . these issues could beneficially form the basis of future studies in this area of analysis . this study has illustrated the common applications of phi in a representative sample of countries and how it has the potential to act as a primary source of funding or fill gaps in shi , statutory phi , or tax - based systems . free market phi ( vhi ) as a primary source is associated with large gaps in uptake and high overall cost . in countries where the public health care system provides universal access to a broad range of services , free - market phi has a predominantly supplemental role in facilitating faster access to health care or in meeting gaps in services . in other countries , phi is complementary to the public system , covering services or reimbursements that are partially funded by the public system . in other countries , controlled - market phi is mandatory but requires risk - equalization mechanisms and a high degree of government intervention to ensure equity , with positive and negative incentives being effective in encouraging its uptake . the uptake of phi depends on many factors that include the makeup and performance of the public health care system , socioeconomic factors such as income , education , and age , and incentives from the government . the effects of phi on equity in financing and access may be either positive or negative depending on the format and role of phi . while developing countries face major challenges in attaining the goal of universal health care , phi is playing a role in many emerging systems and is likely to develop further into the future under a mix of financing arrangements . the findings of this study are intended to be helpful to policymakers as they determine an appropriate mix of arrangements to finance health care and move towards universal coverage .

backgroundsocial and national health insurance schemes are being introduced in many developing countries in moving towards universal health care . however , gaps in coverage are common and can only be met by out - of - pocket payments , general taxation , or private health insurance ( phi ) . this study provides an overview of phi in different health care systems and discusses factors that affect its uptake and equity.methodsa representative sample of countries was identified ( united states , united kingdom , the netherlands , france , australia , and latvia ) that illustrates the principal forms and roles of phi . literature describing each country s health care system was used to summarize how phi is utilized and the factors that affect its uptake and equity.resultsin the united states , phi is a primary source of funding in conjunction with tax - based programs to support vulnerable groups ; in the uk and latvia , phi is used in a supplementary role to universal tax - based systems ; in france and latvia , complementary phi is utilized to cover gaps in public funding ; in the netherlands , phi is supplementary to statutory private and social health insurance ; in australia , the government incentivizes the uptake of complementary phi through tax rebates and penalties . the uptake of phi is influenced by age , income , education , health care system typology , and the incentives or disincentives applied by governments . the effect on equity can either be positive or negative depending on the type of phi adopted and its role within the wider health care system.conclusionphi has many manifestations depending on the type of health care system used and its role within that system . this study has illustrated its common applications and the factors that affect its uptake and equity in different health care systems . the results are anticipated to be helpful in informing how developing countries may utilize phi to meet the aim of achieving universal health care .

Introduction Methods Results The United States The United Kingdom France The Netherlands Latvia Australia Discussion Conclusion

to reduce an inequitable and often catastrophic reliance on out - of - pocket ( oop ) payments to finance health care,1 various forms of social health insurance ( shi ) are being introduced by a number of developing countries in response to the call of the world health organization ( who ) to move towards universal health care.2 shi schemes in developing countries commonly take the form of contributions from employees and employers linked to earnings for the formal sector , and various forms of private or community - based health insurance are potentially available for the informal sector and members of rural communities.3,4 developing countries that have recently introduced or expanded shi include china , vietnam , colombia , mexico , nigeria , ghana , south africa , and tanzania.4,5 although there is evidence that some of these schemes are having a positive impact in reducing oop payments and improving equity in access to health care,6,7 gaps in provision and coverage are common and will need to be addressed.8 such gaps can only be met by one or a combination of the following : ( 1 ) oop payments , ( 2 ) general taxation , or ( 3 ) private health insurance ( phi ) . in some applications , it acts as the primary source of financing , while in other countries it operates in a supplementary role to cover gaps in health care coverage . insurance companies typically address this by screening potential clients regarding preexisting conditions , age , and lifestyle characteristics.12 with this background in mind , the aim of this study is to provide an overview of the application of phi in an informative sample of different health care systems . the factors that affect the uptake of phi are also discussed with the objective of informing policymakers in developing countries regarding how phi can be part of the mix of funding arrangements that can be applied in moving towards universal health care . after reviewing health care systems according to their typology ( tax - based , shi , phi , and mixed public / phi),12 and the application of phi within them,10 the countries selected as representative case studies were : the us , the uk , the netherlands , france , australia , and latvia . pubmed was searched to locate additional studies that reported the factors that affect the uptake of phi . the us is categorized as a mixed system12 as it has a large free - market phi ( vhi ) system as a primary source of financing , which operates in conjunction with tax - funded public programs for specified groups . each state establishes its own eligibility criteria , range of services covered , and tariffs , within broad criteria specified by the federal government.12 however , the poorest individuals are not covered unless they belong to specified groups , such as children less than 6 years of age or pregnant women from families with incomes below 133% of the federal poverty level.12 despite these arrangements , a significant proportion of the us population have no , or inadequate , health insurance.20 in 2010 , 81 million people ( 44% of us adults ) were uninsured or under - insured ( having high medical expenditures relative to their incomes ) : this figure represents an increase of almost one - third from 2003.20 in an attempt to reduce the number of uninsured americans , the affordable care act ( aca ) was introduced in 2010 by the obama administration.20,21 the aim is to expand medicaid coverage to include individuals with incomes below 133% of the federal poverty level , and provide assistance to limit premiums for those with incomes of up to 400% of the federal poverty level.20,21 a recent study suggests that full implementation of the aca could reduce the number of underinsured by up to 70%.20 this case study indicates that the use of nonstatutory and free - market phi as a primary source of funding , with public provision for vulnerable groups , is expensive , complex , and associated with gaps in coverage . achieving universal health care the uk has a tax - based health care system that provides universal health care coverage through its national health service ( nhs ) with supplementary free - market phi ( vhi ) . however , some services that are not covered are funded by user charges , direct oop payments , or through phi.22table 1 shows that total health expenditure is 9.6% of gdp , and public funding accounts for 81.3% of total health care spending . france is a statutory shi country that provides an example of complementary phi in order to meet gaps in social security reimbursements . to cover this gap , the vast majority ( 92% ) of the population has phi ( vhi ) either through their employers or via means - tested vouchers.16,23 only 6.8% of phi is purchased by individuals.23 as can be seen in table 1 , france spends about 12% of its gdp on health care , which is one of highest percentages in the european union ( eu).19,24 most of this ( 73.2% ) is classified as social security funding , and the remainder is general taxation . this scheme covers about 5.3% of the overall population.23,25 the case of france illustrates the added complexity of shi , compared with tax - based universal systems , in that individuals and the government take many additional steps to achieve financial protection in health care and the near - universal coverage that is provided . since being reformed in 2006 , however , substitutive health insurance has been abolished.16 the reformed system now offers a good example of complementary and supplementary phi ( vhi ) to statutory and controlled phi operating in conjunction with an shi component to provide comprehensive coverage . this compares less favorably with the uk ( 62% ) , france ( 33.1% ) , the netherlands ( 38% ) , australia ( 64% ) , and the us ( 25.2%).18 supplementary phi is used to cover health care services and/or prescription drugs which are not financed by the statutory system , such as dentistry for adults , routine health checkups needed for specific job security requirements , physiotherapy and massage , rehabilitation , and some vaccines . actuarially fair premiums are calculated according to companies tariffs , based on the purchaser s age and health status.27 the observed growth in supplementary and complementary phi ( vhi ) and oop expenditure in latvia reflects the inability of public funding to meet an increasing demand for health care services.27 australia offers an example of how incentives are utilized to encourage the uptake of controlled phi for some aspects of care ( hospital ) as an alternative to a universal tax - based system . people can also take out controlled phi to cover or partially cover the financial costs of hospital treatment as private patients to facilitate quicker access to elective surgery with private patient status , or to cover or partially cover dental and other allied health services.16,28 in 2000 , the government introduced lifetime health cover ( lhc ) , an initiative designed to encourage people to take up phi at an early age and maintain their cover throughout their lives.29 under lhc , early uptake of phi is rewarded with lower premiums ( in recognition of the fact that younger people are less likely to require hospital treatment than older people ) , and premiums are eligible for age - related tax rebates of 30%40% , which increase with age.29,30 insurers can not refuse coverage , and premiums are community - rated to equalize risk.29 evidence suggests that these initiatives have increased uptake of phi by 50% , and decreased the average age of purchasers.31 some commentators suggest , however , that the penalty of 2% per year for delaying insurance , introduced as part of the lhc plan , is too low to be effective.32 findings suggest that some insurers flouted community rating systems by screening older consumers into more expensive plans . mixed system12 as it has a large free - market phi ( vhi ) system as a primary source of financing , which operates in conjunction with tax - funded public programs for specified groups . each state establishes its own eligibility criteria , range of services covered , and tariffs , within broad criteria specified by the federal government.12 however , the poorest individuals are not covered unless they belong to specified groups , such as children less than 6 years of age or pregnant women from families with incomes below 133% of the federal poverty level.12 despite these arrangements , a significant proportion of the us population have no , or inadequate , health insurance.20 in 2010 , 81 million people ( 44% of us adults ) were uninsured or under - insured ( having high medical expenditures relative to their incomes ) : this figure represents an increase of almost one - third from 2003.20 in an attempt to reduce the number of uninsured americans , the affordable care act ( aca ) was introduced in 2010 by the obama administration.20,21 the aim is to expand medicaid coverage to include individuals with incomes below 133% of the federal poverty level , and provide assistance to limit premiums for those with incomes of up to 400% of the federal poverty level.20,21 a recent study suggests that full implementation of the aca could reduce the number of underinsured by up to 70%.20 this case study indicates that the use of nonstatutory and free - market phi as a primary source of funding , with public provision for vulnerable groups , is expensive , complex , and associated with gaps in coverage . the uk has a tax - based health care system that provides universal health care coverage through its national health service ( nhs ) with supplementary free - market phi ( vhi ) . however , some services that are not covered are funded by user charges , direct oop payments , or through phi.22table 1 shows that total health expenditure is 9.6% of gdp , and public funding accounts for 81.3% of total health care spending . france is a statutory shi country that provides an example of complementary phi in order to meet gaps in social security reimbursements . to cover this gap , the vast majority ( 92% ) of the population has phi ( vhi ) either through their employers or via means - tested vouchers.16,23 only 6.8% of phi is purchased by individuals.23 as can be seen in table 1 , france spends about 12% of its gdp on health care , which is one of highest percentages in the european union ( eu).19,24 most of this ( 73.2% ) is classified as social security funding , and oop expenditure is low in france ( 7.2% ) , and per capita spending is relatively high ( us$4691 ) . this scheme covers about 5.3% of the overall population.23,25 the case of france illustrates the added complexity of shi , compared with tax - based universal systems , in that individuals and the government take many additional steps to achieve financial protection in health care and the near - universal coverage that is provided . this compares less favorably with the uk ( 62% ) , france ( 33.1% ) , the netherlands ( 38% ) , australia ( 64% ) , and the us ( 25.2%).18 supplementary phi is used to cover health care services and/or prescription drugs which are not financed by the statutory system , such as dentistry for adults , routine health checkups needed for specific job security requirements , physiotherapy and massage , rehabilitation , and some vaccines . actuarially fair premiums are calculated according to companies tariffs , based on the purchaser s age and health status.27 the observed growth in supplementary and complementary phi ( vhi ) and oop expenditure in latvia reflects the inability of public funding to meet an increasing demand for health care services.27 australia offers an example of how incentives are utilized to encourage the uptake of controlled phi for some aspects of care ( hospital ) as an alternative to a universal tax - based system . people can also take out controlled phi to cover or partially cover the financial costs of hospital treatment as private patients to facilitate quicker access to elective surgery with private patient status , or to cover or partially cover dental and other allied health services.16,28 in 2000 , the government introduced lifetime health cover ( lhc ) , an initiative designed to encourage people to take up phi at an early age and maintain their cover throughout their lives.29 under lhc , early uptake of phi is rewarded with lower premiums ( in recognition of the fact that younger people are less likely to require hospital treatment than older people ) , and premiums are eligible for age - related tax rebates of 30%40% , which increase with age.29,30 insurers can not refuse coverage , and premiums are community - rated to equalize risk.29 evidence suggests that these initiatives have increased uptake of phi by 50% , and decreased the average age of purchasers.31 some commentators suggest , however , that the penalty of 2% per year for delaying insurance , introduced as part of the lhc plan , is too low to be effective.32 findings suggest that some insurers flouted community rating systems by screening older consumers into more expensive plans . the case studies presented in this paper have illustrated the wide range of applications and roles that phi plays within a representative sample of countries that utilize , between them , the principal categories of health care systems according to their methods of financing health care . for countries considering the potential of phi in improving equity in financing and access to health care , and addressing gaps in provision , it would be beneficial to consider the factors that affect its uptake and impact on equity , as outlined in the following . achieving universal coverage under these conditions is a challenge which the obama administration is seeking to address.19,20 the uptake of phi is clearly also influenced by health care system typology . the case study of the uk illustrates that long waiting times to access secondary care has been a significant cause of user dissatisfaction with its nhs.33,34 similarly , the increase in phi in latvia during the last decade can largely be attributed to the inability of public funding to meet a rising demand for services coupled with the introduction of regressive oop payments.27 in australia , evidence also confirms that long waiting times for elective procedures has increased the probability of phi uptake.35 in statutory shi and phi systems the issue is not usually long waiting lists or a perceived need to use alternative providers , but rather with gaps in reimbursement or services , as illustrated by france and the netherlands . phi ( vhi ) has been predominantly purchased by high - income groups,10,13,36 which can lead to inequalities in access.10,13,36 in the us , evidence confirms that low income is an important factor contributing to under - insurance,20 and conversely , higher incomes are associated with greater uptake of phi.37 in a uk study covering the period from 1997 to 2000 , income was a principal determinant of uptake of phi,36 and there is evidence to suggest this remains the case.22 evidence regarding access to phi in france also indicates a correlation with income , particularly affecting the elderly.23 socioeconomic status has also been shown to be an important factor determining phi uptake in latvia.27 similarly , an australian study found a strong association between high rates of phi cover and high socioeconomic status.38 proxies of lower income , such as lower educational achievement or presence of mental health problems , have also found to be associated with lower uptake of phi.37,39,40 there is some evidence , however , that phi uptake in lower income groups may increase in the future if initiatives , as exemplified in the case of australia , are actively adopted.41 age has also been shown to be a factor affecting uptake of phi . in the uk , for example , age was identified as a major determinant of phi uptake and may reflect both increasing disposable income and greater concern about acquiring timely access to health care among older working people.36 as a result , older patients in the uk often pay full user charges to obtain access to health care , with one study finding that 30% of people aged over 75 years pay oop user charges for private operations in england and wales.42 in contrast , the australian 45 and up study reported higher uptake of private insurance among younger individuals incentivized by tax and lifetime cover incentives.43 in the us , one study found a graying of the phi market as older , high - income users increasingly predominate.44 however , older people may find free - marked phi difficult to obtain because actuarially fair premiums are higher than in younger people . msas , as a form of incentivized phi , have been particularly popular in asian countries with a strong savings ethos , such as singapore , but their replication in other settings with very different health care systems and socioeconomic characteristics has been shown to be problematic.45 although an in - depth analysis of the impact of phi on equity is beyond the scope of the present study , it is clearly an important consideration for policymakers . although no similar data are available for australia , the use of controlled phi with community - rating , life - time cover premiums mean the application of phi is likely to be progressive , but the financial cost to the government of non - means tested rebates may well dampen this effect estimated in 2011 to be a$4.5 billion ( us$4.7 billion ) annually.16 in terms of equity in access to health care , it should be noted that the benefits provided by some forms of supplementary and complementary phi accrue exclusively to those who are covered by it . furthermore , recent mortality data in the usa suggest that a lack of access to quality medical care appears to be responsible for widening the mortality gap between groups of higher education ( a proxy for income ) and lower education.47 if access to quality care , in terms of early diagnosis and appropriate treatment , is generally available only through phi coverage , then this would suggest expansion of phi to improve the overall quality of health care may be a productive development . the who has quantified this impact in its 2010 report ; oop expenditures of 60%70% of total health expenditure result in 2% of the population becoming impoverished and 3.5% facing financial catastrophe.2 moreover , informal economies , unemployment , self - employment , and aging populations with diminishing workforces may limit the available revenue from social insurance contributions and tax - based funding.13 most of these factors and also the cost of premiums have a major limiting impact in developing countries , which often have a majority of the population living in abject poverty and are striving under many pressures to achieve universal health care.48 in terms of the limitations of our study , we acknowledge that forming strong conclusions from case studies has some shortfalls and we have intentionally selected specific countries with the aim of illustrating the common applications of phi . in developing countries , the impact on equity regarding external donor funding to support weak health care financing may also be substantial as it is reported to be up to 80% in some countries.2 too strong a reliance on donor funding is known to have adverse effects such as dampening the supply - side efforts of governments in financing health care.2 they should seek to increase internal and progressive methods of funding through general taxation and shi where feasible , but also consider the role of phi for specific groups to relieve pressure on public systems , as illustrated in the present case studies . this study has illustrated the common applications of phi in a representative sample of countries and how it has the potential to act as a primary source of funding or fill gaps in shi , statutory phi , or tax - based systems . the uptake of phi depends on many factors that include the makeup and performance of the public health care system , socioeconomic factors such as income , education , and age , and incentives from the government . the effects of phi on equity in financing and access may be either positive or negative depending on the format and role of phi . while developing countries face major challenges in attaining the goal of universal health care , phi is playing a role in many emerging systems and is likely to develop further into the future under a mix of financing arrangements .

the sage method is a highly competent technology that can give a global gene expression profile of a particular type of cell or tissue and also help in identifying a set of specific genes to the cellular conditions by comparing the profiles constructed for a pair of cells that are kept at different conditions [ 1 , 2 , 3 , 4 ] . since the discovery of sage , for several years now , it has been used to provide a comprehensive analysis of a variety of different tissue samples , each usually consisting of millions of cells . the approach has been extended recently to permit analysis of gene expression in substantially fewer cells , thereby allowing analysis of heterogeneous tissues or microanatomical structures . sage data can also be used to complement studies in cases where other gene expression methods may be more convenient or efficient . ( 1 ) a short oligonucleotide sequence tag ( 1011 base pairs ) contains sufficient information to uniquely identify a transcript . these tags are used to identify genes and relative abundance of their transcripts within mrna . ( 2 ) concatenation of short sequence tags allows the efficient analysis of transcripts in a serial manner since sage uses serial processing such that 2550 sage tags are analyzed on each lane of dna sequencer . the resulting sequence data are analyzed to identify each gene expressed in the cell and the levels at which each gene is expressed . this information forms a library that can be used to analyze the differences in gene expression between cells . the frequency of each sage tag in the cloned multimers directly reflects the transcript abundance . therefore , sage results in an accurate picture of gene expression at both the qualitative and the quantitative levels . sage technology has been used in a variety of cell lines and in many systems . the following sections describe the significant studies performed in malarial parasite , yeast , plant , and animal systems . sage is particularly well suited for malarial systems , as the genomes of plasmodium species remain to be fully annotated . by simultaneously and quantitatively analyzing mrna transcript profiles from a given cell population , the successful application of sage in plasmodium falciparum , 3d7 strain parasites , from which a preliminary library of 6880 tags corresponding to 4146 different genes was generated , has been reported recently . it was demonstrated that plasmodium falciparum is amenable to this technique , despite the remarkably high a - t content of its genome . sage tags as short as 10 nucleotides were sufficient to uniquely identify parasite transcripts from both nuclear and mitochondrial genomes . moreover , the skewed a - t content of parasite sequence did not preclude the use of enzymes that are crucial for generating representative sage libraries . finally , a few modifications to dna extraction and cloning steps of the sage protocol proved useful for circumventing specific problems presented by a - t rich genomes . in a related study , sage was applied to the malarial parasite plasmodium falciparum to characterize the comprehensive transcriptional profile of erythrocytic stages . a sage library of approximately 8335 tags representing 4866 different genes was generated from 3d7 strain parasites . basic local alignment search tool analysis of high abundance sage tags revealed that a majority ( 88% ) corresponded to 3d7 sequence , and despite the low complexity of the genome , 70% of these highly abundant tags matched unique loci . characterization of these suggested the major metabolic pathways that are used by the organism under normal culture conditions . furthermore , several tags expressed at high abundance ( 30% of tags matching unique loci of the 3d7 genome ) were derived from previously uncharacterized open reading frames , demonstrating the use of sage in genome annotation . the open platform profiling nature of sage also leads to the important discovery of a novel transcriptional phenomenon in the malarial pathogen : a significant number of highly abundant tags that were derived from annotated genes ( 17% ) corresponded to antisense transcripts . these sage data were validated by two independent means : strand specific rt - pcr and northern analysis , where antisense messages were detected in both asexual and sexual stages . sage analysis has been successfully applied for transcript profiling in yeast . of the genes identified in yeast , 1981 genes had known functions while other 2684 genes were previously uncharacterized . the integration of positional information with gene expression data allowed for the generation of chromosomal expression maps identifying physical regions of transcriptional activity and also identified genes that had not been predicted by sequence information alone . a genome - wide characterization of mrna transcript levels in yeast grown on the fatty acid oleate has been determined using sage . comparison of this sage library with that reported for glucose - grown cells revealed the dramatic adaptive response of yeast to a change in carbon source . in oleate - grown cells , this was exemplified by the huge increase of mrnas encoding the peroxisomal beta - oxidation enzymes required for degradation of fatty acids . the data provide evidence for the existence of redox shuttles across organellar membranes that involve peroxisomal , cytoplasmic , and mitochondrial enzymes . induction of genes under the immediate control of these factors was abolished ; other genes were upregulated , indicating an adaptive response to the changed metabolism imposed by the genetic impairment . analysis of global gene expression in saccharomyces cerevisiae by the sage technique has permitted the identification of at least 302 previously unidentified transcripts from nonannotated open reading frames ( norfs ) . transcription of one of these , norf5/hug1 , is induced by dna damage , and this induction requires mec1 , a homologue of the ataxia telangiectasia mutated ( atm ) gene . hug1 is the first example of a norf with important biological functional properties and defines a novel component of the mec1 checkpoint pathway . in a recent study , 10 genome expression data sets have been analyzed by large - scale cross - referencing against broad structural and functional categories . this analysis enabled to determine features more prevalent in the transcriptome than the genome , that is , those that are common to highly expressed proteins . starting with simplest categories , it has been found that , relative to the genome , the transcriptome is enriched in alanine and glycine and depleted in asparagine and very long proteins . in particular , some enzymatic folds , such as the tim barrel and the g3p dehydrogenase folds , are much more prevalent in the transcriptome than the genome , whereas others , such as the protein - kinase and leucine - zipper folds , are depleted . furthermore , for a given functional category , transcriptome enrichment varies quite substantially between the different expression data sets , with a variation an order of magnitude larger than for the other categories cross - referenced ( eg , amino acids ) . sage was applied for profiling expressed genes in rice seedlings ( oryza sativa l. ) . only 1367 genes ( 23.1% ) matched the rice cdna or est sequences in the dna database . sage showed that most of the highly expressed genes in rice seedlings belong to the category of housekeeping genes . unexpectedly , the most highly expressed gene in rice seedlings was a metallothionein ( mt ) gene , and together with three other messages for mt , it accounts for 2.7% of total gene expression . sage was also applied to identify differentially expressed genes between anaerobically treated and untreated rice seedlings . in combination with microarray analysis , sage serves as a highly efficient tool for the identification and isolation of differentially expressed genes in plant . the global gene expression patterns of arabidopsis pollen using sage were characterized recently . it was interesting to note that the number of unique tags in pollen was low compared with the sage library of the leaf constructed on a similar scale . functional classification of the expressed genes reveals that those involved in cellular biogenesis such as polygalacturonase , pectate lyase , and pectin methylesterase make up more than 40% of the total transcripts . the expression level of the great majority of transcripts was unaffected by cold treatment at 0c for 72 hours , whereas pollen tube growth and seed production were substantially reduced . these results strongly suggest that poor accumulation of proteins that play a role in stress tolerance may be why arabidopsis pollen is cold - sensitive . to characterize gene expression in activated mast cells more comprehensively , the changes in genetic transcripts were surveyed by the method of sage in the rbl-2h3 line of rat mast cells before and after they were stimulated through their receptors with high affinity for immunoglobulin e ( fcepsilonri ) . among the diverse genes that had not been previously associated with mast cells and that were constitutively expressed were those for the cytokine macrophage migration inhibitory factor neurohormone receptors such as growth hormone - releasing factor and melatonin and components of the exocytotic machinery . in addition , several dozen transcripts were differentially expressed in response to antigen - induced clustering of the fcepsilonri . included among these were the genes for preprorelaxin , mitogen - activated protein kinase kinase 3 , and the dual specificity protein phosphatase , rvh6 . sage method was used to systematically analyze transcripts present in a microglial cell line . among the diverse transcripts that had not been previously detected in microglia were those for cytokines , such as endothelial monocyte - activating polypeptide i ( emap i ) , and for cell surface antigens , including adhesion molecules such as cd9 , cd53 , cd107a , cd147 , cd162 and mast cell high affinity ige receptor . in addition , transcripts that were characteristic of hematopoietic cells or mesodermal structures , such as e3 protein , a1 , en-7 , b94 , and ufo were also detected . furthermore , the profile contained a transcript , hn1 , that is important in hematopoietic cells and neurological development suggesting the probable neural differentiation of microglia from the hematopoietic system in development . mrna expression of these genes was confirmed by rt - pcr in primary cultures of microglia . the mouse otx2 gene is a homeobox transcription factor required as early as gastrulation for the proper development of the head . the gene expression profiles were compared in wild - type and otx2(-/- ) 6.5-day post - coitum embryos by using a sage assay adapted to microdissected structures . using sage , a tag expression library from rop-+/+ mouse kidney has been constructed . tag sequences were sorted by abundance , and identity was determined by sequence homology searching . previously characterized transcripts were clustered into functional groups , and those encoding metabolic enzymes , plasma membrane proteins ( transporters / receptors ) , and ribosomal proteins were most abundant . the most common , kidney - specific transcripts were kidney androgen - regulated protein , sodium - phosphate cotransporter , renal cytochrome p-450 , parathyroid hormone receptor , and kidney - specific cadherin . in a recent study , the transcriptome of a highly differentiated mouse clonal cortical collecting duct ( ccd ) principal cell line ( mpkccd(cl4 ) ) and the changes in the transcriptome induced by aldosterone and vasopressin have been analyzed . sage was performed on untreated cells and on cells treated with either aldosterone or vasopressin for 4 hours . statistical comparison of the three sage libraries revealed 34 aits ( aldosterone - induced transcripts ) , 29 arts ( aldosterone - repressed transcripts ) , 48 vits ( vasopressin - induced transcripts ) , and 11 vrts ( vasopressin - repressed transcripts ) . a selection of the differentially expressed , hormone - specific transcripts ( 5 vits , 2 aits , and 1 art ) has been validated in the mpkccd(cl4 ) cell line either by northern blot hybridization or reverse transcription - pcr . the hepatocyte nuclear transcription factor hnf-3-alpha ( vit39 ) , the receptor activity modifying protein ramp3 ( vit48 ) , and the glucocorticoid - induced leucine zipper protein ( gilz ) ( ait28 ) are candidate proteins playing a role in physiological responses of this cell line to vasopressin and aldosterone . the development of cardiovascular diseases such as heart failure involves functional changes that are beneficial short - term , but may be fatal long - term . in a recent study , the current state of genomic research for determination of the transcriptome by the first limited sage analysis of rodent heart gene expression has been described . it has also been discussed that how these results generated with this approach can be applied to the study and treatment of cardiovascular diseases . molecular inventories of the developing mouse neocortex before and after birth were generated using the global gene expression profiling tool sage . the libraries were generated from embryonic day 15 and postnatal day 1 mouse neocortices . the differentially expressed transcripts included genes known to be important in neocortical development ( eg , brain factor 1 , neurod2 , and id2 ) , genes not previously associated with neocortical development ( such as brahma - related gene 1 , receptor for activated c - kinase i , hypermethylated in cancer 2 , and evi9 ) , and genes of unknown identity or function . sage was applied to study differentially expressed genes in mouse brain 14 hours after the induction of focal cerebral ischemia . metallothionein - ii ( mt - ii ) was the most significantly upregulated transcript in the ischemic hemisphere . mt - i and mt - ii are induced by metals , glucocorticoids , and inflammatory signals in a coordinated manner , yet their function remains elusive . mt - i- and mt - ii - deficient mice developed approximately threefold larger infarcts than wild - type mice and a significantly worse neurological outcome . ftl-1 , -3 , and -10 are three murine day 14 fetal thymocyte cell lines produced in order to model developmental stages within early ( cd3-cd4-cd8 ) thymocyte differentiation . in a recent study , the sage method was used to perform a systematic analysis of transcripts present in these cell lines . differentially expressed mrna transcripts representing different gene classes were identified , including t cell functional genes , cytokine receptors , adhesion molecules , and transcription factors . expression of the transcription factors runx2 and phd finger protein 2 and of the igf type 1 receptor was shown to have differentially regulated expression patterns in sorted dn1 - 4 cells . these genes , and others identified by this analysis , are likely to play important roles in the development of t cells . in order to identify genes developmentally regulated in the somatic cells of the testis , sage has been used to generate gene expression profiles from these cells in the fetal and adult mouse testes . to avoid germ cell transcripts , a fetal sage library was generated from germ cell - free fetal w(v)/w(v ) mice and an adult sage library from adult testes depleted of germ cells with busulfan . the differentially regulated genes are likely to provide insight into mechanisms regulating testis function both during development and in the adult animal . sage technology has been utilized to contrast the differential gene expression profile in rat embryo fibroblast cells producing temperature - sensitive p53 tumor suppressor protein at permissive or nonpermissive temperatures . analysis of approximately 15 000 genes revealed that the expression of 14 genes was dependent on functional p53 protein , whereas the expression of 3 genes was significantly higher in cells producing nonfunctional p53 protein . those genes whose expression was increased by functional p53 include ras , u6 snrna , cyclin g , egr-1 , and several novel genes . the expression of actin , tubulin , and hsp70 genes was elevated at the nonpermissive temperature for p53 function . interestingly , the expression of several genes was dependent on a non - temperature - sensitive mutant p53 suggesting altered transcription profiles dependent on specific p53 mutant proteins . these results demonstrate the utility of sage for rapidly and reproducibly evaluating global transcriptional responses within different cell populations . kringle domain , a triple - disulfide - linked domain , is conserved in diverse proteins which play important roles in various biological processes . kremen , a novel member of kringle - containing proteins , has been cloned using a newly developed unique strategy , kringle - sage , which enables comprehensive analysis of kringle - containing proteins . kremen is likely to be a type - i transmembrane protein composed of 473 amino acid residues . kremen has a kringle domain , a wsc domain , and cub domains in the extracellular region , while the intracellular region has no conserved motif involved in signal transduction . in the mouse embryo , the kremen mrna level , which was increased during embryonic development , was localized in the apical ectodermal ridge of limb buds , myotome , and sensory organs ( eg , optic vesicle , otic vesicle , and nasal pit ) . in the adult mouse , kremen mrna was expressed in a variety of tissues with a relatively strong expression in the lung , heart , and skeletal muscle . kremen mrna expression in c2c12 and nie-115 cells increased during respective differentiation into muscular and neural cells . these results suggest a potential role for kremen in the regulation of cellular responses upon extracellular stimulus or cell - cell interaction in neuronal and/or muscle cells . kringle - sage is expected to facilitate further elucidation of structure and functions of kringle proteins . to elucidate the molecular basis of muscle atrophy , the sage method has been performed with control and immobilized muscles of 10 rats . the genes that expressed greater than 0.5% in muscle are involved in the following three functions : ( 1 ) contraction ( troponin i , c , and t ; myosin light chain 13 ; actin ; tropomyosin ; and parvalbumin ) , ( 2 ) energy metabolism ( cytochrome c oxidase i and iii , creatine kinase , glyceraldehyde-3-phosphate - dehydrogenase , phosphoglycerate mutase , atpase 6 , and aldolase a ) , and ( 3 ) housekeeping ( lens epithelial protein ) . muscle atrophy appears to be caused by changes in mrna levels of specific regulators of proteolysis , protein synthesis , and contractile apparatus assembling , such as polyubiquitin , elongation factor 2 , and nebulin . immobilization has produced a decrease more than threefold in gene expression of enzymes involved in energy metabolism , especially atpase , cytochrome c oxidase , nadh dehydrogenase , and protein phosphatase 1 . differential gene expressions of selenoprotein w and uroporphyrinogen decarboxylase , which can be involved in oxidative stress , were also observed . other genes with various functions , such as cholesterol metabolism and growth factors , were also differentially expressed . moreover , novel genes regulated by immobilization were discovered . thus , this study allows a better understanding of global muscle characteristics and the molecular mechanisms of sedentarity and sarcopenia . using the sage method , a gene expression profile of the rat hippocampus a total of 76 790 sage tags were analyzed , allowing identification of 28 748 different tag species , each representing a unique mrna transcript . the tags were divided into different abundancy classes , ranging from tags that were detected over 500 times to tags encountered only once in the 76 790 tags analyzed . the mrna species detected more than 50 times represented 0.3% of the total number of unique tags while accounting for 22% of the total hippocampal mrna mass . the genes expressed at the highest levels were of mitochondrial origin , consistent with a high requirement for energy in neuronal tissue . at a lower level of expression , several neuron - specific transcripts were encountered , encoding various neurotransmitter receptors , transporters , and enzymes involved in neurotransmitter synthesis and turnover , ion channels and pumps , and synaptic components . comparison of relative expression levels demonstrated that glutamate receptors are the most frequent neurotransmitter receptors expressed in the hippocampus , consistent with the important role of glutamatergic neurotransmission in the hippocampus , while gaba receptors were present at approximately ten - fold lower levels . several kinases were present including camkii , which was expressed at high levels , consistent with being the most abundant protein in the spines of hippocampal pyramidal cells . adrenal corticosteroids ( cort ) have a profound effect on the function of the hippocampus . this is mediated in a coordinated manner by mineralocorticoid ( mr ) and glucocorticoid ( gr ) receptors via activation or repression of target genes . using sage , cort - responsive hippocampal genes regulated via mr and/or gr have been identified in a recent study . sage profiles were compared under different conditions of cort exposure , resulting in identification of 203 cort - responsive genes that are involved in many different cellular processes like energy expenditure and cellular metabolism ; protein synthesis and turnover ; signal transduction , neuronal connectivity , and neurotransmission . in situ , hybridization revealed that six randomly chosen cort - responsive genes had distinct expression patterns in neurons of the hippocampus . in addition , using in situ hybridization , it was confirmed that these six genes were indeed regulated by cort , underscoring the validity of the sage data . comparison of mr- and gr - dependent expression profiles revealed that the majority of the cort - responsive genes was regulated either by activated mr or by activated gr , while only a few genes were responsive to both activated mr and gr . this indicates that the molecular basis for the differential effects of activated mr and gr is activation or repression of distinct , yet partially overlapping sets of genes . the putative cort - responsive genes identified in this study will provide insight into the molecular mechanisms underlying the differential and sometimes opposing effects of mr and gr on neuronal excitability , memory formation and behaviour as well as their role in neuronal protection and damage . intraepithelial lymphocytes ( iels ) are abundant , evolutionarily conserved t cells , commonly enriched in t cell receptor ( tcr ) gamma delta expression . however , their primary functional potential and constitutive activation state are incompletely understood . to address this , sage was applied to murine tcr gamma delta+ and tcr alpha beta+ intestinal iels directly ex vivo , identifying 15,574 unique transcripts that collectively portray an activated yet resting , th1-skewed , cytolytic , and immunoregulatory phenotype applicable to multiple subsets of gut iels . expression of granzymes , fas ligand , rantes , prothymosin beta4 , junb , rgs1 , btg1 , and related molecules is high , whereas expression of conventional cytokines and high - affinity cytokine receptors is low . differentially expressed genes readily identify heterogeneity among tcr alpha beta+ iels , whereas differences between resident tcr gamma delta+ iels and tcr alpha beta+ iels are less obvious . although extraocular muscle ( eom ) is a skeletal muscle , aspects of its biology are unlike other striated muscles . in a recent study , sage isolates and sequences 10-bp tags from defined locations in mrna - derived cdna . tag sequence - location was used to extract transcript identity from a curated sage database , and detection frequencies reflected abundance of corresponding mrnas . of the unique tags , 7.8% were detected at high to intermediate levels , 19.3% at lower levels , and 72.9% as single copies ; 40% of the tags matched known expressed sequence tags ( ests ) , most of which ( 85.7% ) represented a unique est . tags without matches in the sage database and those expressed as single copies only were not considered further . sage tags expressed at more than 0.1% of total transcripts reflected several aspects of muscle biology , including sarcomeric structure , energy metabolism , and ribosomal protein expression . genes highly expressed in eom were compared with other existing muscle expression databases to identify conserved and novel patterns in eom . these data provide a normative gene expression database and a novel molecular signature that will facilitate the study of eom development and function and of the mechanisms behind its preferential targeting or sparing in neuromuscular disease . progress in large - scale cdna analysis ( est analysis ) in many organisms is a prerequisite for the useful application of sage , as the annotation of sage tags is based on preexisting est databases . the uniqueness of sage is that it allows transcript profiles to be given as digital data . accordingly , they become suitable for the construction of gene expression databases on computer networks . yeast and cancer transcriptome databases based on sage are already accessible via the internet . in the organisms where transcript data are limited , sage may initially be the most efficient method of identifying new or differentially expressed genes . the sage data analysis could also be used as reference data for the relative expression data obtained by hybridization experiments on cdna arrays , and may ultimately allow comparison of array data between different experiments in different laboratories . sage is also used as a primary discovery engine that can characterize human diseases at the molecular level while illuminating potential targets and markers for therapeutic and diagnostic developments respectively . the ability of sage to define specific transcriptomes will aid in the development of gene therapies whereby cell- or tissue- specific promoters and genes can be utilized to appropriately express and deliver a given therapy . in general , sage alone or in combination with proteomic approaches can accelerate the identification of high - quality drug targets which could be a next generation of therapeutic products . sage , along with other methods , should yield valuable information about the fundamental biology and virulence mechanism of an important plant or human pathogen . in combination with microarray analysis , sage should serve as a highly efficient tool for the identification and isolation of differentially expressed genes in plants and animals .

the serial analysis of gene expression ( sage ) method is based on the isolation of unique sequence tags from individual transcripts and concatenation of tags serially into long dna molecules . sage is an innovative technique that offers the potential of cataloging both the identity and relative frequencies of mrna transcripts in a given rna preparation . it can quantify low - abundance transcripts and reliably detect relatively small differences in transcript abundance between cell populations . sage data can be used to complement studies in cases where other gene expression methods may be more convenient or efficient . sage can be used in a wide variety of applications to identify disease - related genes , to analyze the effect of drugs on tissues , and to provide insights into the disease pathways . the most important application of sage is the identification of differentially expressed genes . in this review , we describe various applications of this powerful technology in malarial parasite , yeast , plant , and animal systems .

INTRODUCTION SAGE STUDIES IN MALARIA PARASITE SAGE IN YEAST STUDIES SAGE IN PLANT STUDIES SAGE IN ANIMAL STUDIES CONCLUSIONS

the sage method is a highly competent technology that can give a global gene expression profile of a particular type of cell or tissue and also help in identifying a set of specific genes to the cellular conditions by comparing the profiles constructed for a pair of cells that are kept at different conditions [ 1 , 2 , 3 , 4 ] . since the discovery of sage , for several years now , it has been used to provide a comprehensive analysis of a variety of different tissue samples , each usually consisting of millions of cells . the approach has been extended recently to permit analysis of gene expression in substantially fewer cells , thereby allowing analysis of heterogeneous tissues or microanatomical structures . sage data can also be used to complement studies in cases where other gene expression methods may be more convenient or efficient . these tags are used to identify genes and relative abundance of their transcripts within mrna . ( 2 ) concatenation of short sequence tags allows the efficient analysis of transcripts in a serial manner since sage uses serial processing such that 2550 sage tags are analyzed on each lane of dna sequencer . the resulting sequence data are analyzed to identify each gene expressed in the cell and the levels at which each gene is expressed . this information forms a library that can be used to analyze the differences in gene expression between cells . the frequency of each sage tag in the cloned multimers directly reflects the transcript abundance . therefore , sage results in an accurate picture of gene expression at both the qualitative and the quantitative levels . sage technology has been used in a variety of cell lines and in many systems . the following sections describe the significant studies performed in malarial parasite , yeast , plant , and animal systems . by simultaneously and quantitatively analyzing mrna transcript profiles from a given cell population , the successful application of sage in plasmodium falciparum , 3d7 strain parasites , from which a preliminary library of 6880 tags corresponding to 4146 different genes was generated , has been reported recently . in a related study , sage was applied to the malarial parasite plasmodium falciparum to characterize the comprehensive transcriptional profile of erythrocytic stages . basic local alignment search tool analysis of high abundance sage tags revealed that a majority ( 88% ) corresponded to 3d7 sequence , and despite the low complexity of the genome , 70% of these highly abundant tags matched unique loci . furthermore , several tags expressed at high abundance ( 30% of tags matching unique loci of the 3d7 genome ) were derived from previously uncharacterized open reading frames , demonstrating the use of sage in genome annotation . the open platform profiling nature of sage also leads to the important discovery of a novel transcriptional phenomenon in the malarial pathogen : a significant number of highly abundant tags that were derived from annotated genes ( 17% ) corresponded to antisense transcripts . these sage data were validated by two independent means : strand specific rt - pcr and northern analysis , where antisense messages were detected in both asexual and sexual stages . of the genes identified in yeast , 1981 genes had known functions while other 2684 genes were previously uncharacterized . the integration of positional information with gene expression data allowed for the generation of chromosomal expression maps identifying physical regions of transcriptional activity and also identified genes that had not been predicted by sequence information alone . a genome - wide characterization of mrna transcript levels in yeast grown on the fatty acid oleate has been determined using sage . the data provide evidence for the existence of redox shuttles across organellar membranes that involve peroxisomal , cytoplasmic , and mitochondrial enzymes . analysis of global gene expression in saccharomyces cerevisiae by the sage technique has permitted the identification of at least 302 previously unidentified transcripts from nonannotated open reading frames ( norfs ) . transcription of one of these , norf5/hug1 , is induced by dna damage , and this induction requires mec1 , a homologue of the ataxia telangiectasia mutated ( atm ) gene . hug1 is the first example of a norf with important biological functional properties and defines a novel component of the mec1 checkpoint pathway . in a recent study , 10 genome expression data sets have been analyzed by large - scale cross - referencing against broad structural and functional categories . furthermore , for a given functional category , transcriptome enrichment varies quite substantially between the different expression data sets , with a variation an order of magnitude larger than for the other categories cross - referenced ( eg , amino acids ) . sage was applied for profiling expressed genes in rice seedlings ( oryza sativa l. ) . sage showed that most of the highly expressed genes in rice seedlings belong to the category of housekeeping genes . unexpectedly , the most highly expressed gene in rice seedlings was a metallothionein ( mt ) gene , and together with three other messages for mt , it accounts for 2.7% of total gene expression . sage was also applied to identify differentially expressed genes between anaerobically treated and untreated rice seedlings . in combination with microarray analysis , sage serves as a highly efficient tool for the identification and isolation of differentially expressed genes in plant . functional classification of the expressed genes reveals that those involved in cellular biogenesis such as polygalacturonase , pectate lyase , and pectin methylesterase make up more than 40% of the total transcripts . these results strongly suggest that poor accumulation of proteins that play a role in stress tolerance may be why arabidopsis pollen is cold - sensitive . to characterize gene expression in activated mast cells more comprehensively , the changes in genetic transcripts were surveyed by the method of sage in the rbl-2h3 line of rat mast cells before and after they were stimulated through their receptors with high affinity for immunoglobulin e ( fcepsilonri ) . in addition , several dozen transcripts were differentially expressed in response to antigen - induced clustering of the fcepsilonri . included among these were the genes for preprorelaxin , mitogen - activated protein kinase kinase 3 , and the dual specificity protein phosphatase , rvh6 . sage method was used to systematically analyze transcripts present in a microglial cell line . in addition , transcripts that were characteristic of hematopoietic cells or mesodermal structures , such as e3 protein , a1 , en-7 , b94 , and ufo were also detected . tag sequences were sorted by abundance , and identity was determined by sequence homology searching . previously characterized transcripts were clustered into functional groups , and those encoding metabolic enzymes , plasma membrane proteins ( transporters / receptors ) , and ribosomal proteins were most abundant . the most common , kidney - specific transcripts were kidney androgen - regulated protein , sodium - phosphate cotransporter , renal cytochrome p-450 , parathyroid hormone receptor , and kidney - specific cadherin . in a recent study , the transcriptome of a highly differentiated mouse clonal cortical collecting duct ( ccd ) principal cell line ( mpkccd(cl4 ) ) and the changes in the transcriptome induced by aldosterone and vasopressin have been analyzed . a selection of the differentially expressed , hormone - specific transcripts ( 5 vits , 2 aits , and 1 art ) has been validated in the mpkccd(cl4 ) cell line either by northern blot hybridization or reverse transcription - pcr . the hepatocyte nuclear transcription factor hnf-3-alpha ( vit39 ) , the receptor activity modifying protein ramp3 ( vit48 ) , and the glucocorticoid - induced leucine zipper protein ( gilz ) ( ait28 ) are candidate proteins playing a role in physiological responses of this cell line to vasopressin and aldosterone . the development of cardiovascular diseases such as heart failure involves functional changes that are beneficial short - term , but may be fatal long - term . in a recent study , the current state of genomic research for determination of the transcriptome by the first limited sage analysis of rodent heart gene expression has been described . molecular inventories of the developing mouse neocortex before and after birth were generated using the global gene expression profiling tool sage . the differentially expressed transcripts included genes known to be important in neocortical development ( eg , brain factor 1 , neurod2 , and id2 ) , genes not previously associated with neocortical development ( such as brahma - related gene 1 , receptor for activated c - kinase i , hypermethylated in cancer 2 , and evi9 ) , and genes of unknown identity or function . sage was applied to study differentially expressed genes in mouse brain 14 hours after the induction of focal cerebral ischemia . metallothionein - ii ( mt - ii ) was the most significantly upregulated transcript in the ischemic hemisphere . mt - i and mt - ii are induced by metals , glucocorticoids , and inflammatory signals in a coordinated manner , yet their function remains elusive . ftl-1 , -3 , and -10 are three murine day 14 fetal thymocyte cell lines produced in order to model developmental stages within early ( cd3-cd4-cd8 ) thymocyte differentiation . in a recent study , the sage method was used to perform a systematic analysis of transcripts present in these cell lines . differentially expressed mrna transcripts representing different gene classes were identified , including t cell functional genes , cytokine receptors , adhesion molecules , and transcription factors . these genes , and others identified by this analysis , are likely to play important roles in the development of t cells . in order to identify genes developmentally regulated in the somatic cells of the testis , sage has been used to generate gene expression profiles from these cells in the fetal and adult mouse testes . the differentially regulated genes are likely to provide insight into mechanisms regulating testis function both during development and in the adult animal . sage technology has been utilized to contrast the differential gene expression profile in rat embryo fibroblast cells producing temperature - sensitive p53 tumor suppressor protein at permissive or nonpermissive temperatures . those genes whose expression was increased by functional p53 include ras , u6 snrna , cyclin g , egr-1 , and several novel genes . the expression of actin , tubulin , and hsp70 genes was elevated at the nonpermissive temperature for p53 function . these results demonstrate the utility of sage for rapidly and reproducibly evaluating global transcriptional responses within different cell populations . kremen , a novel member of kringle - containing proteins , has been cloned using a newly developed unique strategy , kringle - sage , which enables comprehensive analysis of kringle - containing proteins . kremen has a kringle domain , a wsc domain , and cub domains in the extracellular region , while the intracellular region has no conserved motif involved in signal transduction . in the adult mouse , kremen mrna was expressed in a variety of tissues with a relatively strong expression in the lung , heart , and skeletal muscle . kringle - sage is expected to facilitate further elucidation of structure and functions of kringle proteins . the genes that expressed greater than 0.5% in muscle are involved in the following three functions : ( 1 ) contraction ( troponin i , c , and t ; myosin light chain 13 ; actin ; tropomyosin ; and parvalbumin ) , ( 2 ) energy metabolism ( cytochrome c oxidase i and iii , creatine kinase , glyceraldehyde-3-phosphate - dehydrogenase , phosphoglycerate mutase , atpase 6 , and aldolase a ) , and ( 3 ) housekeeping ( lens epithelial protein ) . immobilization has produced a decrease more than threefold in gene expression of enzymes involved in energy metabolism , especially atpase , cytochrome c oxidase , nadh dehydrogenase , and protein phosphatase 1 . differential gene expressions of selenoprotein w and uroporphyrinogen decarboxylase , which can be involved in oxidative stress , were also observed . other genes with various functions , such as cholesterol metabolism and growth factors , were also differentially expressed . using the sage method , a gene expression profile of the rat hippocampus a total of 76 790 sage tags were analyzed , allowing identification of 28 748 different tag species , each representing a unique mrna transcript . the mrna species detected more than 50 times represented 0.3% of the total number of unique tags while accounting for 22% of the total hippocampal mrna mass . at a lower level of expression , several neuron - specific transcripts were encountered , encoding various neurotransmitter receptors , transporters , and enzymes involved in neurotransmitter synthesis and turnover , ion channels and pumps , and synaptic components . comparison of relative expression levels demonstrated that glutamate receptors are the most frequent neurotransmitter receptors expressed in the hippocampus , consistent with the important role of glutamatergic neurotransmission in the hippocampus , while gaba receptors were present at approximately ten - fold lower levels . several kinases were present including camkii , which was expressed at high levels , consistent with being the most abundant protein in the spines of hippocampal pyramidal cells . adrenal corticosteroids ( cort ) have a profound effect on the function of the hippocampus . this is mediated in a coordinated manner by mineralocorticoid ( mr ) and glucocorticoid ( gr ) receptors via activation or repression of target genes . using sage , cort - responsive hippocampal genes regulated via mr and/or gr have been identified in a recent study . sage profiles were compared under different conditions of cort exposure , resulting in identification of 203 cort - responsive genes that are involved in many different cellular processes like energy expenditure and cellular metabolism ; protein synthesis and turnover ; signal transduction , neuronal connectivity , and neurotransmission . the putative cort - responsive genes identified in this study will provide insight into the molecular mechanisms underlying the differential and sometimes opposing effects of mr and gr on neuronal excitability , memory formation and behaviour as well as their role in neuronal protection and damage . expression of granzymes , fas ligand , rantes , prothymosin beta4 , junb , rgs1 , btg1 , and related molecules is high , whereas expression of conventional cytokines and high - affinity cytokine receptors is low . differentially expressed genes readily identify heterogeneity among tcr alpha beta+ iels , whereas differences between resident tcr gamma delta+ iels and tcr alpha beta+ iels are less obvious . in a recent study , sage isolates and sequences 10-bp tags from defined locations in mrna - derived cdna . tag sequence - location was used to extract transcript identity from a curated sage database , and detection frequencies reflected abundance of corresponding mrnas . of the unique tags , 7.8% were detected at high to intermediate levels , 19.3% at lower levels , and 72.9% as single copies ; 40% of the tags matched known expressed sequence tags ( ests ) , most of which ( 85.7% ) represented a unique est . genes highly expressed in eom were compared with other existing muscle expression databases to identify conserved and novel patterns in eom . these data provide a normative gene expression database and a novel molecular signature that will facilitate the study of eom development and function and of the mechanisms behind its preferential targeting or sparing in neuromuscular disease . progress in large - scale cdna analysis ( est analysis ) in many organisms is a prerequisite for the useful application of sage , as the annotation of sage tags is based on preexisting est databases . the uniqueness of sage is that it allows transcript profiles to be given as digital data . accordingly , they become suitable for the construction of gene expression databases on computer networks . yeast and cancer transcriptome databases based on sage are already accessible via the internet . in the organisms where transcript data are limited , sage may initially be the most efficient method of identifying new or differentially expressed genes . the sage data analysis could also be used as reference data for the relative expression data obtained by hybridization experiments on cdna arrays , and may ultimately allow comparison of array data between different experiments in different laboratories . sage is also used as a primary discovery engine that can characterize human diseases at the molecular level while illuminating potential targets and markers for therapeutic and diagnostic developments respectively . the ability of sage to define specific transcriptomes will aid in the development of gene therapies whereby cell- or tissue- specific promoters and genes can be utilized to appropriately express and deliver a given therapy . in general , sage alone or in combination with proteomic approaches can accelerate the identification of high - quality drug targets which could be a next generation of therapeutic products . in combination with microarray analysis , sage should serve as a highly efficient tool for the identification and isolation of differentially expressed genes in plants and animals .

among the lipid nanoparticles , liposomes are widely studied as drug delivery vehicles . liposomes protect the encapsulated drugs from being metabolized during the circulation prior to reaching the target . the us food and drug administration has approved liposome - based formulations for the treatment of several types of cancer . however , upon targeting , the passive release of the encapsulated drugs from the liposomes is often slow . reorganization of the lipid domains has been used as a trigger to enhance , and to control the rate and the extent of contents released from liposomes . among the various triggers , decreased ph in the lysosomes has been widely used as a successful strategy to efficiently release the encapsulated liposomal contents . however , imparting ph sensitivity to liposomes requires the synthesis of specialized lipids with structures that are substantially modified , either due to hydrolysis or due to changes in the protonation states of the lipid head groups , at reduced ph . stabilized gas bubbles are widely used as contrast - enhancing agents for ultrasound imaging of perfused tissues . there are many reports of ultrasound - mediated drug release from nanoparticles , liposomes , and other carriers . although ultrasound waves in the khz frequency efficiently release drugs from the carriers ( due to cavitation and high local temperatures ) , the harmful biological effects associated with low - frequency ultrasound limit the usefulness of such strategies . to make liposomes responsive to high - frequency ultrasound , they need to be coupled with gas pockets . echogenic liposomes ( elips ) entrap small amounts of air along with the hydrophilic drug in their aqueous interior , and are currently being developed as drug delivery vehicles for ultrasound - triggered drug release and simultaneous imaging . although there is uncertainty about the exact location and size of the entrapped air bubbles in the elips , their acoustic characterization has been reported extensively in the literature . we are developing targeted , multimodal liposomes for triggered release of encapsulated contents , and simultaneous ultrasound imaging . furthermore , we are interested in enhancing the contents released from the liposomes by employing diagnostic frequency ( mhz ) ultrasound . we have recently demonstrated the ultrasound - enhanced , extracellular release of liposomal contents mediated by the cancer - cell - secreted enzyme matrix metalloproteinase-9 ( mmp-9 ) . herein , we report a strategy to render liposomes ph sensitive by encapsulating ammonium bicarbonate which generates gas bubbles in situ in response to acidic ph . our strategy does not require the use of ph - sensitive lipids in the liposomal formulations . we hypothesize that , at a reduced ph , the hydronium ions diffuse into the aqueous interior of the liposomes , and produce carbon dioxide bubbles , thereby turning on the echogenicity . as more bubbles are generated , the liposomal bilayer is disturbed , leading to the release of encapsulated contents . we observe that the release was further enhanced by applying ultrasound with a frequency of 1 mhz . to the best of our knowledge there are no reports in the literature of ultrasound enhanced triggered release from ph - tunable echogenic liposomes . we have demonstrated the usefulness of this liposomal delivery system using the panc-1 pancreatic cancer cells . pancreatic cancer is one of the leading causes of cancer - related deaths in both men and women in the united states , with a 5-year survival rate of less than 5% . according to the american cancer society , 38,460 pancreatic cancer related deaths ( almost equally split between men and women ) occurred in united states in 2013 . to demonstrate tunable echogenicity , we prepared the liposomes from 1-palmitoyl-2-oleoyl - sn - glycero-3-phosphocholine ( popc ) , encapsulating 400 mm ammonium bicarbonate along with the self - quenching dye carboxyfluorescein ( 100 mm ) . we reasoned that , for multilamellar liposomes , the outside hydronium ions need to diffuse through several lipid bilayers in order to generate sufficient amounts of co2 gas inside the liposomes . the presence of several lipid bilayers was also expected to decrease the efficiency of the contents released in response to escaping gas bubbles and ultrasonic excitation . hence , we decided to formulate unilamellar liposomes with a narrow size distribution by sonicating and sequentially extruding ( through 800 and 200 nm polycarbonate membrane filters ) the initially formed multilamellar vesicles . we observed ( with dynamic light scattering ) that the average hydrodynamic diameter of the liposomes is 110 15 nm with a polydispersity index of 0.05 ( supporting information , figure s1a ) . these results were corroborated with transmission electron microscopic imaging of the liposomes ( supporting information , figure s1b ) . to demonstrate the tunable echogenicity , we added the liposomes to buffers with different phs ( 7.45.0 ) and recorded the images using a terason t3200 high - frequency ( 1214 mhz ) diagnostic ultrasound transducer . we expected that the generated gas bubbles would reflect the ultrasound , and that the contrast of the images would be more pronounced as the amount of generated gas is increased at a lower ph . we observed that there was a lag time before the liposomes became echogenic , and that the duration of this lag phase decreased with the reduced ph . for example , liposomes in the ph 7.4 buffer did not show any ultrasound contrast in 10 min . at ph 6 , the liposomes became weakly echogenic in 5 min , but at ph 5 , the liposomes were fairly echogenic within 3 min . the ultrasound images for the liposomes in buffers with different ph values after 5 min are shown in figure 1 . figure 1a shows that there is no echo at ph 7.4 ; but as the ph is reduced , progressively stronger echo from the entire cell well is observed ( note that the ultrasound probe is placed at the top of the cell well ) . figures 1b and 1c show how the mean and maximum gray scale values quantitatively change with ph . the echo from the homogeneous suspension of liposomes appears as light bands ( figure 1a ) . they arise due to interference between echoes from subresolution scatterers such as liposomes which themselves are far smaller in size than the ultrasound wavelength . ( a ) ph - dependent diagnostic frequency ultrasound imaging of popc liposomes encapsulating 400 mm ammonium bicarbonate . the dotted white lines represent the regions of interest ( roi ) that were used to calculate the gray scale values . ( b ) mean gray scale values and ( c ) maximum gray scale values for the ultrasound images shown in panel a as a function of ph ( n = 3 ) . we anticipated that the concentration of hydronium ions in the external buffer would affect their diffusion rate inside the liposomes as well as the subsequent generation of co2 bubbles . as the encapsulated ammonium bicarbonate was depleted , the generation of co2 gas slowed down and finally stopped . consistent with this hypothesis , we observed that liposomes in the ph 5 buffer are not echogenic after 20 min ( figure 2 ) . however , we noted that the diameters of the gas bubbles inside the liposomes are likely to be small ( in nanometers ) , and that they may not reflect the ultrasound very well . it is likely that the nanobubbles coalesce in the lipid bilayer of the liposomes , generating larger bubbles , and reflect the ultrasound . popc lipid has a gel low transition temperature ( 2 c ) , and the liposomal bilayer is in the fluid phase under the experimental conditions ( 20 c ) . the loose lipid packing and fluidity of the popc bilayer accommodate the coalescence and the size increase of the gas bubbles . diagnostic frequency ultrasound images of popc liposomes encapsulating 400 mm ammonium bicabonate as a function of frequency and incubation time in a ph 5 buffer . the images were acquired by employing high - frequency ( 1215 mhz ; a , b ) , medium - frequency ( 812 mhz ; c , d ) , and low - frequency ( 48 mhz ; e , f ) ultrasound transducers . we analyzed the ultrasound images shown in figure 1a using the imagej software ( http://rsbweb.nih.gov ) to calculate the mean and maximum gray scale values for region of interest ( roi ) shown in figure 1a . as expected , the mean and maximum gray scale values increase with a decreasing ph . we observed that the highest gray scale value was observed at ph 5 , and it does not increase any more below this ph ( data not shown ) . we also observed a time - dependent decrease in the echogenicity of these liposomes at ph 5.0 ( figure 2 ) . these results demonstrated that liposomes are programmed to reflect the ultrasound only after reaching the acidic microenvironment of cancer cells . having demonstrated ph - tunable echogenicity , we decided to determine if the escaping gas bubbles sufficiently disturb the lipid bilayer to release the encapsulated contents from the liposomes . for this endeavor , we incubated the popc liposomes ( encapsulating carboxyfluorescein and 400 mm ammonium bicarbonate ) in buffers with different ph values ( 7.45.0 ) , and monitored the emission intensity of carboxyfluorescein . however , the emission intensity of carboxyfluorescein is quenched as the ph is lowered . to correct for this decreased emission intensity , we measured the absorption spectra of carboxyfluorescein as a function of ph , and we determined the isosbestic point to be 460 nm . subsequently , the dye solution was prepared in buffers with ph of 7.4 , 6.0 , and 5.0 ; the solution was excited at 460 nm , and the emission spectra were recorded . we then monitored the emission of the dye at 497 nm ( excitation : 460 nm ) for 2 h. the correction factors were calculated at each ph as a function of time , and all emission intensities were appropriately corrected for calculating the percentage released ( supporting information ) . when the liposomes were incubated in acidic buffers , there was a time lag before dye release ( figure 3 ) . however , the liposomes continued to leak the contents for a considerably long time ( 23 h ) . the continued leakage indicates that the disturbances in the lipid bilayers created by the escaping gas bubbles either are not sealed or take a long time to heal . representative release profiles of carboxyfluorescein from popc liposomes encapsulating 400 mm ammonium bicarbonate . the liposomes were incubated in buffers with ph 7.4 ( blue circles ) , ph 6.0 ( purple triangles ) , and ph 5.0 ( green stars ) . the lines are generated by connecting the observed data points . while the liposomes at a ph of 7.4 ( control ) released only 15% of the encapsulated dye in 2 h , at a ph of 5 , the release increased to 55% ( figure 3 ) . when we encapsulated sodium bicarbonate in the liposomes ( instead of ammonium bicarbonate ) , the amount of the content release decreased . in 2 h , we observed that the sodium bicarbonate encapsulated liposomes released 40% of the encapsulated dye ( at ph = 5.0 ; supporting information , figures s3 and s4 ) . for both of these liposomal formulations , the rate of contents release decreased considerably after 2 h. in 3 h at ph 5.0 , the ammonium bicarbonate encapsulated liposomes released 75% of the contents , and the sodium bicarbonate encapsulated liposomes released 44% of the contents ( figure 4a ) . decreasing the amount of encapsulated ammonium bicarbonate ( from 400 mm to 200 mm ) also reduced the amount of contents release from the liposomes ( figure 4b ) . release of encapsulated carboxyfluorescein from popc liposomes as a function of ph encapsulating ( a ) 400 mm ammonium bicarbonate and ( b ) 200 mm ammonium bicarbonate after 3 h ( n = 3 ) . the acidic decomposition of ammonium bicarbonate generates nh3 and co2 , while sodium bicarbonate produces the sodium salt of the buffer , h2o , and co2 . the ammonia gas will react with the hydronium ions in the liposome interior , leading to a reduction in proton concentration . the resultant proton gradient will facilitate the diffusion of more hydronium ions into the liposomal lumen and generate more co2 gas and ammonia . amount of generated gas decreases by reducing the concentration of encapsulated ammonium bicarbonate ( from 400 to 200 mm ) , leading to a reduction in contents release from the liposomes ( figure 4b ) . as an additional control , we prepared the popc liposomes without encapsulating any gas precursor and studied the contents release as a function of ph . we observed minimal release ( < 10% ) of the encapsulated carboxyfluorescein at ph 7.4 and 6.0 . however , at ph 5.0 , about 20% of the dye was released in 2 h ( figure s5 , supporting information ) . we do not have an explanation for this observation yet . in these liposome formulations the popc molecules contain the saturated palmitoyl and the unsaturated oleoyl groups . due to the presence of an alkene in the z - configuration , this lipid does not form a tight bilayer , and the gel transition temperature is also low ( 2 c ) . it is possible that the loose packing of the popc lipids will likely allow the escaping co2 bubbles to coalesce inside the hydrophobic bilayer of the liposomes . the resulting larger gas bubbles will disturb the bilayer while escaping , allowing the contents to leak . to determine if the lipid packing in the liposomal bilayer and the gel transition temperature affect the contents released , we prepared two batches of dspc ( 1,2-distearoyl - sn - glycero-3-phosphocholine ) liposomes that encapsulate ammonium and sodium bicarbonate respectively ( 400 mm each ) . the dspc molecules contain two saturated stearoyl groups and form a tight bilayer with a melting temperature of 56 c . we hypothesized that the tightly packed lipid molecules in the bilayer would hinder the coalescence of the escaping co2 bubbles generated inside the aqueous core of the liposomes . in addition , the rate of diffusion of the hydronium ions across the lipid bilayer would be slower compared to the popc bilayer . we observed that both ammonium bicarbonate and sodium bicarbonate encapsulated dspc liposomes released less than 5% of their contents after incubation for 2 h at a ph of 5.0 ( supporting information , table s1 ) . we employed tapping mode atomic force microscopic imaging to determine if the escaping gas bubbles caused any structural changes ( shape and surface morphology ) to the ammonium bicarbonate encapsulated popc liposomes . after preparation , the ammonium bicarbonate encapsulated liposomes ( ph = 7.4 buffer ) were spherical with an average diameter around 100 nm ( figure 5a ) . however , after incubating in a ph 5.0 buffer for an hour , the liposomes fused , and the majority of the structures showed irregular shapes with sizes up to 800 nm ( figure 5b ) . these results demonstrated that the escaping gas bubbles caused permanent changes to the liposomes morphology , leading to leakage of the encapsulated contents . ( b ) liposomes containing 400 mm ammonium bicarbonate after incubation in ph 5 buffer for an hour . ( c ) buffer containing liposomes after incubation in a ph 5 buffer for an hour . we reasoned that the released gas bubbles inside the liposomes would allow an additional control on the contents released when employing high - frequency ultrasound . to test this hypothesis , we incubated the ammonium bicarbonate encapsulated ( 400 mm ) popc liposomes in buffers with ph of 6.0 ( figure 6a ) and 5.0 ( figure 6b ) , and after 5 min , we exposed them to continuous wave ultrasound ( 1 mhz , 2 w / cm ) for 5 min . when incubated in a ph 5.0 buffer , 80% of the encapsulated contents were released from the liposomes in 2 h ( compared to 55% released in the absence of ultrasound ; figure 6b ) . the corresponding content releases were considerably lower in ph 6.0 buffer ( figure 6a ) . decreasing the concentration of encapsulated ammonium bicarbonate ( from 400 mm to 200 mm ) reduced the amount of contents released upon the application of ultrasound , to 45% ( figure 7 ) . we also observed that the applied ultrasound exerted a maximum effect when applied within 515 min of incubating the liposomes with the ph 5 buffer . it is likely that the generated co2 bubbles escape from the liposomes within 15 min , and after that time , liposomes become less responsive to ultrasound . during the imaging studies , we observed a decrease in the echogenicity of the liposomes after 15 min of incubation in the ph 5 buffer ( figure 2 ) . ultrasound - enhanced ( 1 mhz , cw , 2 w / cm , 5 min ) , ph - triggered release from popc liposomes encapsulating 400 mm ammonium bicarbonate at ph = 6.0 ( a ) and ph = 5.0 ( b ) . orange bars : release after 2 h with ultrasound application ( n = 3 ) . ultrasound - enhanced ( 1 mhz , cw , 2 w / cm , 5 min ) , ph - triggered release from popc liposomes encapsulating 200 of mm ammonium bicarbonate at ph = 6.0 ( a ) and ph = 5.0 ( b ) . orange bars : release after 2 h with ultrasound application ( n = 3 ) . when the ammonium bicarbonate encapsulated popc liposomes were incubated in ph 6 buffer , we observed that applying the ultrasound enhanced the release by 1520% . contrary to the ph 5 experimental results , this enhancement in contents release was not strongly dependent on the time when the ultrasound was applied ( figures 6a and 7a ) . at a ph of 6 , the concentration of hydronium ions was 10 times less compared to that at a ph of 5 . the lower hydronium ion concentration at a ph of 5 contributed to a slow generation of gas bubbles inside the liposomes , and it took longer to consume the encapsulated ammonium bicarbonate . these two factors likely contributed to the results observed with ultrasound at a ph of 6.0 . we observed that applying ultrasound increased liposome solutions temperature from 25 to 30 c . it is possible that a thermal effect along with the mechanical effect could be responsible for the content release . to determine if this temperature change influenced the contents released from liposomes , we repeated the studies ( in a ph 5 buffer ) in a large ice bath . the results from these two experiments were identical , indicating that the temperature increase did not influence the contents released from our ph - sensitive liposomes . however , we note that one can not rule out the possibility of local hot spots being generated in the liposomes themselves even when they are in the ice bath . such temperature hotspot can only be created by the mechanical compression of the air cavity entrapped in the liposomes . having optimized the ultrasound - enhanced release from the ph - sensitive liposomes , we proceeded to demonstrate the effectiveness of the strategy in cellular studies . to demonstrate efficient cellular internalization , we prepared liposomes incorporating 1 mol % 1,2-distearoyl - sn - glycero-3-phosphoethanolamine - n-[folate(polyethylene glycol)-5000 ] ( ammonium salt , commercially available from avanti polar lipids ) and popc encapsulating 100 mm carboxyfluorescein . we selected the folate receptor overexpressing pancreatic ductal carcinoma cells ( panc-1 ) for our cellular studies . after incubating with the liposomes , we imaged the cells by employing a confocal fluorescence microscope . we noticed that liposomes incorporating 1 mol % of the folate lipid were taken up more effectively by the panc-1 cells compared to the liposomes without the folate lipid ( figure 8) . if the cells had a higher expression of the folate receptor , the internalization rate was faster . for example , the breast cancer cell line mcf-7 internalized the folate lipid containing liposomes faster compared to the panc-1 cells ( supporting information , figure s6 ) . fluorescence microscopic images for the uptake of ph - tunable , echogenic popc liposomes encapsulating carboxyfluorescein by the folate receptor overexpressing panc-1 cancer cells . images were obtained using different filters : brightfield ( bf ) , fluorescein isothiocyanate ( fitc , green fluorescence ) , and 4,6-diamino-2-phenylindole ( dapi , blue fluorescence ) . ( a ) nontargeted liposomes after 3 h of incubation ( magnification : 20 ) . ( b ) nontargeted liposomes after 6 h of incubation ( magnification : 20 ) . ( c ) folate - targeted liposomes after 3 h of incubation ( magnification : 20 ) . ( d ) folate - targeted liposomes after 6 h of incubation ( magnification : 20 ) . after confirming cellular internalization , we encapsulated the anticancer drug doxorubicin in the popc liposomes and studied its release in the cytosol of the panc-1 cells ( in the absence and presence of applied diagnostic frequency ultrasound ) . although gemcitabine is the standard chemotherapeutic drug for pancreatic cancer , doxorubicin is currently being tested as a possible adjuvant therapy . we noted , a priori , that some literature reports question the safety of ultrasound for healthy tissues surrounding a tumor . to determine if the ultrasound has any deleterious effects for the normal cells , we seeded the panc-1 cells onto transwell inserts consisting of two chambers . diagnostic frequency ultrasound is reported to pass through the insert and reach the lower chamber . in this experimental design , the panc-1 cells in the upper chamber represented the tumor tissue , receiving direct exposure to the liposomes as well as the applied ultrasound . the cells in the lower chamber represented the neighboring tissue , which may be indirectly exposed to the treatment ( figure 9a ) . the pore size for the transwell insert was 400 nm , and the average diameter for the liposomes was 110 nm . hence , we expected that some liposomes and ultrasound waves would pass through the membrane to reach the lower chamber . ( a ) panc-1 cell viability studies using live ( green ) and dead ( red ) cell staining of different treatment groups ( n = 3 ) . the upper chamber cells received direct exposure , whereas the lower chamber cells received indirect exposure to popc liposomes and ultrasound . ( 1 ) folate - targeted doxorubicin liposomes ( encapsulating ammonium bicarbonate ) + ultrasound . ( 2 ) nontargeted doxorubicin liposomes ( encapsulating ammonium bicarbonate ) + ultrasound . ( 4 ) folate - targeted liposomes ( encapsulating ammonium bicarbonate but no doxorubicin ) + ultrasound . ( 5 ) folate - targeted doxorubicin liposomes ( no ammonium bicarbonate encapsulation ) + ultrasound . ( 6 ) ultrasound only . ( 7 ) no treatment ( control ) . the final doxorubicin concentration used was 25 g / ml . ( b ) cell viability of the upper chamber ( orange bars ) and lower chamber ( violet bars ) . upon reaching confluency , we exposed the upper chamber s cells to various combinations of targeted / nontargeted doxorubicin - encapsulated liposomes and ultrasound ( applied between 15 and 20 min of incubation ; figure 9a ) . subsequently , we placed the cells in an incubator for 6 h and stained to visualize the live and dead cells . we observed that indirect exposure to any of the treatments did not cause cell death in the lower chamber ( figures 9a and 9b ) . on the other hand , direct exposure to folate - targeted or nontargeted , ph - tunable , doxorubicin - encapsulated liposomes and ultrasound led to significant cell death in the upper chamber ( figures 9a and 9b ) . we observed that the folate - targeted , doxorubicin and ammonium bicarbonate encapsulated popc liposomes ( figure 9a-1 ) were more toxic ( 14% cell viability ) compared to the corresponding liposomes without bicarbonate encapsulation ( cell viability 25% ; figure 9a-5 ) . it was reported that the cavitation force of exploding co2 bubbles in the lysosomes mechanically disrupts the membranes , leading to the release of lysosomal proteolytic enzymes in the cytosol and to cell death . contrary to this report , we observed less than 5% cell death in the presence of liposomes that only encapsulate ammonium bicarbonate ( i.e. , without doxorubicin ; figure 9a-4 ) . these results indicated that , in our experiments , cavitation induced by co2 bubbles enhanced the toxicity of the liposomal formulations . clearly , the folate - targeted doxorubicin liposomes ( encapsulating ammonium bicarbonate ) in the presence of applied ultrasound were most effective in killing the panc-1 cells ( figures 9a-1 and 9b , group 1 ) . this combination reduced the cell viability to 14% ( figure 9b , group 1 ) . interestingly , the free doxorubicin in the presence of applied ultrasound was more effective compared to liposomal doxorubicin ( without folate ) in inducing cell death ( figures 9a-2 and 9a-3 ) . it is likely that sonoporation by the ultrasound is contributing to this effect . in the absence of any microbubbles , the pores formed in the cell membranes by the applied ultrasound are likely to be small and transient . possibly , the sizes of these transient pores are large enough to allow doxorubicin molecules to cross the cell membranes . these observations are consistent with literature reports demonstrating a higher uptake of smaller particles compared to larger ones upon sonoporation . we observed that , under our experimental conditions , direct or indirect exposure to the ultrasound does not induce cell death ( figure 9a-6 ) . our objective was not to compare the efficacy of free doxorubicin in the absence and presence of applied diagnostic - frequency ultrasound . our goal was to determine the effectiveness of the ammonium bicarbonate and doxorubicin coencapsulated liposomes when ultrasound is applied . numerous literature reports demonstrate the efficacy of doxorubicin for panc-1 cells ( without the applied ultrasound ) ; hence , we did not include this control experiment . panc-1 is a metastatic pancreatic cancer cell line known to secrete matrix metalloproteinase ( mmp ) -2 and -9 enzymes in the extracellular matrix . these two proteolytic enzymes are responsible for the hydrolysis of the extracellular matrix , leading to the migration and metastasis of cancer cells . ultrasound treatment can loosen the extracellular material surrounding a tumor , resulting in the dissemination of cancer cells into the bloodstream . this action leads to increased migration and metastasis of the cancer cells when exposed to ultrasound . to determine if our experimental conditions contribute to such effects , we conducted migration assays of the panc-1 cells in the presence of applied ultrasound . for this endeavor after 6 h , we exposed the cells to ultrasound ( 1 mhz , 5 min ) , incubated them overnight , and determined their migration . we observed that there was no significant difference ( p > 0.01 , n = 5 ) in the migration ability of the ultrasound - exposed cells compared to the control samples ( no ultrasound exposure ) . these results suggested that , within our experimental parameters , the migration of the panc-1 cells remained unaffected by the applied ultrasound . further in vivo validation studies with the ammonium bicarbonate encapsulated liposomes are in progress , and the results will be reported in the future . we have successfully demonstrated the proof - of - concept for a new strategy to release liposomal contents in response to reduced ph . with our design , the liposomes encapsulate the gas precursor , ammonium bicarbonate , and do not incorporate ph - sensitive lipids in the bilayer . when incubated in buffers of acidic ph , co2 gas bubbles are generated , thus , inducing echogenicity to the liposomes . the escaping gas bubbles cause structural changes to the liposomes , and release the encapsulated contents ( up to 56% ) . the content release is further enhanced by the simultaneous application of diagnostic - frequency ultrasound ( 1 mhz , 5 min ; 80% release ) . the fluidity of the liposomal membranes plays a crucial role in the contents released . by incorporating a folate lipid in the bilayer , we have successfully targeted the liposomes to pancreatic cancer cells that overexpress the folate receptor on the surface . liposome - encapsulated doxorubicin is efficiently released in the cancer cells , and the release is enhanced by the simultaneous application of diagnostic frequency ultrasound . while the ultrasound was innocuous , the combination of doxorubicin released from the liposomes and ultrasound reduced the viability of pancreatic cancer cells to 14% . with further developments , these liposomes have the potential to be an excellent option for ultrasound image guided , targeted drug delivery at tumor sites .

liposomes are representative lipid nanoparticles widely used for delivering anticancer drugs , dna fragments , or sirna to cancer cells . upon targeting , various internal and external triggers have been used to increase the rate for contents release from the liposomes . among the internal triggers , decreased ph within the cellular lysosomes has been successfully used to enhance the rate for releasing contents . however , imparting ph sensitivity to liposomes requires the synthesis of specialized lipids with structures that are substantially modified at a reduced ph . herein , we report an alternative strategy to render liposomes ph sensitive by encapsulating a precursor which generates gas bubbles in situ in response to acidic ph . the disturbance created by the escaping gas bubbles leads to the rapid release of the encapsulated contents from the liposomes . atomic force microscopic studies indicate that the liposomal structure is destroyed at a reduced ph . the gas bubbles also render the liposomes echogenic , allowing ultrasound imaging . to demonstrate the applicability of this strategy , we have successfully targeted doxorubicin - encapsulated liposomes to the pancreatic ductal carcinoma cells that overexpress the folate receptor on the surface . in response to the decreased ph in the lysosomes , the encapsulated anticancer drug is efficiently released . contents released from these liposomes are further enhanced by the application of continuous wave ultrasound ( 1 mhz ) , resulting in substantially reduced viability for the pancreatic cancer cells ( 14% ) .

Introduction Experimental Section Results and Discussion Conclusions

among the lipid nanoparticles , liposomes are widely studied as drug delivery vehicles . however , upon targeting , the passive release of the encapsulated drugs from the liposomes is often slow . reorganization of the lipid domains has been used as a trigger to enhance , and to control the rate and the extent of contents released from liposomes . among the various triggers , decreased ph in the lysosomes has been widely used as a successful strategy to efficiently release the encapsulated liposomal contents . however , imparting ph sensitivity to liposomes requires the synthesis of specialized lipids with structures that are substantially modified , either due to hydrolysis or due to changes in the protonation states of the lipid head groups , at reduced ph . stabilized gas bubbles are widely used as contrast - enhancing agents for ultrasound imaging of perfused tissues . although ultrasound waves in the khz frequency efficiently release drugs from the carriers ( due to cavitation and high local temperatures ) , the harmful biological effects associated with low - frequency ultrasound limit the usefulness of such strategies . although there is uncertainty about the exact location and size of the entrapped air bubbles in the elips , their acoustic characterization has been reported extensively in the literature . we are developing targeted , multimodal liposomes for triggered release of encapsulated contents , and simultaneous ultrasound imaging . furthermore , we are interested in enhancing the contents released from the liposomes by employing diagnostic frequency ( mhz ) ultrasound . we have recently demonstrated the ultrasound - enhanced , extracellular release of liposomal contents mediated by the cancer - cell - secreted enzyme matrix metalloproteinase-9 ( mmp-9 ) . herein , we report a strategy to render liposomes ph sensitive by encapsulating ammonium bicarbonate which generates gas bubbles in situ in response to acidic ph . we hypothesize that , at a reduced ph , the hydronium ions diffuse into the aqueous interior of the liposomes , and produce carbon dioxide bubbles , thereby turning on the echogenicity . as more bubbles are generated , the liposomal bilayer is disturbed , leading to the release of encapsulated contents . we observe that the release was further enhanced by applying ultrasound with a frequency of 1 mhz . to the best of our knowledge there are no reports in the literature of ultrasound enhanced triggered release from ph - tunable echogenic liposomes . we have demonstrated the usefulness of this liposomal delivery system using the panc-1 pancreatic cancer cells . pancreatic cancer is one of the leading causes of cancer - related deaths in both men and women in the united states , with a 5-year survival rate of less than 5% . to demonstrate tunable echogenicity , we prepared the liposomes from 1-palmitoyl-2-oleoyl - sn - glycero-3-phosphocholine ( popc ) , encapsulating 400 mm ammonium bicarbonate along with the self - quenching dye carboxyfluorescein ( 100 mm ) . the presence of several lipid bilayers was also expected to decrease the efficiency of the contents released in response to escaping gas bubbles and ultrasonic excitation . we observed ( with dynamic light scattering ) that the average hydrodynamic diameter of the liposomes is 110 15 nm with a polydispersity index of 0.05 ( supporting information , figure s1a ) . to demonstrate the tunable echogenicity , we added the liposomes to buffers with different phs ( 7.45.0 ) and recorded the images using a terason t3200 high - frequency ( 1214 mhz ) diagnostic ultrasound transducer . we expected that the generated gas bubbles would reflect the ultrasound , and that the contrast of the images would be more pronounced as the amount of generated gas is increased at a lower ph . we observed that there was a lag time before the liposomes became echogenic , and that the duration of this lag phase decreased with the reduced ph . figure 1a shows that there is no echo at ph 7.4 ; but as the ph is reduced , progressively stronger echo from the entire cell well is observed ( note that the ultrasound probe is placed at the top of the cell well ) . we anticipated that the concentration of hydronium ions in the external buffer would affect their diffusion rate inside the liposomes as well as the subsequent generation of co2 bubbles . consistent with this hypothesis , we observed that liposomes in the ph 5 buffer are not echogenic after 20 min ( figure 2 ) . however , we noted that the diameters of the gas bubbles inside the liposomes are likely to be small ( in nanometers ) , and that they may not reflect the ultrasound very well . it is likely that the nanobubbles coalesce in the lipid bilayer of the liposomes , generating larger bubbles , and reflect the ultrasound . popc lipid has a gel low transition temperature ( 2 c ) , and the liposomal bilayer is in the fluid phase under the experimental conditions ( 20 c ) . the loose lipid packing and fluidity of the popc bilayer accommodate the coalescence and the size increase of the gas bubbles . having demonstrated ph - tunable echogenicity , we decided to determine if the escaping gas bubbles sufficiently disturb the lipid bilayer to release the encapsulated contents from the liposomes . however , the liposomes continued to leak the contents for a considerably long time ( 23 h ) . the continued leakage indicates that the disturbances in the lipid bilayers created by the escaping gas bubbles either are not sealed or take a long time to heal . while the liposomes at a ph of 7.4 ( control ) released only 15% of the encapsulated dye in 2 h , at a ph of 5 , the release increased to 55% ( figure 3 ) . when we encapsulated sodium bicarbonate in the liposomes ( instead of ammonium bicarbonate ) , the amount of the content release decreased . in 2 h , we observed that the sodium bicarbonate encapsulated liposomes released 40% of the encapsulated dye ( at ph = 5.0 ; supporting information , figures s3 and s4 ) . for both of these liposomal formulations , the rate of contents release decreased considerably after 2 h. in 3 h at ph 5.0 , the ammonium bicarbonate encapsulated liposomes released 75% of the contents , and the sodium bicarbonate encapsulated liposomes released 44% of the contents ( figure 4a ) . decreasing the amount of encapsulated ammonium bicarbonate ( from 400 mm to 200 mm ) also reduced the amount of contents release from the liposomes ( figure 4b ) . amount of generated gas decreases by reducing the concentration of encapsulated ammonium bicarbonate ( from 400 to 200 mm ) , leading to a reduction in contents release from the liposomes ( figure 4b ) . as an additional control , we prepared the popc liposomes without encapsulating any gas precursor and studied the contents release as a function of ph . we observed minimal release ( < 10% ) of the encapsulated carboxyfluorescein at ph 7.4 and 6.0 . it is possible that the loose packing of the popc lipids will likely allow the escaping co2 bubbles to coalesce inside the hydrophobic bilayer of the liposomes . to determine if the lipid packing in the liposomal bilayer and the gel transition temperature affect the contents released , we prepared two batches of dspc ( 1,2-distearoyl - sn - glycero-3-phosphocholine ) liposomes that encapsulate ammonium and sodium bicarbonate respectively ( 400 mm each ) . we hypothesized that the tightly packed lipid molecules in the bilayer would hinder the coalescence of the escaping co2 bubbles generated inside the aqueous core of the liposomes . in addition , the rate of diffusion of the hydronium ions across the lipid bilayer would be slower compared to the popc bilayer . we employed tapping mode atomic force microscopic imaging to determine if the escaping gas bubbles caused any structural changes ( shape and surface morphology ) to the ammonium bicarbonate encapsulated popc liposomes . after preparation , the ammonium bicarbonate encapsulated liposomes ( ph = 7.4 buffer ) were spherical with an average diameter around 100 nm ( figure 5a ) . however , after incubating in a ph 5.0 buffer for an hour , the liposomes fused , and the majority of the structures showed irregular shapes with sizes up to 800 nm ( figure 5b ) . these results demonstrated that the escaping gas bubbles caused permanent changes to the liposomes morphology , leading to leakage of the encapsulated contents . we reasoned that the released gas bubbles inside the liposomes would allow an additional control on the contents released when employing high - frequency ultrasound . to test this hypothesis , we incubated the ammonium bicarbonate encapsulated ( 400 mm ) popc liposomes in buffers with ph of 6.0 ( figure 6a ) and 5.0 ( figure 6b ) , and after 5 min , we exposed them to continuous wave ultrasound ( 1 mhz , 2 w / cm ) for 5 min . when incubated in a ph 5.0 buffer , 80% of the encapsulated contents were released from the liposomes in 2 h ( compared to 55% released in the absence of ultrasound ; figure 6b ) . decreasing the concentration of encapsulated ammonium bicarbonate ( from 400 mm to 200 mm ) reduced the amount of contents released upon the application of ultrasound , to 45% ( figure 7 ) . it is likely that the generated co2 bubbles escape from the liposomes within 15 min , and after that time , liposomes become less responsive to ultrasound . during the imaging studies , we observed a decrease in the echogenicity of the liposomes after 15 min of incubation in the ph 5 buffer ( figure 2 ) . ultrasound - enhanced ( 1 mhz , cw , 2 w / cm , 5 min ) , ph - triggered release from popc liposomes encapsulating 400 mm ammonium bicarbonate at ph = 6.0 ( a ) and ph = 5.0 ( b ) . ultrasound - enhanced ( 1 mhz , cw , 2 w / cm , 5 min ) , ph - triggered release from popc liposomes encapsulating 200 of mm ammonium bicarbonate at ph = 6.0 ( a ) and ph = 5.0 ( b ) . contrary to the ph 5 experimental results , this enhancement in contents release was not strongly dependent on the time when the ultrasound was applied ( figures 6a and 7a ) . the lower hydronium ion concentration at a ph of 5 contributed to a slow generation of gas bubbles inside the liposomes , and it took longer to consume the encapsulated ammonium bicarbonate . to determine if this temperature change influenced the contents released from liposomes , we repeated the studies ( in a ph 5 buffer ) in a large ice bath . the results from these two experiments were identical , indicating that the temperature increase did not influence the contents released from our ph - sensitive liposomes . however , we note that one can not rule out the possibility of local hot spots being generated in the liposomes themselves even when they are in the ice bath . such temperature hotspot can only be created by the mechanical compression of the air cavity entrapped in the liposomes . having optimized the ultrasound - enhanced release from the ph - sensitive liposomes , we proceeded to demonstrate the effectiveness of the strategy in cellular studies . we selected the folate receptor overexpressing pancreatic ductal carcinoma cells ( panc-1 ) for our cellular studies . we noticed that liposomes incorporating 1 mol % of the folate lipid were taken up more effectively by the panc-1 cells compared to the liposomes without the folate lipid ( figure 8) . if the cells had a higher expression of the folate receptor , the internalization rate was faster . for example , the breast cancer cell line mcf-7 internalized the folate lipid containing liposomes faster compared to the panc-1 cells ( supporting information , figure s6 ) . fluorescence microscopic images for the uptake of ph - tunable , echogenic popc liposomes encapsulating carboxyfluorescein by the folate receptor overexpressing panc-1 cancer cells . after confirming cellular internalization , we encapsulated the anticancer drug doxorubicin in the popc liposomes and studied its release in the cytosol of the panc-1 cells ( in the absence and presence of applied diagnostic frequency ultrasound ) . in this experimental design , the panc-1 cells in the upper chamber represented the tumor tissue , receiving direct exposure to the liposomes as well as the applied ultrasound . ( 1 ) folate - targeted doxorubicin liposomes ( encapsulating ammonium bicarbonate ) + ultrasound . upon reaching confluency , we exposed the upper chamber s cells to various combinations of targeted / nontargeted doxorubicin - encapsulated liposomes and ultrasound ( applied between 15 and 20 min of incubation ; figure 9a ) . we observed that indirect exposure to any of the treatments did not cause cell death in the lower chamber ( figures 9a and 9b ) . on the other hand , direct exposure to folate - targeted or nontargeted , ph - tunable , doxorubicin - encapsulated liposomes and ultrasound led to significant cell death in the upper chamber ( figures 9a and 9b ) . we observed that the folate - targeted , doxorubicin and ammonium bicarbonate encapsulated popc liposomes ( figure 9a-1 ) were more toxic ( 14% cell viability ) compared to the corresponding liposomes without bicarbonate encapsulation ( cell viability 25% ; figure 9a-5 ) . it was reported that the cavitation force of exploding co2 bubbles in the lysosomes mechanically disrupts the membranes , leading to the release of lysosomal proteolytic enzymes in the cytosol and to cell death . clearly , the folate - targeted doxorubicin liposomes ( encapsulating ammonium bicarbonate ) in the presence of applied ultrasound were most effective in killing the panc-1 cells ( figures 9a-1 and 9b , group 1 ) . in the absence of any microbubbles , the pores formed in the cell membranes by the applied ultrasound are likely to be small and transient . numerous literature reports demonstrate the efficacy of doxorubicin for panc-1 cells ( without the applied ultrasound ) ; hence , we did not include this control experiment . panc-1 is a metastatic pancreatic cancer cell line known to secrete matrix metalloproteinase ( mmp ) -2 and -9 enzymes in the extracellular matrix . these two proteolytic enzymes are responsible for the hydrolysis of the extracellular matrix , leading to the migration and metastasis of cancer cells . ultrasound treatment can loosen the extracellular material surrounding a tumor , resulting in the dissemination of cancer cells into the bloodstream . this action leads to increased migration and metastasis of the cancer cells when exposed to ultrasound . to determine if our experimental conditions contribute to such effects , we conducted migration assays of the panc-1 cells in the presence of applied ultrasound . for this endeavor after 6 h , we exposed the cells to ultrasound ( 1 mhz , 5 min ) , incubated them overnight , and determined their migration . we observed that there was no significant difference ( p > 0.01 , n = 5 ) in the migration ability of the ultrasound - exposed cells compared to the control samples ( no ultrasound exposure ) . these results suggested that , within our experimental parameters , the migration of the panc-1 cells remained unaffected by the applied ultrasound . further in vivo validation studies with the ammonium bicarbonate encapsulated liposomes are in progress , and the results will be reported in the future . we have successfully demonstrated the proof - of - concept for a new strategy to release liposomal contents in response to reduced ph . with our design , the liposomes encapsulate the gas precursor , ammonium bicarbonate , and do not incorporate ph - sensitive lipids in the bilayer . when incubated in buffers of acidic ph , co2 gas bubbles are generated , thus , inducing echogenicity to the liposomes . the escaping gas bubbles cause structural changes to the liposomes , and release the encapsulated contents ( up to 56% ) . the content release is further enhanced by the simultaneous application of diagnostic - frequency ultrasound ( 1 mhz , 5 min ; 80% release ) . the fluidity of the liposomal membranes plays a crucial role in the contents released . by incorporating a folate lipid in the bilayer , we have successfully targeted the liposomes to pancreatic cancer cells that overexpress the folate receptor on the surface . liposome - encapsulated doxorubicin is efficiently released in the cancer cells , and the release is enhanced by the simultaneous application of diagnostic frequency ultrasound . while the ultrasound was innocuous , the combination of doxorubicin released from the liposomes and ultrasound reduced the viability of pancreatic cancer cells to 14% .

parental diabetes status was determined from a longitudinal study of health conducted within the gila river ( pima ) indian community in which residents 5 years old have been invited for research examinations consisting of the measurement of height and weight and a 75-g oral glucose tolerance test ( ogtt ) approximately every 2 years . diabetes was diagnosed according to world health organization 1999 criteria ( 7 ) or by review of the medical record if made in a clinical setting . we defined three groups of offspring according to parental diabetes status : 1 ) offspring of diabetic fathers ( odf ) consisting of subjects whose father developed diabetes at < 35 years of age , but whose mother remained nondiabetic until > 35 years of age ; 2 ) offspring of mothers with early onset of diabetes ( omed ) consisting of subjects whose mother developed diabetes at < 35 years of age , but after the date of birth of the subject documented by a normal ogtt following delivery ( mean sd time between subject 's date of birth and mothers date of diagnosis , 8.9 4.2 years ) , and whose fathers remained nondiabetic until > 35 years of age ; and 3 ) a control group ( con ) consisting of offspring whose parents were known to be nondiabetic until > 50 years of age . the third group can be considered a control group because of previous evidence that offspring of parents who do not develop diabetes until age > 50 years have a risk for developing diabetes comparable with that of offspring whose parents never developed diabetes ( 8) . for additional characterization of the parents , we determined the diabetic cumulative incidence ( ci ) score of each parent in the sample ( 8) . briefly , the diabetes score was derived by first calculating the specific cumulative incidence of diabetes as a function of age ( cia ) in the pima population . if diabetes was present , then the score is calculated by 1 cia at the age of first diagnosis of diabetes . if diabetes was not present , then score is calculated as 0 cia at the time of the last biennial examination . the score thus contains information on whether an individual developed diabetes and the age of onset of diabetes and is positive if diabetes was ever present and greater if diabetes developed at an earlier age . conversely , a negative score is calculated if the individual was nondiabetic at the last biennial examination and is most negative in those who remain nondiabetic into old age . volunteers also participated in a study of metabolic determinants for the development of type 2 diabetes and obesity . at the time of their participation , all subjects were in good health and without evidence of diabetes ( by ogtt ) , as determined by a comprehensive medical evaluation including medical history , physical examination , and routine laboratory testing . subjects were admitted for 1015 days to the clinical research unit of the national institute of diabetes and digestive and kidney diseases in phoenix , arizona , and were provided a standard weight - maintaining energy needs diet containing 50% of calories as carbohydrate , 30% as fat , and 20% as protein for at least 3 days before metabolic testing . after at least 3 days , volunteers underwent a 75-g ogtt after a 12-h overnight fast to exclude diabetes . only subjects who were full - heritage pima indian or related tohono o'odham were included in the analysis . body composition was measured by dual - energy x - ray absorptiometry using a total body scanner ( dpx - l ; lunar , madison , wi ) . percentage body fat ( % bf ) , fat mass ( fm ) , and fat - free mass ( ffm ) were calculated as previously described ( 9 ) . insulin action was assessed at physiological insulin concentrations during the hyperinsulinemic - euglycemic clamp technique ( 10 ) . briefly , after an overnight fast , a primed ( 30 ci ) continuous ( 0.3 ci / min ) infusion of [ 3-h ] glucose was started to determine endogenous glucose production ( egp ) . at least 2 h after starting the isotope infusion , a primed continuous intravenous insulin infusion was administered for 100 min at a constant rate of 40 mu / m / min . blood samples for measurement of [ 3-h ] glucose specific activity were collected at the end of the basal period and every 10 min during the final 40 min of insulin infusion . under basal ( i.e. , fasting ) conditions , egp was calculated as the [ 3-h ] glucose infusion rate divided by the steady - state plasma 3-[h ] glucose specific activity . the rate of total insulin - stimulated glucose disposal was calculated for the last 40 min of the insulin infusion and was corrected for the rate of egp calculated from the steele equation ( 11 ) . individual variation in plasma glucose and insulin concentrations during the clamp were taken into account in the calculation of insulin action ( m - low ) ( 12 ) . all measurements derived from the clamp were normalized to estimated metabolic body size ( embs ) , which is directly derived from ffm but takes into account the fact that the intercept of the relationship between ffm and resting metabolic rate is not zero ( 17.7 kg in our laboratory [ i.e. , embs = ffm + 17.7 kg ] ) ( 12 ) . insulin secretion was measured as the response to a 25-g intravenous glucose tolerance test ( ivgtt ) . the air was calculated as the average increment in plasma insulin concentration above basal in samples obtained 3 , 4 , and 5 min after the bolus injection of glucose ( 13 ) . at 0730 h , subjects were fed a mixed - meal breakfast ( consisting of a bacon - and - egg sandwich on toast accompanied by orange juice ) containing 10% of calories from protein , 45% from fat , and 45% from carbohydrates and providing 20% of daily energy requirements for each subject . the meal was consumed within 15 min , and subjects rested quietly in bed throughout the study . blood samples for insulin and glucose were drawn before and 30 , 60 , 90 , 120 , 150 , and 180 min after initiation of the meal test . plasma glucose concentrations were measured by the glucose oxidize method ( beckman instruments , fullerton , ca ) . plasma insulin concentrations were measured by the herbert modification of the method of yalow and berson ( 14 ) , by an automated auto - analyzer ( icn radiochemicals , costa mesa , ca ) , or by an automated immunoassay ( access , beckman instruments ) . all studies were approved by the institutional review board of the national institute of diabetes and digestive and kidney disease . statistical analyses were performed using the procedures of the sas statistical package ( version 8.2 ; sas institute , cary , nc ) . air and m - low were log10 transformed to normalize their distributions before parametric analyses . unless otherwise specified , all data are expressed as means sd . general , anthropometric , and metabolic characteristics in table 1 were evaluated using student 's t test , anova , or analyses for continuous and categorical variables , respectively . since even mild degrees of glucose intolerance can be associated with impairments in insulin secretion ( 15 ) only subjects with normal glucose tolerance were included for further analyses of air . the glucose and insulin responses during the ogtt , ivgtt , and meal test by group were compared by ancova using the mixed procedure in sas . the general linear models were used to adjust % bf for age and sex ; m - low ( mg kg estimated metabolic body size ( embs ) min ) for age , sex , and % bf , and air for age , sex , % bf , and m - low ( 10 ) . adjusted least - squares means ( lsmeans ) were used to compare groups by parental diabetes status using post hoc t test , with tukey 's honestly significant difference adjustment for multiple comparisons . for all groups , further analyses were conducted and adjusted for the same covariates using general estimating equations to account for family membership . general , anthropometric , and body composition parameters of subjects according to the age of diabetes onset in the parents * birth weight in odf group available only in seven subjects . p < 0.05 odf vs. con ( anova ; sex differences analysis ) . bgo , basal glucose output ; igt , impaired glucose tolerance ; ngt , normal glucose tolerance . parental diabetes status was determined from a longitudinal study of health conducted within the gila river ( pima ) indian community in which residents 5 years old have been invited for research examinations consisting of the measurement of height and weight and a 75-g oral glucose tolerance test ( ogtt ) approximately every 2 years . diabetes was diagnosed according to world health organization 1999 criteria ( 7 ) or by review of the medical record if made in a clinical setting . we defined three groups of offspring according to parental diabetes status : 1 ) offspring of diabetic fathers ( odf ) consisting of subjects whose father developed diabetes at < 35 years of age , but whose mother remained nondiabetic until > 35 years of age ; 2 ) offspring of mothers with early onset of diabetes ( omed ) consisting of subjects whose mother developed diabetes at < 35 years of age , but after the date of birth of the subject documented by a normal ogtt following delivery ( mean sd time between subject 's date of birth and mothers date of diagnosis , 8.9 4.2 years ) , and whose fathers remained nondiabetic until > 35 years of age ; and 3 ) a control group ( con ) consisting of offspring whose parents were known to be nondiabetic until > 50 years of age . the third group can be considered a control group because of previous evidence that offspring of parents who do not develop diabetes until age > 50 years have a risk for developing diabetes comparable with that of offspring whose parents never developed diabetes ( 8) . for additional characterization of the parents , we determined the diabetic cumulative incidence ( ci ) score of each parent in the sample ( 8) . briefly , the diabetes score was derived by first calculating the specific cumulative incidence of diabetes as a function of age ( cia ) in the pima population . if diabetes was present , then the score is calculated by 1 cia at the age of first diagnosis of diabetes . if diabetes was not present , then score is calculated as 0 cia at the time of the last biennial examination . the score thus contains information on whether an individual developed diabetes and the age of onset of diabetes and is positive if diabetes was ever present and greater if diabetes developed at an earlier age . conversely , a negative score is calculated if the individual was nondiabetic at the last biennial examination and is most negative in those who remain nondiabetic into old age . volunteers also participated in a study of metabolic determinants for the development of type 2 diabetes and obesity . at the time of their participation , all subjects were in good health and without evidence of diabetes ( by ogtt ) , as determined by a comprehensive medical evaluation including medical history , physical examination , and routine laboratory testing . subjects were admitted for 1015 days to the clinical research unit of the national institute of diabetes and digestive and kidney diseases in phoenix , arizona , and were provided a standard weight - maintaining energy needs diet containing 50% of calories as carbohydrate , 30% as fat , and 20% as protein for at least 3 days before metabolic testing . after at least 3 days , volunteers underwent a 75-g ogtt after a 12-h overnight fast to exclude diabetes . only subjects who were full - heritage pima indian or related tohono o'odham were included in the analysis . body composition was measured by dual - energy x - ray absorptiometry using a total body scanner ( dpx - l ; lunar , madison , wi ) . percentage body fat ( % bf ) , fat mass ( fm ) , and fat - free mass ( ffm ) were calculated as previously described ( 9 ) . insulin action was assessed at physiological insulin concentrations during the hyperinsulinemic - euglycemic clamp technique ( 10 ) . briefly , after an overnight fast , a primed ( 30 ci ) continuous ( 0.3 ci / min ) infusion of [ 3-h ] glucose was started to determine endogenous glucose production ( egp ) . at least 2 h after starting the isotope infusion , a primed continuous intravenous insulin infusion was administered for 100 min at a constant rate of 40 mu / m / min . blood samples for measurement of [ 3-h ] glucose specific activity were collected at the end of the basal period and every 10 min during the final 40 min of insulin infusion . under basal ( i.e. , fasting ) conditions , egp was calculated as the [ 3-h ] glucose infusion rate divided by the steady - state plasma 3-[h ] glucose specific activity . the rate of total insulin - stimulated glucose disposal was calculated for the last 40 min of the insulin infusion and was corrected for the rate of egp calculated from the steele equation ( 11 ) . individual variation in plasma glucose and insulin concentrations during the clamp were taken into account in the calculation of insulin action ( m - low ) ( 12 ) . all measurements derived from the clamp were normalized to estimated metabolic body size ( embs ) , which is directly derived from ffm but takes into account the fact that the intercept of the relationship between ffm and resting metabolic rate is not zero ( 17.7 kg in our laboratory [ i.e. , embs = ffm + 17.7 kg ] ) ( 12 ) . insulin secretion was measured as the response to a 25-g intravenous glucose tolerance test ( ivgtt ) . the air was calculated as the average increment in plasma insulin concentration above basal in samples obtained 3 , 4 , and 5 min after the bolus injection of glucose ( 13 ) . at 0730 h , subjects were fed a mixed - meal breakfast ( consisting of a bacon - and - egg sandwich on toast accompanied by orange juice ) containing 10% of calories from protein , 45% from fat , and 45% from carbohydrates and providing 20% of daily energy requirements for each subject . the meal was consumed within 15 min , and subjects rested quietly in bed throughout the study . blood samples for insulin and glucose were drawn before and 30 , 60 , 90 , 120 , 150 , and 180 min after initiation of the meal test . plasma glucose concentrations were measured by the glucose oxidize method ( beckman instruments , fullerton , ca ) . plasma insulin concentrations were measured by the herbert modification of the method of yalow and berson ( 14 ) , by an automated auto - analyzer ( icn radiochemicals , costa mesa , ca ) , or by an automated immunoassay ( access , beckman instruments ) . all studies were approved by the institutional review board of the national institute of diabetes and digestive and kidney disease . statistical analyses were performed using the procedures of the sas statistical package ( version 8.2 ; sas institute , cary , nc ) . air and m - low were log10 transformed to normalize their distributions before parametric analyses . unless otherwise specified , all data are expressed as means sd . general , anthropometric , and metabolic characteristics in table 1 were evaluated using student 's t test , anova , or analyses for continuous and categorical variables , respectively . since even mild degrees of glucose intolerance can be associated with impairments in insulin secretion ( 15 ) only subjects with normal glucose tolerance were included for further analyses of air . the glucose and insulin responses during the ogtt , ivgtt , and meal test by group were compared by ancova using the mixed procedure in sas . the general linear models were used to adjust % bf for age and sex ; m - low ( mg kg estimated metabolic body size ( embs ) min ) for age , sex , and % bf , and air for age , sex , % bf , and m - low ( 10 ) . adjusted least - squares means ( lsmeans ) were used to compare groups by parental diabetes status using post hoc t test , with tukey 's honestly significant difference adjustment for multiple comparisons . for all groups , further analyses were conducted and adjusted for the same covariates using general estimating equations to account for family membership . general , anthropometric , and body composition parameters of subjects according to the age of diabetes onset in the parents * birth weight in odf group available only in seven subjects . p < 0.05 odf vs. con ( anova ; sex differences analysis ) . bgo , basal glucose output ; igt , impaired glucose tolerance ; ngt , normal glucose tolerance . general , anthropometric , and metabolic characteristics of the study population are shown in table 1 . we found 10 odf ; 8 of 10 fathers from the odf group had diabetes preconception , and 2 fathers had diagnoses of diabetes 3 and 4 years after the offspring birth . onset age of diabetes for fathers was lower in the odf group than for mothers in the omed group ( 26.6 4.2 vs. 30.4 3.0 years , p = 0.02 ) . this slight difference in age was due to exclusion of mothers in the omed group who did not have a nondiabetic ogtt following childbirth . consistent with this age difference , fathers ' ci score in the odf group was higher than mothers ' ci score in the omed group ( p = 0.04 , table 1 ) . there was no significant difference between mothers ci score in the odf group and fathers ci score in the omed group , indicating no difference in relative age of onset of diabetes in the opposite parent . the ci score was comparable between mothers and fathers in the con group ( table 1 ) . the birth weight and height was not different among the three groups ( table 1 ) . after adjustment for age and sex , % bf remained lower in the odf group than in the con group ( lsmeans [ 95% ci ] : 27.3% [ 23.331.3 ] in odf vs. 35.4% [ 32.238.5 ] in con and 32.4% [ 28.836.1 ] in omed , p = 0.04 ) . the ffm , adjusted for age and sex , tended to be lower in odf in comparison to con and omed , but did not reach statistical significance ( 57.9 kg [ 50.465.3 ] in odf vs. 65.8 kg [ 60.071.7 ] in con , and 67.2 kg [ 60.474.1 ] in omed , p = 0.1 ) . odfs had lower fasting glucose , but 2-h plasma glucose concentrations were comparable with those of cons or omeds ( p = 0.01 and p = 0.1 , respectively , anova ; table 1 ) . despite similar fasting plasma insulin concentrations and glucose responses during the ogtt , plasma insulin concentrations at 30 and 120 min during ogtt ( ins30 , ins120 ) were lower in the odf group than in the con and omed groups ( f = 4.38 , p = 0.02 ; f = 3.81 , p = 0.04 , respectively ; fig . 1 ) . after adjustment for age , sex , 30 min glucose concentration , and m - low , only ins30 remained lower in the odf group than in both the con and omed groups ( lsmeans [ 95% ci ] : 722 pmol / l [ 1321,582 ] in odf vs. 2,699 plasma glucose and insulin concentrations during ogtt ( a and b ) , during ivgtt ( c and d ) , and during meal test ( e and f ) in odf , con , and omed . statistical significance as revealed by two - way anova for the time and group factors and their interactions ( time group ) . ogtt : post hoc tests for significant differences in time versus group interactions ( effect of time [ p < 0.0001 for all models ] ) ; effect of group ( glucose p = 0.02 , insulin p = 0.001 ) ; effect of time groups interaction ( glucose p = 0.3 , insulin p = 0.1 ) . ivgtt : post hoc tests for significant differences in time versus group interactions ( effect of time [ p < 0.0001 for all models ] ) ; effect of group ( glucose p = 0.02 , insulin p < 0.0001 ) ; effect of time groups interaction , ( glucose p = 0.3 , insulin p < 0.0001 ) . ivgtt data available only in six subjects in the odf , six in the con , and seven in the omed group . meal test : post hoc tests for significant differences in time versus group interactions ( effect of time [ p < 0.0001 for all models ] ) ; effect of group ( glucose p = 0.2 , insulin p < 0.0001 ) ; effect of time groups interaction ( glucose p = 0.3 , insulin p = 0.002 ) . meal test data available only in six subjects in the odf , eight in the con , and eleven in the omed group . intravenous glucose administration resulted in a comparable increase in plasma glucose levels in all three groups . the insulin responses were significantly lower in odf than in con subjects ( effect of diagnosis time p < 0.0001 ; anova ; fig . 1 ) . in addition , log - transformed air was lower in the odf group than the con group ( table 1 ) , and it remained significant after adjustment for age , sex , % bf , and m - low ( lsmeans [ 95% ci ] : 2.08 pmol / l ( 1.962.20 ) in odf vs. 2.55 pmol [ 2.242.75 ] in con and 2.40 pmol / l [ 2.132.55 ] in omed , p < 0.05 ) . during the meal test the insulin responses during the meal test were significantly lower in odf than in con subjects ( effect of diagnosis time sex p = 0.02 ; fig . plasma insulin concentrations at 30 min of the meal test were slightly lower in the odf group compared to both the con and omed groups , but it did not reach statistical significance prior to adjustment ( 1,084 462 pmol / l in odf vs. 2090 1,081 1 ) , or following adjustment for age , sex , 30-min plasma glucose concentration , and m - low ( lsmeans [ 95% ci ] : 1,422 pmol / l [ 4892,355 ] in odf vs. 2,087 pmol / l ( 1,3722,803 ) in con and 1,492 pmol / l [ 1,0591924 ] in omed , p = 0.1 ) . odfs were on average leaner ( having a lower % bf ) ; therefore the mean log transformed m - lows were higher in the odf group than in the con and omed groups ( table 1 ) , but after adjustment for age , sex , and % bf , m - lows were comparable in all three groups ( lsmeans [ 95% ci ] : 2.68 [ 1.803.55 ] mg kg embs min in odf vs. 2.34 [ 1.732.96 ] mg kg embs min in con , 2.28 [ 1.872.68 ] mg kg embs min in omed , p = 0.4 ) . our analyses indicate that adult nondiabetic offspring of fathers with early onset of diabetes are leaner than offspring of either mothers with early onset of type 2 diabetes or control subjects ( neither parent developed type 2 diabetes by age 50 years ) . odfs had lower insulin secretion , demonstrated by multiple tests ( ivgtt , ogtt , and meal test ) , but after adjusting for body size , odfs showed comparable insulin action in vivo . previous studies have shown that the offspring of individuals with early - onset type 2 diabetes are at increased risk for developing diabetes ( 5,8 ) . some studies report that the risk associated with maternal and paternal early onset of diabetes is approximately additive ( 16 ) , while others do not ( 17 ) . the present analysis was undertaken to clarify the separate effects of parental diabetes on the offspring 's phenotype . to our knowledge , this is the first study to show a paternal influence on adult body composition . while previous studies have shown that offspring of diabetic fathers have lower birth weights ( 3,18 ) , these studies did not explore parameters for body composition at later ages . the general effect of family history on offspring phenotype has been extensively examined , but attempts to separate different parental effects have been more limited . lindsay et al . ( 3 ) found that paternal history of diabetes was associated with lower birth weight . in fact , only those with lbw and paternal history of diabetes had increased diabetes risk , and lbw predicted diabetes risk in the father . our results are in agreement with a previous study also in pima indians ( 5 ) in which lower air was associated with earlier onset of diabetes in either parent . in that study , early onset was defined as age of onset below the median age for the cohort ( age < 41 years for mothers and < 46 years for fathers ) . in those whose mothers developed diabetes at < 35 years of age ( exclusive of exposure to diabetes in pregnancy ) , insulin secretion rate was also lower than in those whose parents developed type 2 diabetes at a later age . however , it is not clear to what extent differences in glucose tolerance ( i.e. , greater impaired glucose tolerance in group of offspring of the mothers with early onset of type 2 diabetes ) may have affected these results . our results extend and add to these findings as we are able to demonstrate in different tests ( ivgtt , ogtt , and meal tests ) done on different days a consistently lower air . during the ogtt , the 30-min insulin remained lower in odf compared to both omed and con , even after adjustment for age , sex , 30-min glucose , and m - low . in contrast , the 120-min insulin was no longer significantly lower after the same adjustments . these results indicate that the primary effect of early paternal diabetes is on early insulin secretion , rather than insulin action . this is supported by the relatively lower fasting plasma glucose in odfs and the lack of a difference in m - low after adjustment for body size . imprinting is the expression of only a single copy of a gene depending on parent - of - origin and is commonly found in genes that affect fetal growth . there are at least two distinct mechanisms through which epigenetic information can be inherited : dna methylation ( commonly associated with gene silencing and a contributor to x chromosomal inactivation ) and histone modification ( 19 ) . numerous genes have been found to be imprinted , and some of them are also involved in longitudinal and skeletal growth and may also be involved in organ growth ( 20 ) . notable among them are genes such as igf-2 , a growth hormone signaling gene ( 21 ) . in the human liver , the major production source of circulating igf-2 , the igf-2 gene is maternally imprinted during fetal life , whereas postnatally igf-2 is primarily transcribed from the p1 promoter , which is biallelically active in the liver . in other adult tissue ( including the pancreas ) , igf-2 is mainly transcribed from the paternally active promoters ( 22 ) . animal studies have demonstrated that genes involved directly in pancreatic -cell development might be imprinted , e.g. , the gene for insulin receptor subunit sur1 ( 6,21 ) . another study demonstrates that genes essential to pancreatic development , such as pancreatic homeobox transcription factor-1 ( pdx-1 ) are susceptible to epigenetic modifications ( 23 ) . in humans , an independent association between paternal insulin resistance and cord insulin concentrations ( 24 ) also indicates indirect evidence of imprinting . genes related to adipocyte development , specifically preadipocyte factor 1 ( pref1 ) , necdin , and paternally expressed gene 1 ( peg1 ) can be imprinted ( 6 ) . pref1 ( an inhibitor of adipogenesis ) is expressed from the paternally inherited chromosome , and imprinting of pref1 is under complex control by both maternal and paternal alleles ( 6 ) . although a study in mice overexpressing pref1 showed reduced fat content ( 25 ) , additional studies are required to elucidate the potential role of epigenetic effects in adipose tissue development in humans . because of our stringent group criteria , our sample size is small . however , our findings of a leaner phenotype in the odf group are robust and consistent with the previous findings of lbw in this group . our results could have been affected by the fact that these individuals were studied in adulthood , and so any individuals who developed type 2 diabetes prior to age 18 years were excluded . however , this would have , if anything , reduced our power to see group differences , particularly in an important diabetes risk factor such as air . although the age of onset of diabetes in the parents was quite young , there was a slight ( 3.8 4.0 years ) but statistically significant difference in the age of onset of diabetes in parents of the odf versus omed group . however , the air in odf was markedly lower across different tests on different days , implying that the paternal effect was more important than this relatively small difference in age of diagnosis . in conclusion , the results of the present analyses indicate that offspring of fathers with early - onset diabetes are leaner and have lower early insulin secretion compared to individuals in whom both parents remained without diabetes up to 50 years of age . insulin action , after adjustment for body size , was comparable in all three groups of offspring . these findings indicate an important role of paternal heritability in body composition and -cell dysfunction . paternal transmission patterns for susceptibility to diabetes indicate that epigenetic mechanisms are involved in the predisposition to diabetes . whether epigenetic markers are associated with parent - of - origin effects needs further investigation .

objectivepaternal and maternal type 2 diabetes , exclusive of gestational diabetes , may influence risk factors in the offspring differently ( through possible epigenetic effects of parental diabetes ) and are difficult to identify without accurate dates of diagnosis . we aimed to examine a metabolic phenotype in three different groups of offspring to see distinct paternal versus maternal effects.research design and methodswe examined body composition and insulin action ( m ) in nondiabetic subjects and insulin secretion tested via acute insulin response ( air ) in normal glucose - tolerant full - heritage pima indian adults categorized by disparate parental diabetes status : 1 ) offspring of fathers with early - onset diabetes ( age < 35 years ) and nondiabetic mothers ( odf ; n = 10 ) , 2 ) offspring of mothers with early - onset diabetes ( age < 35 years ) , not exposed to diabetes in utero with nondiabetic fathers ( omed ; n = 11 ) , and 3 ) a control group of offspring of parents without diabetes until > 50 years of age ( con ; n = 15).resultsodfs were leaner than cons and omeds ( percent of body fat [ % bf ] : least - squares means adjusted for age and sex [ 95% ci ] : 27.3 [ 23.331.3 ] in odfs vs. 35.4 [ 32.238.5 ] in cons and 32.4 [ 28.836.1 ] in omeds , p = 0.04 ) . odfs were more insulin sensitive ( had a higher m ) than omeds or cons , but not after adjustment for age , sex , and % bf . air adjusted for m , age , sex , and % bf was lower in odfs versus cons and omeds ( p < 0.05).conclusionsadult odfs were leaner and had lower early insulin secretion , despite being equally insulin sensitive after adjustment for body fat compared to the other groups , indicating a paternal imprinted effect .

RESEARCH DESIGN AND METHODS Determination of parental diabetes status Study subjects Dual-energy X-ray absorptiometry Measurement of insulin action Insulin secretion Meal test Analytic measurements Statistical analysis RESULTS CONCLUSIONS

parental diabetes status was determined from a longitudinal study of health conducted within the gila river ( pima ) indian community in which residents 5 years old have been invited for research examinations consisting of the measurement of height and weight and a 75-g oral glucose tolerance test ( ogtt ) approximately every 2 years . we defined three groups of offspring according to parental diabetes status : 1 ) offspring of diabetic fathers ( odf ) consisting of subjects whose father developed diabetes at < 35 years of age , but whose mother remained nondiabetic until > 35 years of age ; 2 ) offspring of mothers with early onset of diabetes ( omed ) consisting of subjects whose mother developed diabetes at < 35 years of age , but after the date of birth of the subject documented by a normal ogtt following delivery ( mean sd time between subject 's date of birth and mothers date of diagnosis , 8.9 4.2 years ) , and whose fathers remained nondiabetic until > 35 years of age ; and 3 ) a control group ( con ) consisting of offspring whose parents were known to be nondiabetic until > 50 years of age . the third group can be considered a control group because of previous evidence that offspring of parents who do not develop diabetes until age > 50 years have a risk for developing diabetes comparable with that of offspring whose parents never developed diabetes ( 8) . briefly , the diabetes score was derived by first calculating the specific cumulative incidence of diabetes as a function of age ( cia ) in the pima population . volunteers also participated in a study of metabolic determinants for the development of type 2 diabetes and obesity . at the time of their participation , all subjects were in good health and without evidence of diabetes ( by ogtt ) , as determined by a comprehensive medical evaluation including medical history , physical examination , and routine laboratory testing . subjects were admitted for 1015 days to the clinical research unit of the national institute of diabetes and digestive and kidney diseases in phoenix , arizona , and were provided a standard weight - maintaining energy needs diet containing 50% of calories as carbohydrate , 30% as fat , and 20% as protein for at least 3 days before metabolic testing . only subjects who were full - heritage pima indian or related tohono o'odham were included in the analysis . percentage body fat ( % bf ) , fat mass ( fm ) , and fat - free mass ( ffm ) were calculated as previously described ( 9 ) . insulin action was assessed at physiological insulin concentrations during the hyperinsulinemic - euglycemic clamp technique ( 10 ) . individual variation in plasma glucose and insulin concentrations during the clamp were taken into account in the calculation of insulin action ( m - low ) ( 12 ) . all measurements derived from the clamp were normalized to estimated metabolic body size ( embs ) , which is directly derived from ffm but takes into account the fact that the intercept of the relationship between ffm and resting metabolic rate is not zero ( 17.7 kg in our laboratory [ i.e. at 0730 h , subjects were fed a mixed - meal breakfast ( consisting of a bacon - and - egg sandwich on toast accompanied by orange juice ) containing 10% of calories from protein , 45% from fat , and 45% from carbohydrates and providing 20% of daily energy requirements for each subject . since even mild degrees of glucose intolerance can be associated with impairments in insulin secretion ( 15 ) only subjects with normal glucose tolerance were included for further analyses of air . the glucose and insulin responses during the ogtt , ivgtt , and meal test by group were compared by ancova using the mixed procedure in sas . the general linear models were used to adjust % bf for age and sex ; m - low ( mg kg estimated metabolic body size ( embs ) min ) for age , sex , and % bf , and air for age , sex , % bf , and m - low ( 10 ) . adjusted least - squares means ( lsmeans ) were used to compare groups by parental diabetes status using post hoc t test , with tukey 's honestly significant difference adjustment for multiple comparisons . for all groups , further analyses were conducted and adjusted for the same covariates using general estimating equations to account for family membership . general , anthropometric , and body composition parameters of subjects according to the age of diabetes onset in the parents * birth weight in odf group available only in seven subjects . p < 0.05 odf vs. con ( anova ; sex differences analysis ) . parental diabetes status was determined from a longitudinal study of health conducted within the gila river ( pima ) indian community in which residents 5 years old have been invited for research examinations consisting of the measurement of height and weight and a 75-g oral glucose tolerance test ( ogtt ) approximately every 2 years . we defined three groups of offspring according to parental diabetes status : 1 ) offspring of diabetic fathers ( odf ) consisting of subjects whose father developed diabetes at < 35 years of age , but whose mother remained nondiabetic until > 35 years of age ; 2 ) offspring of mothers with early onset of diabetes ( omed ) consisting of subjects whose mother developed diabetes at < 35 years of age , but after the date of birth of the subject documented by a normal ogtt following delivery ( mean sd time between subject 's date of birth and mothers date of diagnosis , 8.9 4.2 years ) , and whose fathers remained nondiabetic until > 35 years of age ; and 3 ) a control group ( con ) consisting of offspring whose parents were known to be nondiabetic until > 50 years of age . the third group can be considered a control group because of previous evidence that offspring of parents who do not develop diabetes until age > 50 years have a risk for developing diabetes comparable with that of offspring whose parents never developed diabetes ( 8) . briefly , the diabetes score was derived by first calculating the specific cumulative incidence of diabetes as a function of age ( cia ) in the pima population . volunteers also participated in a study of metabolic determinants for the development of type 2 diabetes and obesity . at the time of their participation , all subjects were in good health and without evidence of diabetes ( by ogtt ) , as determined by a comprehensive medical evaluation including medical history , physical examination , and routine laboratory testing . subjects were admitted for 1015 days to the clinical research unit of the national institute of diabetes and digestive and kidney diseases in phoenix , arizona , and were provided a standard weight - maintaining energy needs diet containing 50% of calories as carbohydrate , 30% as fat , and 20% as protein for at least 3 days before metabolic testing . only subjects who were full - heritage pima indian or related tohono o'odham were included in the analysis . percentage body fat ( % bf ) , fat mass ( fm ) , and fat - free mass ( ffm ) were calculated as previously described ( 9 ) . insulin action was assessed at physiological insulin concentrations during the hyperinsulinemic - euglycemic clamp technique ( 10 ) . the rate of total insulin - stimulated glucose disposal was calculated for the last 40 min of the insulin infusion and was corrected for the rate of egp calculated from the steele equation ( 11 ) . individual variation in plasma glucose and insulin concentrations during the clamp were taken into account in the calculation of insulin action ( m - low ) ( 12 ) . blood samples for insulin and glucose were drawn before and 30 , 60 , 90 , 120 , 150 , and 180 min after initiation of the meal test . since even mild degrees of glucose intolerance can be associated with impairments in insulin secretion ( 15 ) only subjects with normal glucose tolerance were included for further analyses of air . the glucose and insulin responses during the ogtt , ivgtt , and meal test by group were compared by ancova using the mixed procedure in sas . the general linear models were used to adjust % bf for age and sex ; m - low ( mg kg estimated metabolic body size ( embs ) min ) for age , sex , and % bf , and air for age , sex , % bf , and m - low ( 10 ) . adjusted least - squares means ( lsmeans ) were used to compare groups by parental diabetes status using post hoc t test , with tukey 's honestly significant difference adjustment for multiple comparisons . for all groups , further analyses were conducted and adjusted for the same covariates using general estimating equations to account for family membership . general , anthropometric , and body composition parameters of subjects according to the age of diabetes onset in the parents * birth weight in odf group available only in seven subjects . p < 0.05 odf vs. con ( anova ; sex differences analysis ) . general , anthropometric , and metabolic characteristics of the study population are shown in table 1 . we found 10 odf ; 8 of 10 fathers from the odf group had diabetes preconception , and 2 fathers had diagnoses of diabetes 3 and 4 years after the offspring birth . onset age of diabetes for fathers was lower in the odf group than for mothers in the omed group ( 26.6 4.2 vs. 30.4 3.0 years , p = 0.02 ) . this slight difference in age was due to exclusion of mothers in the omed group who did not have a nondiabetic ogtt following childbirth . consistent with this age difference , fathers ' ci score in the odf group was higher than mothers ' ci score in the omed group ( p = 0.04 , table 1 ) . there was no significant difference between mothers ci score in the odf group and fathers ci score in the omed group , indicating no difference in relative age of onset of diabetes in the opposite parent . the ci score was comparable between mothers and fathers in the con group ( table 1 ) . after adjustment for age and sex , % bf remained lower in the odf group than in the con group ( lsmeans [ 95% ci ] : 27.3% [ 23.331.3 ] in odf vs. 35.4% [ 32.238.5 ] in con and 32.4% [ 28.836.1 ] in omed , p = 0.04 ) . the ffm , adjusted for age and sex , tended to be lower in odf in comparison to con and omed , but did not reach statistical significance ( 57.9 kg [ 50.465.3 ] in odf vs. 65.8 kg [ 60.071.7 ] in con , and 67.2 kg [ 60.474.1 ] in omed , p = 0.1 ) . odfs had lower fasting glucose , but 2-h plasma glucose concentrations were comparable with those of cons or omeds ( p = 0.01 and p = 0.1 , respectively , anova ; table 1 ) . despite similar fasting plasma insulin concentrations and glucose responses during the ogtt , plasma insulin concentrations at 30 and 120 min during ogtt ( ins30 , ins120 ) were lower in the odf group than in the con and omed groups ( f = 4.38 , p = 0.02 ; f = 3.81 , p = 0.04 , respectively ; fig . after adjustment for age , sex , 30 min glucose concentration , and m - low , only ins30 remained lower in the odf group than in both the con and omed groups ( lsmeans [ 95% ci ] : 722 pmol / l [ 1321,582 ] in odf vs. 2,699 plasma glucose and insulin concentrations during ogtt ( a and b ) , during ivgtt ( c and d ) , and during meal test ( e and f ) in odf , con , and omed . ogtt : post hoc tests for significant differences in time versus group interactions ( effect of time [ p < 0.0001 for all models ] ) ; effect of group ( glucose p = 0.02 , insulin p = 0.001 ) ; effect of time groups interaction ( glucose p = 0.3 , insulin p = 0.1 ) . ivgtt : post hoc tests for significant differences in time versus group interactions ( effect of time [ p < 0.0001 for all models ] ) ; effect of group ( glucose p = 0.02 , insulin p < 0.0001 ) ; effect of time groups interaction , ( glucose p = 0.3 , insulin p < 0.0001 ) . ivgtt data available only in six subjects in the odf , six in the con , and seven in the omed group . meal test : post hoc tests for significant differences in time versus group interactions ( effect of time [ p < 0.0001 for all models ] ) ; effect of group ( glucose p = 0.2 , insulin p < 0.0001 ) ; effect of time groups interaction ( glucose p = 0.3 , insulin p = 0.002 ) . meal test data available only in six subjects in the odf , eight in the con , and eleven in the omed group . the insulin responses were significantly lower in odf than in con subjects ( effect of diagnosis time p < 0.0001 ; anova ; fig . in addition , log - transformed air was lower in the odf group than the con group ( table 1 ) , and it remained significant after adjustment for age , sex , % bf , and m - low ( lsmeans [ 95% ci ] : 2.08 pmol / l ( 1.962.20 ) in odf vs. 2.55 pmol [ 2.242.75 ] in con and 2.40 pmol / l [ 2.132.55 ] in omed , p < 0.05 ) . during the meal test the insulin responses during the meal test were significantly lower in odf than in con subjects ( effect of diagnosis time sex p = 0.02 ; fig . plasma insulin concentrations at 30 min of the meal test were slightly lower in the odf group compared to both the con and omed groups , but it did not reach statistical significance prior to adjustment ( 1,084 462 pmol / l in odf vs. 2090 1,081 1 ) , or following adjustment for age , sex , 30-min plasma glucose concentration , and m - low ( lsmeans [ 95% ci ] : 1,422 pmol / l [ 4892,355 ] in odf vs. 2,087 pmol / l ( 1,3722,803 ) in con and 1,492 pmol / l [ 1,0591924 ] in omed , p = 0.1 ) . odfs were on average leaner ( having a lower % bf ) ; therefore the mean log transformed m - lows were higher in the odf group than in the con and omed groups ( table 1 ) , but after adjustment for age , sex , and % bf , m - lows were comparable in all three groups ( lsmeans [ 95% ci ] : 2.68 [ 1.803.55 ] mg kg embs min in odf vs. 2.34 [ 1.732.96 ] mg kg embs min in con , 2.28 [ 1.872.68 ] mg kg embs min in omed , p = 0.4 ) . our analyses indicate that adult nondiabetic offspring of fathers with early onset of diabetes are leaner than offspring of either mothers with early onset of type 2 diabetes or control subjects ( neither parent developed type 2 diabetes by age 50 years ) . odfs had lower insulin secretion , demonstrated by multiple tests ( ivgtt , ogtt , and meal test ) , but after adjusting for body size , odfs showed comparable insulin action in vivo . previous studies have shown that the offspring of individuals with early - onset type 2 diabetes are at increased risk for developing diabetes ( 5,8 ) . the present analysis was undertaken to clarify the separate effects of parental diabetes on the offspring 's phenotype . to our knowledge , this is the first study to show a paternal influence on adult body composition . while previous studies have shown that offspring of diabetic fathers have lower birth weights ( 3,18 ) , these studies did not explore parameters for body composition at later ages . in fact , only those with lbw and paternal history of diabetes had increased diabetes risk , and lbw predicted diabetes risk in the father . our results are in agreement with a previous study also in pima indians ( 5 ) in which lower air was associated with earlier onset of diabetes in either parent . in that study , early onset was defined as age of onset below the median age for the cohort ( age < 41 years for mothers and < 46 years for fathers ) . in those whose mothers developed diabetes at < 35 years of age ( exclusive of exposure to diabetes in pregnancy ) , insulin secretion rate was also lower than in those whose parents developed type 2 diabetes at a later age . , greater impaired glucose tolerance in group of offspring of the mothers with early onset of type 2 diabetes ) may have affected these results . our results extend and add to these findings as we are able to demonstrate in different tests ( ivgtt , ogtt , and meal tests ) done on different days a consistently lower air . during the ogtt , the 30-min insulin remained lower in odf compared to both omed and con , even after adjustment for age , sex , 30-min glucose , and m - low . these results indicate that the primary effect of early paternal diabetes is on early insulin secretion , rather than insulin action . this is supported by the relatively lower fasting plasma glucose in odfs and the lack of a difference in m - low after adjustment for body size . there are at least two distinct mechanisms through which epigenetic information can be inherited : dna methylation ( commonly associated with gene silencing and a contributor to x chromosomal inactivation ) and histone modification ( 19 ) . genes related to adipocyte development , specifically preadipocyte factor 1 ( pref1 ) , necdin , and paternally expressed gene 1 ( peg1 ) can be imprinted ( 6 ) . pref1 ( an inhibitor of adipogenesis ) is expressed from the paternally inherited chromosome , and imprinting of pref1 is under complex control by both maternal and paternal alleles ( 6 ) . although a study in mice overexpressing pref1 showed reduced fat content ( 25 ) , additional studies are required to elucidate the potential role of epigenetic effects in adipose tissue development in humans . however , our findings of a leaner phenotype in the odf group are robust and consistent with the previous findings of lbw in this group . our results could have been affected by the fact that these individuals were studied in adulthood , and so any individuals who developed type 2 diabetes prior to age 18 years were excluded . although the age of onset of diabetes in the parents was quite young , there was a slight ( 3.8 4.0 years ) but statistically significant difference in the age of onset of diabetes in parents of the odf versus omed group . in conclusion , the results of the present analyses indicate that offspring of fathers with early - onset diabetes are leaner and have lower early insulin secretion compared to individuals in whom both parents remained without diabetes up to 50 years of age . insulin action , after adjustment for body size , was comparable in all three groups of offspring . these findings indicate an important role of paternal heritability in body composition and -cell dysfunction . paternal transmission patterns for susceptibility to diabetes indicate that epigenetic mechanisms are involved in the predisposition to diabetes .

the world health survey indicated that in india the prevalence of psychosis was between 0.7% and 3.6% , while the treatment rates were only 36 - 85% . in addition to the high mental health burden and large treatment gap of mental disorders there are significant limitations in the availability and integration of mental health services in the public health sector . hence , the ministry of health and family welfare ( mohfw ) india and its public health institutes are collaborating to develop local solutions to build and strengthen the national community mental health system . there is an urgent need for developing appropriate community mental health models in india that can be replicated and scaled up to increase access to appropriate care for people with mental illness . the district mental health program ( dmhp ) is a community - based mental health service delivery program initiated in 1996 under the national mental health plan and were now active in 123 of the 652 districts of india . the program aspires to increase access to comprehensive mental health care service by developing partnerships between the district mental health team , the primary health care ( phc ) teams , community - based organizations , nongovernmental organizations ( ngos ) , service users , family groups and various government departments . in the beginning , the project focused on early diagnosis and treatment , training of phc staff , and information , education and communication activities with the core clinical team . mental health promotional activities such as life skills education and counseling in schools , counseling services in colleges , workplace stress management , suicide prevention services , as well as a managerial team for implementing various dmhp activities have been added . the major achievements of the program have been the development of community mental health services in the most underserved areas . to decentralize mental health care in the community using the public health infrastructure and other resources , mental health training , awareness and services are provided in collaboration with various community - based partners . in terms of challenges , the training needs for phc staff are enormous due to the large numbers of phc workers in india . a shortage of qualified mental health human resources , as well as little involvement of other primary care health workers in the mental health services has meant difficulty in recruiting the district mental health teams . lack of coordination between health and medical education departments has resulted in conflict in program implementation . stigmas attached to mental illness remain widely prevalent , and still pose formidable barriers between the mentally ill and community mental health services . mental health remains a comparatively neglected area , given the lowest priority in social and development planning . there is still much to be done to integrate mental health into the mainstream public and general health . as a strategy to build the dmhp and increase accessibility of essential community mental health services , mohfw and its public health institutes have collaborated with asia australia mental health ( aamh ) on innovative community mental health development project . the project began in 2011 and aims to develop locally sustainable best practice community mental health models to inform policy and practice at local district , state and national level . four pilot sites were identified , and project activities are focused on developing local capacity to prevent , treat and rehabilitate people with mental disorders through integrating mental health care into public health . the project collaborates with international partners under a formal agreement with technical expertise provided by the university of melbourne ( aamh ) . a bilateral community mental health advisory committee chaired by the director general of health services ( dghs ) , mohfw provides the oversight of the overall project . four different pilot sites with a capacity to provide leadership for a national program and to develop best practice models were selected . these reflect the diverse conditions and needs across india including models for urban setting ( chandigarh ) , rural tribal setting ( gumla ) , mixed urban setting ( ramanathapuram ) and low - health resource settings ( panchmahal ) . each of the four sites is linked to local tertiary care mental health institute for technical and managerial support . the pilot projects involved relevant public sectors and community stakeholders ( such as state departments , health institutes , phc providers , community agencies and ngo 's ) in the development and implementation of the program . to more effectively measure and communicate project aims , processes , and potential outcomes , the advisory committee worked with the project team to establish common procedures for each site [ table 1 ] and key principles for developing best practice community mental health models [ table 2 ] . common methodology for model development key principles for developing community mental health model of care gumla district , ranchi institute of neuro psychiatry and allied sciences ( rinpas ) ( jharkhand - east region ) the rinpas is situated in the tribal heartland of jharkhand state embracing santhal pargana and chotanagpur . district gumla , almost 90 km away from capital city ranchi , comprises of about 10.25 lakhs population . it is estimated that more than 75% of the population belonging to a tribal community , with unique cultural , religious , and social practices . as well as the district hospital , there is a district mental health center ( dmhp with psychiatrist , clinical psychologist , psychiatrist social worker , and psychiatric nurse ) , 13 phc centers , 11 community healthcare centers and 242 health sub centerssome of the main areas of concern identified by the mental health services at the start of this project included a failure to understand the indigenous social and emotional context of the presenting problems , a high rate of unaddressed mental health needs which affect the physical health and overall socioeconomic development , as well as pervasive and harmful myths and misconceptions in the community surrounding mental illness . the project planned to cover two blocks with the goal of delivering proper treatment , follow - up and sociovocational rehabilitation that will benefit tribal people affected with mental illness and their caregiversthe planned approach aims to engage the local stakeholders ( panchayat leaders , health workers , voluntary workers , faith healers , general practitioners ) in a culturally appropriate way , encourage joint planning , and mobilize community mental health resources . the project aims to increase culturally appropriate mental health promotion , improve convergence with other health and administrative sectors and increase numbers of people identifying with the tribal backgrounds employed in health and education centers and training in mental health the rinpas is situated in the tribal heartland of jharkhand state embracing santhal pargana and chotanagpur . district gumla , almost 90 km away from capital city ranchi , comprises of about 10.25 lakhs population . it is estimated that more than 75% of the population belonging to a tribal community , with unique cultural , religious , and social practices . as well as the district hospital , there is a district mental health center ( dmhp with psychiatrist , clinical psychologist , psychiatrist social worker , and psychiatric nurse ) , 13 phc centers , 11 community healthcare centers and 242 health sub centers some of the main areas of concern identified by the mental health services at the start of this project included a failure to understand the indigenous social and emotional context of the presenting problems , a high rate of unaddressed mental health needs which affect the physical health and overall socioeconomic development , as well as pervasive and harmful myths and misconceptions in the community surrounding mental illness . the project planned to cover two blocks with the goal of delivering proper treatment , follow - up and sociovocational rehabilitation that will benefit tribal people affected with mental illness and their caregivers the planned approach aims to engage the local stakeholders ( panchayat leaders , health workers , voluntary workers , faith healers , general practitioners ) in a culturally appropriate way , encourage joint planning , and mobilize community mental health resources . the project aims to increase culturally appropriate mental health promotion , improve convergence with other health and administrative sectors and increase numbers of people identifying with the tribal backgrounds employed in health and education centers and training in mental health chandigarh district , chandigarh government medical college ( north region ) chandigarh has an area of 114 km and a population of 1.05 million , including an estimated 90% urban residents . as a joint capital of both punjab and haryana states , chandigarh has 1 district hospital , 2 community health centers , 1 poly clinic , 30 civil dispensaries , 20 ayush dispensaries , and 17 sub centersin the chandigarh district , it has been identified that existing dmhp model may not have sustainable sources after funding after the 5 years current central government arrangement and the implementation of the dmhp has been in the nature of a parallel program rather than integrated , especially at the grassroots levelsthe department of psychiatry , government medical college and hospital has been working into the community for the last 14 years , providing community mental health services through community camps , community out - reach clinics in the periphery of chandigarh , half way home , crisis intervention team , suicide prevention helpline and providing technical assistance to self - help groupsthrough this work , it has been seen that there are insufficient social and vocational rehabilitation services for the persons with chronic and severe forms of mental disorders and one of the main goals of this pilot project is to explore initiatives for capacity building and empowerment of consumers and caregiversas an overarching principle , this particular pilot site identified the importance of including prevention and positive mental health promotion into the mental health approach chandigarh has an area of 114 km and a population of 1.05 million , including an estimated 90% urban residents . as a joint capital of both punjab and haryana states , chandigarh has 1 district hospital , 2 community health centers , 1 poly clinic , 30 civil dispensaries , 20 ayush dispensaries , and 17 sub centers in the chandigarh district , it has been identified that existing dmhp model may not have sustainable sources after funding after the 5 years current central government arrangement and the implementation of the dmhp has been in the nature of a parallel program rather than integrated , especially at the grassroots levels the department of psychiatry , government medical college and hospital has been working into the community for the last 14 years , providing community mental health services through community camps , community out - reach clinics in the periphery of chandigarh , half way home , crisis intervention team , suicide prevention helpline and providing technical assistance to self - help groups through this work , it has been seen that there are insufficient social and vocational rehabilitation services for the persons with chronic and severe forms of mental disorders and one of the main goals of this pilot project is to explore initiatives for capacity building and empowerment of consumers and caregivers as an overarching principle , this particular pilot site identified the importance of including prevention and positive mental health promotion into the mental health approach ramanathapuram district , madurai medical college ( tamil nadu - south region ) ramanathapuram district is an administrative district of tamil nadu state in southern india with a population of 1,337,560 . there are 45 phcs , seven taluk hospitals and one district headquarters hospital in ramnad district tamil nadu . this district also has 56 private hospitals , including the three siddha , homeopathy and unanni medicines hospitalsthe dmhp has been in operation in ramnad district since , 2001 . at this time , resources were put into training public health staff , but there have been no ongoing mental health education for the 300 medical doctors and 700 health workers working in the districtfor this region , the ramanathapuram dmhp is supported by the m. s. chellamuthu trust and research foundation , madurai . this leading ngo has a wide range of activities , including treatment , psycho - social rehabilitation , vocational training and placements aiming to assist people with mental health problems to recover and reintegrate with the mainstream community through this work , facilities and infrastructure have been identified as inadequate for comprehensive care and universal coverage , particularly in rural areas . myths and misconception about mental illness also contribute to poor access of existing services , including the lack awareness about the availability of , or the effectiveness of treatmentthe ramanathapuram district project highlighted the potential for services creating shared responsibility with village panchayats and primary health center teams created a better link for specialist mental health services and community services . the project also identified the capacity to actively engage religious persons , native healers and spiritual guides who can assist with providing support for people experiencing mental health problems and bring a wider expertise in mental health advocacy workthere is another innovative experiment in creating a teleconference facility by which we are linking the block level hospitals with district headquarters hospital and thereby we are increasing the accessibility , affordability and decreasing the stigma to a greater extend . ramanathapuram district is an administrative district of tamil nadu state in southern india with a population of 1,337,560 . there are 45 phcs , seven taluk hospitals and one district headquarters hospital in ramnad district tamil nadu . this district also has 56 private hospitals , including the three siddha , homeopathy and unanni medicines hospitals the dmhp has been in operation in ramnad district since , 2001 . at this time , resources were put into training public health staff , but there have been no ongoing mental health education for the 300 medical doctors and 700 health workers working in the district for this region , the ramanathapuram dmhp is supported by the m. s. chellamuthu trust and research foundation , madurai . this leading ngo has a wide range of activities , including treatment , psycho - social rehabilitation , vocational training and placements aiming to assist people with mental health problems to recover and reintegrate with the mainstream community through this work , facilities and infrastructure have been identified as inadequate for comprehensive care and universal coverage , particularly in rural areas . myths and misconception about mental illness also contribute to poor access of existing services , including the lack awareness about the availability of , or the effectiveness of treatment the ramanathapuram district project highlighted the potential for services creating shared responsibility with village panchayats and primary health center teams created a better link for specialist mental health services and community services . the project also identified the capacity to actively engage religious persons , native healers and spiritual guides who can assist with providing support for people experiencing mental health problems and bring a wider expertise in mental health advocacy work there is another innovative experiment in creating a teleconference facility by which we are linking the block level hospitals with district headquarters hospital and thereby we are increasing the accessibility , affordability and decreasing the stigma to a greater extend . panchmahal district , ahmedabad mental hospital ( gujarat - west region ) gujarat , with a population of 60,383,638 ( census-2011 ) has a district hospital , 13 community health centers , 63 phc centers , 400 sub centers , 21 ayurveda centers , and 9 homeopathic centersthe dmhp has been extended to eight districts of gujarat , linking district general / civil hospitals to adjoining hospitals for mental health or department of psychiatry of medical colleges . the department of health and family welfare , government of gujarat commissioned work which led to the 2004 mission report titled priorities for mental health sector development in gujarat . this report provided a broad canvas of strategies which need to be pursued to address issues facing this sector in the context of gujarat in 12 strategic directions including : resource mobilization and allocation , strengthening the ethics of care , addressing stigma , and integration of mental health with health and other sectors department of health and family welfare , government of gujarat also released a 12 5 year plan 2012 - 2017 , which includes the commitment to strengthening the mental health program to provide better medical services to the mentally ill patients , protect their right and rehabilitate them in the societyin line with these state strategies , the hospital for mental health , ahmedabad focused the community project on utilizing available human resources at the village / taluka level for case identification and counseling and well - established referral linkages with both public and private agencies . the project sought to build on foundations of existing sources of rapport , influence and capacity to mobilize the community at the village level . including these stakeholders and expertise in mainstream mental health delivery will help reduce the stigma as well as address crucial gaps in mh services . the project aims to increase mental health awareness , sensitization and use of local mental health services and its impact on the explanatory models used to understand mental illness . gujarat , with a population of 60,383,638 ( census-2011 ) has a district hospital , 13 community health centers , 63 phc centers , 400 sub centers , 21 ayurveda centers , and 9 homeopathic centers the dmhp has been extended to eight districts of gujarat , linking district general / civil hospitals to adjoining hospitals for mental health or department of psychiatry of medical colleges . the department of health and family welfare , government of gujarat commissioned work which led to the 2004 mission report titled priorities for mental health sector development in gujarat . this report provided a broad canvas of strategies which need to be pursued to address issues facing this sector in the context of gujarat in 12 strategic directions including : resource mobilization and allocation , strengthening the ethics of care , addressing stigma , and integration of mental health with health and other sectors department of health and family welfare , government of gujarat also released a 12 5 year plan 2012 - 2017 , which includes the commitment to strengthening the mental health program to provide better medical services to the mentally ill patients , protect their right and rehabilitate them in the society in line with these state strategies , the hospital for mental health , ahmedabad focused the community project on utilizing available human resources at the village / taluka level for case identification and counseling and well - established referral linkages with both public and private agencies . the project sought to build on foundations of existing sources of rapport , influence and capacity to mobilize the community at the village level . including these stakeholders and expertise in mainstream mental health delivery will help reduce the stigma as well as address crucial gaps in mh services . the project aims to increase mental health awareness , sensitization and use of local mental health services and its impact on the explanatory models used to understand mental illness . appropriate and trained human resources are critical to developing collaborative arrangements for effective community based mental health services . a skilled workforce has been recognized as key to strong community participation , linkages with the voluntary sector and civil society initiatives , with academic institutions from inter - disciplinary backgrounds . the national institute of health and family welfare ( nihfw ) , dghs , mohfw and aamh have jointly designed structured training for community mental health workforce . the training explores methods to develop human resources for community - based services , and importantly to develop a broad understanding of the role of the mental health professional . it incorporates the core principles of recovery - oriented practice including : supporting personal recovery and promoting well - being ; delivering services informed by evidence and consistent with the social model of health ; proactive and purposeful engagement to build trusting relationships . this ensures practice is sensitive to the needs of families , friends and careers ; and working in partnership with key stakeholders . innovative training programs thus not only need to build on existing training resources but also develop new skill sets relevant to the principles and approach of the project . an example of the training delivered is the 5 days training program delivered in november 2013 , which included 20 participants from the 4 pilot sites . participants included medical officers and program managers who were responsible for the implementation of community mental health projects . the training started with topics that would set the context , by reflecting on global trends and best practice in asia pacific . the interactive workshops sought to broaden the perspectives of key stakeholders on the burden of mental illness in india and factors influencing progression of mental illness and rates of recovery , as well as efforts to respond to the challenges of mental illness and the mental health treatment gap . an essential strategy was to develop a shared understanding and consistent approach to recovery - oriented and community - based mental health service delivery in india . expertise on management strategies for mental illnesses complemented participants discussions and workshops on a multidisciplinary approach and local considerations . material included establishment of rehabilitation services within communities as well as the roles of families , self - help groups , caregivers associations , and other specialized ngo 's . the philosophy of recovery was also seen to positively influence stronger methods for measuring success . the training examined existing guidelines for monitoring and evaluation for each site with discussion on various aspects such as project indicators , data collection , data compilation , and report writing . the training sessions also examined effective management of common mental disorders including treatment seeking behaviors and treatment compliance , common childhood and adolescent psychiatric problems , addiction , abuses of addictive substances , and aged mental health problems . the workshops were centered on clinicians creating positive futures for people suffering from mental illness . expertise shared at the training incorporated information , education and communication strategies for mental health promotion . four different pilot sites with a capacity to provide leadership for a national program and to develop best practice models were selected . these reflect the diverse conditions and needs across india including models for urban setting ( chandigarh ) , rural tribal setting ( gumla ) , mixed urban setting ( ramanathapuram ) and low - health resource settings ( panchmahal ) . each of the four sites is linked to local tertiary care mental health institute for technical and managerial support . the pilot projects involved relevant public sectors and community stakeholders ( such as state departments , health institutes , phc providers , community agencies and ngo 's ) in the development and implementation of the program . to more effectively measure and communicate project aims , processes , and potential outcomes , the advisory committee worked with the project team to establish common procedures for each site [ table 1 ] and key principles for developing best practice community mental health models [ table 2 ] . gumla district , ranchi institute of neuro psychiatry and allied sciences ( rinpas ) ( jharkhand - east region ) the rinpas is situated in the tribal heartland of jharkhand state embracing santhal pargana and chotanagpur . district gumla , almost 90 km away from capital city ranchi , comprises of about 10.25 lakhs population . it is estimated that more than 75% of the population belonging to a tribal community , with unique cultural , religious , and social practices . as well as the district hospital , there is a district mental health center ( dmhp with psychiatrist , clinical psychologist , psychiatrist social worker , and psychiatric nurse ) , 13 phc centers , 11 community healthcare centers and 242 health sub centerssome of the main areas of concern identified by the mental health services at the start of this project included a failure to understand the indigenous social and emotional context of the presenting problems , a high rate of unaddressed mental health needs which affect the physical health and overall socioeconomic development , as well as pervasive and harmful myths and misconceptions in the community surrounding mental illness . the project planned to cover two blocks with the goal of delivering proper treatment , follow - up and sociovocational rehabilitation that will benefit tribal people affected with mental illness and their caregiversthe planned approach aims to engage the local stakeholders ( panchayat leaders , health workers , voluntary workers , faith healers , general practitioners ) in a culturally appropriate way , encourage joint planning , and mobilize community mental health resources . the project aims to increase culturally appropriate mental health promotion , improve convergence with other health and administrative sectors and increase numbers of people identifying with the tribal backgrounds employed in health and education centers and training in mental health the rinpas is situated in the tribal heartland of jharkhand state embracing santhal pargana and chotanagpur . district gumla , almost 90 km away from capital city ranchi , comprises of about 10.25 lakhs population . it is estimated that more than 75% of the population belonging to a tribal community , with unique cultural , religious , and social practices . as well as the district hospital , there is a district mental health center ( dmhp with psychiatrist , clinical psychologist , psychiatrist social worker , and psychiatric nurse ) , 13 phc centers , 11 community healthcare centers and 242 health sub centers some of the main areas of concern identified by the mental health services at the start of this project included a failure to understand the indigenous social and emotional context of the presenting problems , a high rate of unaddressed mental health needs which affect the physical health and overall socioeconomic development , as well as pervasive and harmful myths and misconceptions in the community surrounding mental illness . the project planned to cover two blocks with the goal of delivering proper treatment , follow - up and sociovocational rehabilitation that will benefit tribal people affected with mental illness and their caregivers the planned approach aims to engage the local stakeholders ( panchayat leaders , health workers , voluntary workers , faith healers , general practitioners ) in a culturally appropriate way , encourage joint planning , and mobilize community mental health resources . the project aims to increase culturally appropriate mental health promotion , improve convergence with other health and administrative sectors and increase numbers of people identifying with the tribal backgrounds employed in health and education centers and training in mental health chandigarh district , chandigarh government medical college ( north region ) chandigarh has an area of 114 km and a population of 1.05 million , including an estimated 90% urban residents . as a joint capital of both punjab and haryana states , chandigarh has 1 district hospital , 2 community health centers , 1 poly clinic , 30 civil dispensaries , 20 ayush dispensaries , and 17 sub centersin the chandigarh district , it has been identified that existing dmhp model may not have sustainable sources after funding after the 5 years current central government arrangement and the implementation of the dmhp has been in the nature of a parallel program rather than integrated , especially at the grassroots levelsthe department of psychiatry , government medical college and hospital has been working into the community for the last 14 years , providing community mental health services through community camps , community out - reach clinics in the periphery of chandigarh , half way home , crisis intervention team , suicide prevention helpline and providing technical assistance to self - help groupsthrough this work , it has been seen that there are insufficient social and vocational rehabilitation services for the persons with chronic and severe forms of mental disorders and one of the main goals of this pilot project is to explore initiatives for capacity building and empowerment of consumers and caregiversas an overarching principle , this particular pilot site identified the importance of including prevention and positive mental health promotion into the mental health approach chandigarh has an area of 114 km and a population of 1.05 million , including an estimated 90% urban residents . as a joint capital of both punjab and haryana states , chandigarh has 1 district hospital , 2 community health centers , 1 poly clinic , 30 civil dispensaries , 20 ayush dispensaries , and 17 sub centers in the chandigarh district , it has been identified that existing dmhp model may not have sustainable sources after funding after the 5 years current central government arrangement and the implementation of the dmhp has been in the nature of a parallel program rather than integrated , especially at the grassroots levels the department of psychiatry , government medical college and hospital has been working into the community for the last 14 years , providing community mental health services through community camps , community out - reach clinics in the periphery of chandigarh , half way home , crisis intervention team , suicide prevention helpline and providing technical assistance to self - help groups through this work , it has been seen that there are insufficient social and vocational rehabilitation services for the persons with chronic and severe forms of mental disorders and one of the main goals of this pilot project is to explore initiatives for capacity building and empowerment of consumers and caregivers as an overarching principle , this particular pilot site identified the importance of including prevention and positive mental health promotion into the mental health approach ramanathapuram district , madurai medical college ( tamil nadu - south region ) ramanathapuram district is an administrative district of tamil nadu state in southern india with a population of 1,337,560 . there are 45 phcs , seven taluk hospitals and one district headquarters hospital in ramnad district tamil nadu . this district also has 56 private hospitals , including the three siddha , homeopathy and unanni medicines hospitalsthe dmhp has been in operation in ramnad district since , 2001 . at this time , resources were put into training public health staff , but there have been no ongoing mental health education for the 300 medical doctors and 700 health workers working in the districtfor this region , the ramanathapuram dmhp is supported by the m. s. chellamuthu trust and research foundation , madurai . this leading ngo has a wide range of activities , including treatment , psycho - social rehabilitation , vocational training and placements aiming to assist people with mental health problems to recover and reintegrate with the mainstream community through this work , facilities and infrastructure have been identified as inadequate for comprehensive care and universal coverage , particularly in rural areas . myths and misconception about mental illness also contribute to poor access of existing services , including the lack awareness about the availability of , or the effectiveness of treatmentthe ramanathapuram district project highlighted the potential for services creating shared responsibility with village panchayats and primary health center teams created a better link for specialist mental health services and community services . the project also identified the capacity to actively engage religious persons , native healers and spiritual guides who can assist with providing support for people experiencing mental health problems and bring a wider expertise in mental health advocacy workthere is another innovative experiment in creating a teleconference facility by which we are linking the block level hospitals with district headquarters hospital and thereby we are increasing the accessibility , affordability and decreasing the stigma to a greater extend . ramanathapuram district is an administrative district of tamil nadu state in southern india with a population of 1,337,560 . there are 45 phcs , seven taluk hospitals and one district headquarters hospital in ramnad district tamil nadu . this district also has 56 private hospitals , including the three siddha , homeopathy and unanni medicines hospitals the dmhp has been in operation in ramnad district since , 2001 . at this time , resources were put into training public health staff , but there have been no ongoing mental health education for the 300 medical doctors and 700 health workers working in the district for this region , the ramanathapuram dmhp is supported by the m. s. chellamuthu trust and research foundation , madurai . this leading ngo has a wide range of activities , including treatment , psycho - social rehabilitation , vocational training and placements aiming to assist people with mental health problems to recover and reintegrate with the mainstream community through this work , facilities and infrastructure have been identified as inadequate for comprehensive care and universal coverage , particularly in rural areas . myths and misconception about mental illness also contribute to poor access of existing services , including the lack awareness about the availability of , or the effectiveness of treatment the ramanathapuram district project highlighted the potential for services creating shared responsibility with village panchayats and primary health center teams created a better link for specialist mental health services and community services . the project also identified the capacity to actively engage religious persons , native healers and spiritual guides who can assist with providing support for people experiencing mental health problems and bring a wider expertise in mental health advocacy work there is another innovative experiment in creating a teleconference facility by which we are linking the block level hospitals with district headquarters hospital and thereby we are increasing the accessibility , affordability and decreasing the stigma to a greater extend . panchmahal district , ahmedabad mental hospital ( gujarat - west region ) gujarat , with a population of 60,383,638 ( census-2011 ) has a district hospital , 13 community health centers , 63 phc centers , 400 sub centers , 21 ayurveda centers , and 9 homeopathic centersthe dmhp has been extended to eight districts of gujarat , linking district general / civil hospitals to adjoining hospitals for mental health or department of psychiatry of medical colleges . the department of health and family welfare , government of gujarat commissioned work which led to the 2004 mission report titled priorities for mental health sector development in gujarat . this report provided a broad canvas of strategies which need to be pursued to address issues facing this sector in the context of gujarat in 12 strategic directions including : resource mobilization and allocation , strengthening the ethics of care , addressing stigma , and integration of mental health with health and other sectors department of health and family welfare , government of gujarat also released a 12 5 year plan 2012 - 2017 , which includes the commitment to strengthening the mental health program to provide better medical services to the mentally ill patients , protect their right and rehabilitate them in the societyin line with these state strategies , the hospital for mental health , ahmedabad focused the community project on utilizing available human resources at the village / taluka level for case identification and counseling and well - established referral linkages with both public and private agencies . the project sought to build on foundations of existing sources of rapport , influence and capacity to mobilize the community at the village level . including these stakeholders and expertise in mainstream mental health delivery will help reduce the stigma as well as address crucial gaps in mh services . the project aims to increase mental health awareness , sensitization and use of local mental health services and its impact on the explanatory models used to understand mental illness . gujarat , with a population of 60,383,638 ( census-2011 ) has a district hospital , 13 community health centers , 63 phc centers , 400 sub centers , 21 ayurveda centers , and 9 homeopathic centers the dmhp has been extended to eight districts of gujarat , linking district general / civil hospitals to adjoining hospitals for mental health or department of psychiatry of medical colleges . the department of health and family welfare , government of gujarat commissioned work which led to the 2004 mission report titled priorities for mental health sector development in gujarat . this report provided a broad canvas of strategies which need to be pursued to address issues facing this sector in the context of gujarat in 12 strategic directions including : resource mobilization and allocation , strengthening the ethics of care , addressing stigma , and integration of mental health with health and other sectors department of health and family welfare , government of gujarat also released a 12 5 year plan 2012 - 2017 , which includes the commitment to strengthening the mental health program to provide better medical services to the mentally ill patients , protect their right and rehabilitate them in the society in line with these state strategies , the hospital for mental health , ahmedabad focused the community project on utilizing available human resources at the village / taluka level for case identification and counseling and well - established referral linkages with both public and private agencies . the project sought to build on foundations of existing sources of rapport , influence and capacity to mobilize the community at the village level . including these stakeholders and expertise in mainstream mental health delivery will help reduce the stigma as well as address crucial gaps in mh services . the project aims to increase mental health awareness , sensitization and use of local mental health services and its impact on the explanatory models used to understand mental illness . appropriate and trained human resources are critical to developing collaborative arrangements for effective community based mental health services . a skilled workforce has been recognized as key to strong community participation , linkages with the voluntary sector and civil society initiatives , with academic institutions from inter - disciplinary backgrounds . the national institute of health and family welfare ( nihfw ) , dghs , mohfw and aamh have jointly designed structured training for community mental health workforce . the training explores methods to develop human resources for community - based services , and importantly to develop a broad understanding of the role of the mental health professional . it incorporates the core principles of recovery - oriented practice including : supporting personal recovery and promoting well - being ; delivering services informed by evidence and consistent with the social model of health ; proactive and purposeful engagement to build trusting relationships . this ensures practice is sensitive to the needs of families , friends and careers ; and working in partnership with key stakeholders . innovative training programs thus not only need to build on existing training resources but also develop new skill sets relevant to the principles and approach of the project . an example of the training delivered is the 5 days training program delivered in november 2013 , which included 20 participants from the 4 pilot sites . participants included medical officers and program managers who were responsible for the implementation of community mental health projects . the training started with topics that would set the context , by reflecting on global trends and best practice in asia pacific . the interactive workshops sought to broaden the perspectives of key stakeholders on the burden of mental illness in india and factors influencing progression of mental illness and rates of recovery , as well as efforts to respond to the challenges of mental illness and the mental health treatment gap . an essential strategy was to develop a shared understanding and consistent approach to recovery - oriented and community - based mental health service delivery in india . expertise on management strategies for mental illnesses complemented participants discussions and workshops on a multidisciplinary approach and local considerations . material included establishment of rehabilitation services within communities as well as the roles of families , self - help groups , caregivers associations , and other specialized ngo 's . the philosophy of recovery was also seen to positively influence stronger methods for measuring success . the training examined existing guidelines for monitoring and evaluation for each site with discussion on various aspects such as project indicators , data collection , data compilation , and report writing . the training sessions also examined effective management of common mental disorders including treatment seeking behaviors and treatment compliance , common childhood and adolescent psychiatric problems , addiction , abuses of addictive substances , and aged mental health problems . the workshops were centered on clinicians creating positive futures for people suffering from mental illness . expertise shared at the training incorporated information , education and communication strategies for mental health promotion . with an enormous mental health burden and inadequate treatment access in india , innovative solutions in community mental health care based on local conditions are urgently needed . local community resources are increasingly being recognized as a valuable part of the health system but are often neglected in the medical model of care . solutions to address the high - burden need to acknowledge that culture and traditional care , family and community structures are important elements in mental health care . further cultural aspects of the ways mental illnesses and disorders are viewed , presented and treated must be considered when applying an intervention model that is acceptable to the local community . as one model of care can not be applied to all persons or for all parts of india the project thus has developed different models and trialed across four pilot sites , to help increase access to appropriate community mental health care . india today has a framework for mental health care in the public , private and voluntary sectors and the challenges for the future will be improving the interfaces between mental health services and the wider community . thus , building partnerships with the local communities and partners like families and careers or traditional healers are not only essential for a comprehensive community mental health system but it can have important benefits to patients . developing these partnerships , which is promoted in the project , needs to be based upon mutual trust , respect , good communication , accountability and collaborative work . apart from the input of people with mental disorders and their families , care plans need to consider all relevant stakeholders , and through consultation employ their abilities and resources efficiently . just relying on increasing mental health hospitals or specialists workforce , though important to a certain extent in meeting the needs of those with acute and serious mental illness , is neither sustainable nor sufficient . if qualified mental health personnel are insufficient to meet the high demands , then short - term skill - based training for general and primary health staff can be conducted . they can be linked to specialist professionals either in the public or private sector for specialist referral services . training the phc team can be streamlined with standard training material and detailed operational guidelines . integration of primary care mental health training with the district training program will avoid any potential conflict with training for other programs . these task - shifting training that incorporate recovery and community oriented approaches are being implemented within the project sites . challenges in expanding the dmhp to all districts in india remained and one strategic approach would be to develop regional training resource centers such as through the local tertiary care mental health institutes to support the program . it is critical to examine the role of the existing mental health professionals and administrators in facilitating such the change from a hospital - based service system to a community - oriented service . through effective and unique training programs , clinicians can be better equipped to apply leadership skills and qualities to support better patient recovery outcomes . further the integration of mental health into socioeconomic and health policies requires sensitization of relevant administrators and staff at various sectors including social welfare , health , education , employment , and other development agencies . the community mental health project is an innovative and exciting opportunity to create positive change in meeting the challenges of community mental health care in india . it recognizes that no one single model of care can be applied all communities in the country and that locally appropriate models working in close partnership with local communities is required . targeted and skill - based training programs are useful to build local leadership capacity in implementing quality and culturally appropriate community mental health services .

the ministry of health and family welfare and its public health institutes are collaborating with asia australia mental health on an innovative community mental health development project designed to enhance initiatives under the district mental health program and increase accessibility of essential community mental health services . the project is an exciting opportunity to create positive change in meeting the challenges of community mental health care in india . it recognizes that no one single model of care can be applied to all the community in the country and that locally appropriate models working in close partnership with local communities is required . targeted and skill - based training programs are useful to build local leadership capacity in implementing quality and culturally appropriate community mental health services .

INTRODUCTION DISTRICT MENTAL HEALTH PROGRAM THE COMMUNITY MENTAL HEALTH DEVELOPMENT PROJECT Development of the project Pilot project sites and partners Building human resource capacity DISCUSSION CONCLUSION

in addition to the high mental health burden and large treatment gap of mental disorders there are significant limitations in the availability and integration of mental health services in the public health sector . hence , the ministry of health and family welfare ( mohfw ) india and its public health institutes are collaborating to develop local solutions to build and strengthen the national community mental health system . there is an urgent need for developing appropriate community mental health models in india that can be replicated and scaled up to increase access to appropriate care for people with mental illness . the district mental health program ( dmhp ) is a community - based mental health service delivery program initiated in 1996 under the national mental health plan and were now active in 123 of the 652 districts of india . the program aspires to increase access to comprehensive mental health care service by developing partnerships between the district mental health team , the primary health care ( phc ) teams , community - based organizations , nongovernmental organizations ( ngos ) , service users , family groups and various government departments . the major achievements of the program have been the development of community mental health services in the most underserved areas . to decentralize mental health care in the community using the public health infrastructure and other resources , mental health training , awareness and services are provided in collaboration with various community - based partners . a shortage of qualified mental health human resources , as well as little involvement of other primary care health workers in the mental health services has meant difficulty in recruiting the district mental health teams . as a strategy to build the dmhp and increase accessibility of essential community mental health services , mohfw and its public health institutes have collaborated with asia australia mental health ( aamh ) on innovative community mental health development project . a bilateral community mental health advisory committee chaired by the director general of health services ( dghs ) , mohfw provides the oversight of the overall project . common methodology for model development key principles for developing community mental health model of care gumla district , ranchi institute of neuro psychiatry and allied sciences ( rinpas ) ( jharkhand - east region ) the rinpas is situated in the tribal heartland of jharkhand state embracing santhal pargana and chotanagpur . as well as the district hospital , there is a district mental health center ( dmhp with psychiatrist , clinical psychologist , psychiatrist social worker , and psychiatric nurse ) , 13 phc centers , 11 community healthcare centers and 242 health sub centerssome of the main areas of concern identified by the mental health services at the start of this project included a failure to understand the indigenous social and emotional context of the presenting problems , a high rate of unaddressed mental health needs which affect the physical health and overall socioeconomic development , as well as pervasive and harmful myths and misconceptions in the community surrounding mental illness . the project planned to cover two blocks with the goal of delivering proper treatment , follow - up and sociovocational rehabilitation that will benefit tribal people affected with mental illness and their caregiversthe planned approach aims to engage the local stakeholders ( panchayat leaders , health workers , voluntary workers , faith healers , general practitioners ) in a culturally appropriate way , encourage joint planning , and mobilize community mental health resources . the project aims to increase culturally appropriate mental health promotion , improve convergence with other health and administrative sectors and increase numbers of people identifying with the tribal backgrounds employed in health and education centers and training in mental health the rinpas is situated in the tribal heartland of jharkhand state embracing santhal pargana and chotanagpur . as well as the district hospital , there is a district mental health center ( dmhp with psychiatrist , clinical psychologist , psychiatrist social worker , and psychiatric nurse ) , 13 phc centers , 11 community healthcare centers and 242 health sub centers some of the main areas of concern identified by the mental health services at the start of this project included a failure to understand the indigenous social and emotional context of the presenting problems , a high rate of unaddressed mental health needs which affect the physical health and overall socioeconomic development , as well as pervasive and harmful myths and misconceptions in the community surrounding mental illness . the project planned to cover two blocks with the goal of delivering proper treatment , follow - up and sociovocational rehabilitation that will benefit tribal people affected with mental illness and their caregivers the planned approach aims to engage the local stakeholders ( panchayat leaders , health workers , voluntary workers , faith healers , general practitioners ) in a culturally appropriate way , encourage joint planning , and mobilize community mental health resources . the project aims to increase culturally appropriate mental health promotion , improve convergence with other health and administrative sectors and increase numbers of people identifying with the tribal backgrounds employed in health and education centers and training in mental health chandigarh district , chandigarh government medical college ( north region ) chandigarh has an area of 114 km and a population of 1.05 million , including an estimated 90% urban residents . as a joint capital of both punjab and haryana states , chandigarh has 1 district hospital , 2 community health centers , 1 poly clinic , 30 civil dispensaries , 20 ayush dispensaries , and 17 sub centersin the chandigarh district , it has been identified that existing dmhp model may not have sustainable sources after funding after the 5 years current central government arrangement and the implementation of the dmhp has been in the nature of a parallel program rather than integrated , especially at the grassroots levelsthe department of psychiatry , government medical college and hospital has been working into the community for the last 14 years , providing community mental health services through community camps , community out - reach clinics in the periphery of chandigarh , half way home , crisis intervention team , suicide prevention helpline and providing technical assistance to self - help groupsthrough this work , it has been seen that there are insufficient social and vocational rehabilitation services for the persons with chronic and severe forms of mental disorders and one of the main goals of this pilot project is to explore initiatives for capacity building and empowerment of consumers and caregiversas an overarching principle , this particular pilot site identified the importance of including prevention and positive mental health promotion into the mental health approach chandigarh has an area of 114 km and a population of 1.05 million , including an estimated 90% urban residents . as a joint capital of both punjab and haryana states , chandigarh has 1 district hospital , 2 community health centers , 1 poly clinic , 30 civil dispensaries , 20 ayush dispensaries , and 17 sub centers in the chandigarh district , it has been identified that existing dmhp model may not have sustainable sources after funding after the 5 years current central government arrangement and the implementation of the dmhp has been in the nature of a parallel program rather than integrated , especially at the grassroots levels the department of psychiatry , government medical college and hospital has been working into the community for the last 14 years , providing community mental health services through community camps , community out - reach clinics in the periphery of chandigarh , half way home , crisis intervention team , suicide prevention helpline and providing technical assistance to self - help groups through this work , it has been seen that there are insufficient social and vocational rehabilitation services for the persons with chronic and severe forms of mental disorders and one of the main goals of this pilot project is to explore initiatives for capacity building and empowerment of consumers and caregivers as an overarching principle , this particular pilot site identified the importance of including prevention and positive mental health promotion into the mental health approach ramanathapuram district , madurai medical college ( tamil nadu - south region ) ramanathapuram district is an administrative district of tamil nadu state in southern india with a population of 1,337,560 . at this time , resources were put into training public health staff , but there have been no ongoing mental health education for the 300 medical doctors and 700 health workers working in the district for this region , the ramanathapuram dmhp is supported by the m. s. chellamuthu trust and research foundation , madurai . the department of health and family welfare , government of gujarat commissioned work which led to the 2004 mission report titled priorities for mental health sector development in gujarat . this report provided a broad canvas of strategies which need to be pursued to address issues facing this sector in the context of gujarat in 12 strategic directions including : resource mobilization and allocation , strengthening the ethics of care , addressing stigma , and integration of mental health with health and other sectors department of health and family welfare , government of gujarat also released a 12 5 year plan 2012 - 2017 , which includes the commitment to strengthening the mental health program to provide better medical services to the mentally ill patients , protect their right and rehabilitate them in the societyin line with these state strategies , the hospital for mental health , ahmedabad focused the community project on utilizing available human resources at the village / taluka level for case identification and counseling and well - established referral linkages with both public and private agencies . the project aims to increase mental health awareness , sensitization and use of local mental health services and its impact on the explanatory models used to understand mental illness . the department of health and family welfare , government of gujarat commissioned work which led to the 2004 mission report titled priorities for mental health sector development in gujarat . this report provided a broad canvas of strategies which need to be pursued to address issues facing this sector in the context of gujarat in 12 strategic directions including : resource mobilization and allocation , strengthening the ethics of care , addressing stigma , and integration of mental health with health and other sectors department of health and family welfare , government of gujarat also released a 12 5 year plan 2012 - 2017 , which includes the commitment to strengthening the mental health program to provide better medical services to the mentally ill patients , protect their right and rehabilitate them in the society in line with these state strategies , the hospital for mental health , ahmedabad focused the community project on utilizing available human resources at the village / taluka level for case identification and counseling and well - established referral linkages with both public and private agencies . the project aims to increase mental health awareness , sensitization and use of local mental health services and its impact on the explanatory models used to understand mental illness . the national institute of health and family welfare ( nihfw ) , dghs , mohfw and aamh have jointly designed structured training for community mental health workforce . as well as the district hospital , there is a district mental health center ( dmhp with psychiatrist , clinical psychologist , psychiatrist social worker , and psychiatric nurse ) , 13 phc centers , 11 community healthcare centers and 242 health sub centerssome of the main areas of concern identified by the mental health services at the start of this project included a failure to understand the indigenous social and emotional context of the presenting problems , a high rate of unaddressed mental health needs which affect the physical health and overall socioeconomic development , as well as pervasive and harmful myths and misconceptions in the community surrounding mental illness . the project planned to cover two blocks with the goal of delivering proper treatment , follow - up and sociovocational rehabilitation that will benefit tribal people affected with mental illness and their caregiversthe planned approach aims to engage the local stakeholders ( panchayat leaders , health workers , voluntary workers , faith healers , general practitioners ) in a culturally appropriate way , encourage joint planning , and mobilize community mental health resources . the project aims to increase culturally appropriate mental health promotion , improve convergence with other health and administrative sectors and increase numbers of people identifying with the tribal backgrounds employed in health and education centers and training in mental health the rinpas is situated in the tribal heartland of jharkhand state embracing santhal pargana and chotanagpur . as well as the district hospital , there is a district mental health center ( dmhp with psychiatrist , clinical psychologist , psychiatrist social worker , and psychiatric nurse ) , 13 phc centers , 11 community healthcare centers and 242 health sub centers some of the main areas of concern identified by the mental health services at the start of this project included a failure to understand the indigenous social and emotional context of the presenting problems , a high rate of unaddressed mental health needs which affect the physical health and overall socioeconomic development , as well as pervasive and harmful myths and misconceptions in the community surrounding mental illness . the project planned to cover two blocks with the goal of delivering proper treatment , follow - up and sociovocational rehabilitation that will benefit tribal people affected with mental illness and their caregivers the planned approach aims to engage the local stakeholders ( panchayat leaders , health workers , voluntary workers , faith healers , general practitioners ) in a culturally appropriate way , encourage joint planning , and mobilize community mental health resources . the project aims to increase culturally appropriate mental health promotion , improve convergence with other health and administrative sectors and increase numbers of people identifying with the tribal backgrounds employed in health and education centers and training in mental health chandigarh district , chandigarh government medical college ( north region ) chandigarh has an area of 114 km and a population of 1.05 million , including an estimated 90% urban residents . as a joint capital of both punjab and haryana states , chandigarh has 1 district hospital , 2 community health centers , 1 poly clinic , 30 civil dispensaries , 20 ayush dispensaries , and 17 sub centersin the chandigarh district , it has been identified that existing dmhp model may not have sustainable sources after funding after the 5 years current central government arrangement and the implementation of the dmhp has been in the nature of a parallel program rather than integrated , especially at the grassroots levelsthe department of psychiatry , government medical college and hospital has been working into the community for the last 14 years , providing community mental health services through community camps , community out - reach clinics in the periphery of chandigarh , half way home , crisis intervention team , suicide prevention helpline and providing technical assistance to self - help groupsthrough this work , it has been seen that there are insufficient social and vocational rehabilitation services for the persons with chronic and severe forms of mental disorders and one of the main goals of this pilot project is to explore initiatives for capacity building and empowerment of consumers and caregiversas an overarching principle , this particular pilot site identified the importance of including prevention and positive mental health promotion into the mental health approach chandigarh has an area of 114 km and a population of 1.05 million , including an estimated 90% urban residents . as a joint capital of both punjab and haryana states , chandigarh has 1 district hospital , 2 community health centers , 1 poly clinic , 30 civil dispensaries , 20 ayush dispensaries , and 17 sub centers in the chandigarh district , it has been identified that existing dmhp model may not have sustainable sources after funding after the 5 years current central government arrangement and the implementation of the dmhp has been in the nature of a parallel program rather than integrated , especially at the grassroots levels the department of psychiatry , government medical college and hospital has been working into the community for the last 14 years , providing community mental health services through community camps , community out - reach clinics in the periphery of chandigarh , half way home , crisis intervention team , suicide prevention helpline and providing technical assistance to self - help groups through this work , it has been seen that there are insufficient social and vocational rehabilitation services for the persons with chronic and severe forms of mental disorders and one of the main goals of this pilot project is to explore initiatives for capacity building and empowerment of consumers and caregivers as an overarching principle , this particular pilot site identified the importance of including prevention and positive mental health promotion into the mental health approach ramanathapuram district , madurai medical college ( tamil nadu - south region ) ramanathapuram district is an administrative district of tamil nadu state in southern india with a population of 1,337,560 . at this time , resources were put into training public health staff , but there have been no ongoing mental health education for the 300 medical doctors and 700 health workers working in the district for this region , the ramanathapuram dmhp is supported by the m. s. chellamuthu trust and research foundation , madurai . the department of health and family welfare , government of gujarat commissioned work which led to the 2004 mission report titled priorities for mental health sector development in gujarat . this report provided a broad canvas of strategies which need to be pursued to address issues facing this sector in the context of gujarat in 12 strategic directions including : resource mobilization and allocation , strengthening the ethics of care , addressing stigma , and integration of mental health with health and other sectors department of health and family welfare , government of gujarat also released a 12 5 year plan 2012 - 2017 , which includes the commitment to strengthening the mental health program to provide better medical services to the mentally ill patients , protect their right and rehabilitate them in the societyin line with these state strategies , the hospital for mental health , ahmedabad focused the community project on utilizing available human resources at the village / taluka level for case identification and counseling and well - established referral linkages with both public and private agencies . the department of health and family welfare , government of gujarat commissioned work which led to the 2004 mission report titled priorities for mental health sector development in gujarat . this report provided a broad canvas of strategies which need to be pursued to address issues facing this sector in the context of gujarat in 12 strategic directions including : resource mobilization and allocation , strengthening the ethics of care , addressing stigma , and integration of mental health with health and other sectors department of health and family welfare , government of gujarat also released a 12 5 year plan 2012 - 2017 , which includes the commitment to strengthening the mental health program to provide better medical services to the mentally ill patients , protect their right and rehabilitate them in the society in line with these state strategies , the hospital for mental health , ahmedabad focused the community project on utilizing available human resources at the village / taluka level for case identification and counseling and well - established referral linkages with both public and private agencies . the national institute of health and family welfare ( nihfw ) , dghs , mohfw and aamh have jointly designed structured training for community mental health workforce . the interactive workshops sought to broaden the perspectives of key stakeholders on the burden of mental illness in india and factors influencing progression of mental illness and rates of recovery , as well as efforts to respond to the challenges of mental illness and the mental health treatment gap . with an enormous mental health burden and inadequate treatment access in india , innovative solutions in community mental health care based on local conditions are urgently needed . as one model of care can not be applied to all persons or for all parts of india the project thus has developed different models and trialed across four pilot sites , to help increase access to appropriate community mental health care . india today has a framework for mental health care in the public , private and voluntary sectors and the challenges for the future will be improving the interfaces between mental health services and the wider community . if qualified mental health personnel are insufficient to meet the high demands , then short - term skill - based training for general and primary health staff can be conducted . challenges in expanding the dmhp to all districts in india remained and one strategic approach would be to develop regional training resource centers such as through the local tertiary care mental health institutes to support the program . the community mental health project is an innovative and exciting opportunity to create positive change in meeting the challenges of community mental health care in india . it recognizes that no one single model of care can be applied all communities in the country and that locally appropriate models working in close partnership with local communities is required . targeted and skill - based training programs are useful to build local leadership capacity in implementing quality and culturally appropriate community mental health services .

c57bl/6j ins2 mice were purchased from jackson laboratory ( bar harbor , me , usa ) , ins2 female mice were bred to wild - type ( wt ) male mice in our animal facilities . retinas of male and female mice were examined ; however , only males were used in the morphometric retinal analyses because disease progression in females is slower and less uniform . diabetes onset was verified by measuring urine glucose using the diastic reagent strips for urinalysis ( bayer , mishawaka , in , usa ) and blood glucose using a blood glucose monitoring system ( abbott laboratories , alameda , ca , usa ) . all mice received food and water ad libitum and were maintained on a 12-hour light - dark cycle . body weight and blood glucose were measured at time of death , and data are provided in tables 1 and 2 . average weights and blood glucose levels of mice used in the delayed ( + ) -ptz administration study average weights and blood glucose levels of mice used in the analysis of ins2- sig1r mice two studies were conducted . in the first study , we examined the effects of delaying ( + ) -ptz injection ( after onset of diabetes ) on retinal neuronal death in diabetic retinopathy . , we determined whether the retinal neurodegeneration of ins2 mice would be accelerated if the mice lacked sigma1r , and we asked whether the retinal phenotype would involve vasculopathy when sigma1r was absent . c57bl/6-ins2j mice were bred with sig1r mice ( the generation of which has been previously described ) to produce ins2/sig1r mice , which were crossed with sig1r mice to generate ins2/sig1rmice . in addition , we confirmed that the mice did not carry the rd8 ( crb1 ) mutation , which manifests as disrupted morphology including retinal rosettes . mice were maintained for either 12 or 16 weeks , after which they were subjected to functional and morphologic analysis . maintenance of animals adhered to the institutional guidelines for the humane treatment of animals following our institutional animal care and use committee ( iacuc)-approved protocol and to the arvo statement for the use of animals in ophthalmic and vision research . mice were administered ( + ) -ptz either 4 or 8 weeks after onset of diabetes ( thus , one group of ins2 mice was injected beginning at age 8 weeks and a second was injected beginning at age 12 weeks ) . additional groups of mice included ins2 noninjected , wt ( ins2 ) noninjected , and wt ( ins2 ) injected beginning at 8 weeks ( table 1 ) . louis , mo , usa ) was dissolved initially in dimethyl sulfoxide ( dmso ) and diluted with 0.01 m pbs , and it was administered via intraperitoneal injection at a dosage 0.5 mg kg two times per week for 22 weeks . mice were euthanized at 25 weeks , as was the case in the previous study in which ins2 mice were administered ( + ) -ptz at diabetes onset . retinas were evaluated morphometrically ( described below ) . to evaluate retinal morphology in vivo , spectral - domain ( sd)-oct imaging was performed in ins2/sig1rmice . mice were anesthetized using a rodent anesthesia cocktail ( ketamine hydrochloride / xylazine hydrochloride solution [ 80:12 mm / ml ] ; sigma - aldrich corp . ) . pupils were dilated with 1% tropicamide ( bausch and lomb , tampa , fl , usa ) followed by application of genteal lubricant eye gel , 10 g ( alcon , ft . the sd - oct images were obtained using the bioptigen spectral - domain ophthalmic imaging system ( sd ois ; bioptigen envisu r2200 ; bioptigen , inc . , durham , nc , usa ) . imaging included averaged single b - scan and volume intensity scans ( vips ) with images centered on the optic nerve head . postimaging analysis included auto segmentation report analysis and manual assessment of all retinal layers using invivovuetm diver 2.4 software ( bioptigen , inc . ) . fundus imaging was performed in ins2/sig1rmice using the micron iii camera ( phoenix research laboratories , inc . , pleasanton , ca , usa ) in mice that had been anesthetized as described for sd - oct . following fundus imaging , fluorescein angiography was performed following a 20-l 10% fluorescein lite ( apollo ophthalmic , newport beach , ca , usa ) intraperitoneal injection , which permitted visualization of retinal vasculature . eyes were removed immediately and were either oriented in tissue - tek oct ( miles laboratories , elkhart , in , usa ) and were frozen slowly by immersion in liquid nitrogen ( without fixation ) or were fixed in 4% paraformaldehyde/2% glutaraldehyde in 0.1 m cacodylate buffer and embedded in jb-4 embedding compound ( ems , hatfield , pa , usa ) per our method . sections were stained with hematoxylin and eosin ( h&e ) and used for systematic morphometric analysis using a zeiss axioplan2 fluorescent microscope equipped with hrm camera and the axiovision 4.6.3 program ( carl zeiss , oberkochen , germany ) . measurements included the thickness of the ( 1 ) total retina , ( 2 ) inner plexiform layer ( ipl ) , ( 3 ) inl , ( 4 ) outer plexiform layer ( opl ) , and ( 5 ) the photoreceptor inner / outer segments . the number of rows of photoreceptor cell nuclei in the outer nuclear layer ( onl ) was counted . the number of cell bodies in the gcl was quantified by counting cells from the temporal ora serrata to the nasal ora serrata and expressing the data as number of cells per 100 m length of retina . sections analyzed were from the center of the eyeball ; thus , the optic nerve was visible . three measurements were made on each side ( temporal and nasal ) of the optic nerve at 200- to 300-m intervals , resulting in a total of six measurements obtained per eye . per mouse , at least two sections were analyzed for the left and right eye . glial fibrillary acidic protein ( gfap ) is a 54-kda intermediate filament protein that is a major constituent of retinal astrocytes ; under retinal stress conditions , gfap expression is up - regulated in retinal mller glial cells in a process termed gliosis . triton x-100 , and incubated with power block , after which sections were incubated with rabbit anti - gfap ( 1:500 ; dako , carpentaria , ca , usa ) overnight at 4c . sections were washed and incubated triton x-100 , followed by incubation with secondary antibody alexa fluor 488conjugated donkey anti - rabbit igg ( 1:1,000 ; invitrogen , carlsbad , ca , usa ) for 1 hour at room temperature . sections were washed three times with pbs - triton x-100 and coverslipped with fluoroshield with 4,6-diamidino-2-phenylindole ( dapi ) . sections were examined by epifluorescence using a zeiss axioplan-2 microscope , equipped with the axiovision program , an hrm camera , and filters to detect fitc and dapi . fluorescence intensity was analyzed using metamorph image analysis software ( molecular devices , sunnyvale , ca , usa ) . additional immunofluorescence experiments were performed to detect dying cells using the apoptag fluorescein in situ apoptosis detection kit ( tunel assay ; emd millipore , billerica , ma , usa ) per our published methods . analysis of variance was used to determine whether there were significant differences in morphologic measurements in mice administered ( + ) -ptz at the two time intervals compared with noninjected animals . for oct measurements and cell counting in gcl studies , 2-way anova was used to determine whether there were significant differences between wt , ins2 , and ins2/sig1rmouse groups at the two ages studied . statistical analysis was conducted using the graphpad prism software ( version 6 ; graphpad software , inc . , la jolla , ca , usa ) . c57bl/6j ins2 mice were purchased from jackson laboratory ( bar harbor , me , usa ) , ins2 female mice were bred to wild - type ( wt ) male mice in our animal facilities . retinas of male and female mice were examined ; however , only males were used in the morphometric retinal analyses because disease progression in females is slower and less uniform . diabetes onset was verified by measuring urine glucose using the diastic reagent strips for urinalysis ( bayer , mishawaka , in , usa ) and blood glucose using a blood glucose monitoring system ( abbott laboratories , alameda , ca , usa ) . all mice received food and water ad libitum and were maintained on a 12-hour light - dark cycle . body weight and blood glucose were measured at time of death , and data are provided in tables 1 and 2 . average weights and blood glucose levels of mice used in the delayed ( + ) -ptz administration study average weights and blood glucose levels of mice used in the analysis of ins2- sig1r mice two studies were conducted . in the first study , we examined the effects of delaying ( + ) -ptz injection ( after onset of diabetes ) on retinal neuronal death in diabetic retinopathy . , we determined whether the retinal neurodegeneration of ins2 mice would be accelerated if the mice lacked sigma1r , and we asked whether the retinal phenotype would involve vasculopathy when sigma1r was absent . c57bl/6-ins2j mice were bred with sig1r mice ( the generation of which has been previously described ) to produce ins2/sig1r mice , which were crossed with sig1r mice to generate ins2/sig1rmice . in addition , we confirmed that the mice did not carry the rd8 ( crb1 ) mutation , which manifests as disrupted morphology including retinal rosettes . mice were maintained for either 12 or 16 weeks , after which they were subjected to functional and morphologic analysis . maintenance of animals adhered to the institutional guidelines for the humane treatment of animals following our institutional animal care and use committee ( iacuc)-approved protocol and to the arvo statement for the use of animals in ophthalmic and vision research . mice were administered ( + ) -ptz either 4 or 8 weeks after onset of diabetes ( thus , one group of ins2 mice was injected beginning at age 8 weeks and a second was injected beginning at age 12 weeks ) . additional groups of mice included ins2 noninjected , wt ( ins2 ) noninjected , and wt ( ins2 ) injected beginning at 8 weeks ( table 1 ) . louis , mo , usa ) was dissolved initially in dimethyl sulfoxide ( dmso ) and diluted with 0.01 m pbs , and it was administered via intraperitoneal injection at a dosage 0.5 mg kg two times per week for 22 weeks . mice were euthanized at 25 weeks , as was the case in the previous study in which ins2 mice were administered ( + ) -ptz at diabetes onset . to evaluate retinal morphology in vivo , spectral - domain ( sd)-oct imaging was performed in ins2/sig1rmice . mice were anesthetized using a rodent anesthesia cocktail ( ketamine hydrochloride / xylazine hydrochloride solution [ 80:12 mm / ml ] ; sigma - aldrich corp . ) . pupils were dilated with 1% tropicamide ( bausch and lomb , tampa , fl , usa ) followed by application of genteal lubricant eye gel , 10 g ( alcon , ft . the sd - oct images were obtained using the bioptigen spectral - domain ophthalmic imaging system ( sd ois ; bioptigen envisu r2200 ; bioptigen , inc . , durham , nc , usa ) . imaging included averaged single b - scan and volume intensity scans ( vips ) with images centered on the optic nerve head . postimaging analysis included auto segmentation report analysis and manual assessment of all retinal layers using invivovuetm diver 2.4 software ( bioptigen , inc . ) . fundus imaging was performed in ins2/sig1rmice using the micron iii camera ( phoenix research laboratories , inc . , pleasanton , ca , usa ) in mice that had been anesthetized as described for sd - oct . following fundus imaging , fluorescein angiography was performed following a 20-l 10% fluorescein lite ( apollo ophthalmic , newport beach , ca , usa ) intraperitoneal injection , which permitted visualization of retinal vasculature . eyes were removed immediately and were either oriented in tissue - tek oct ( miles laboratories , elkhart , in , usa ) and were frozen slowly by immersion in liquid nitrogen ( without fixation ) or were fixed in 4% paraformaldehyde/2% glutaraldehyde in 0.1 m cacodylate buffer and embedded in jb-4 embedding compound ( ems , hatfield , pa , usa ) per our method . sections were stained with hematoxylin and eosin ( h&e ) and used for systematic morphometric analysis using a zeiss axioplan2 fluorescent microscope equipped with hrm camera and the axiovision 4.6.3 program ( carl zeiss , oberkochen , germany ) . measurements included the thickness of the ( 1 ) total retina , ( 2 ) inner plexiform layer ( ipl ) , ( 3 ) inl , ( 4 ) outer plexiform layer ( opl ) , and ( 5 ) the photoreceptor inner / outer segments . the number of rows of photoreceptor cell nuclei in the outer nuclear layer ( onl ) was counted . the number of cell bodies in the gcl was quantified by counting cells from the temporal ora serrata to the nasal ora serrata and expressing the data as number of cells per 100 m length of retina . sections analyzed were from the center of the eyeball ; thus , the optic nerve was visible . three measurements were made on each side ( temporal and nasal ) of the optic nerve at 200- to 300-m intervals , resulting in a total of six measurements obtained per eye . per mouse , glial fibrillary acidic protein ( gfap ) is a 54-kda intermediate filament protein that is a major constituent of retinal astrocytes ; under retinal stress conditions , gfap expression is up - regulated in retinal mller glial cells in a process termed gliosis . triton x-100 , and incubated with power block , after which sections were incubated with rabbit anti - gfap ( 1:500 ; dako , carpentaria , ca , usa ) overnight at 4c . sections were washed and incubated triton x-100 , followed by incubation with secondary antibody alexa fluor 488conjugated donkey anti - rabbit igg ( 1:1,000 ; invitrogen , carlsbad , ca , usa ) for 1 hour at room temperature . sections were washed three times with pbs - triton x-100 and coverslipped with fluoroshield with 4,6-diamidino-2-phenylindole ( dapi ) . sections were examined by epifluorescence using a zeiss axioplan-2 microscope , equipped with the axiovision program , an hrm camera , and filters to detect fitc and dapi . fluorescence intensity was analyzed using metamorph image analysis software ( molecular devices , sunnyvale , ca , usa ) . additional immunofluorescence experiments were performed to detect dying cells using the apoptag fluorescein in situ apoptosis detection kit ( tunel assay ; emd millipore , billerica , ma , usa ) per our published methods . analysis of variance was used to determine whether there were significant differences in morphologic measurements in mice administered ( + ) -ptz at the two time intervals compared with noninjected animals . for oct measurements and cell counting in gcl studies , 2-way anova was used to determine whether there were significant differences between wt , ins2 , and ins2/sig1rmouse groups at the two ages studied . statistical analysis was conducted using the graphpad prism software ( version 6 ; graphpad software , inc . , la jolla , ca , usa ) . earlier studies demonstrated that twice weekly administration of ( + ) -ptz at the 0.5-mg kg dosage reduced apoptotic neuron death characteristic of ins2mice and preserved retinal architecture , when treatment started at diabetes onset . we used this same ( + ) -ptz dosage and treatment regimen in the present study , but initiated injections either 4 or 8 weeks after diabetes onset , which meant that injections began at age 8 or 12 weeks in ins2mice . representative photomicrographs of retinas of wt mice receiving either no ( + ) -ptz injection or receiving ( + ) -ptz twice weekly beginning at age 8 weeks are shown ( fig . 1 ) . there was no deleterious effect on retinal architecture when wt mice were administered the sigma1r ligand ( fig . 1 ) and no differences in morphometric analysis of retinal layers ( data not shown ) representative photomicrographs of h&e - stained retinas of control ( c57bl/6 ) mice ( wt ) that received ( a ) no ( + ) -ptz injection or ( b ) 0.5 mg kg ( + ) -ptz injection twice weekly beginning at 8 weeks . gcl , ganglion cell layer ; ipl , inner plexiform layer ; inl , inner nuclear layer ; onl , outer nuclear layer ; rpe , retinal pigment epithelial layer . in the case of the ins2 mice that received no ( + ) -ptz injections , the retinas showed mild disruption . figures 2a and 2b show representative photomicrographs of retinas from two of the mice in the nontreated group . the alterations in the inl by 25 weeks in the ins2 mice have been reported . the nuclear layers did not have their characteristic uniform stratified appearance , but this was not attributable to presence of the rd8 mutation . mice that had been injected intraperitoneally beginning at 4 or 8 weeks after onset of diabetes onset with 0.5 mg kg ( + ) -ptz twice weekly showed preservation of retinal architecture ( figs . the retinas were subjected to morphometric analysis , and the total retinal thickness did not differ between the wt mice , ins2-noninjected mice , and ins2-injected mice beginning at 4 or 8 weeks ( fig . 2 g ) . nor did the thickness of the individual layers ( ipl , inl , opl , and onl ) differ significantly between groups ( data not shown ) . however , when the numbers of cells in the gcl were quantified , there was a significant difference between the wt mice and the ins2-noninjected mice ( fig . wild - type mice had approximately 13 cells per 100 m length of retina , whereas the ins2-noninjected mice had approximately 9 cells for the same retinal length ( fig . for the mice treated with sigma1r ligand at either 4 or 8 weeks after onset of diabetes , there were approximately 11 cells per 100 m retinal length , which was significantly greater than the ins2-noninjected mice . although the numbers of cells in the gcl of ( + ) -ptz - treated ins2 mice were not equal to that of the wt mice , there was clear preservation of these cells compared with diabetic mice not treated with the sigma1r ligand . we also noted that there was waveform irregularity of the onl accompanied by shallow retinal detachment and irregularly elongated photoreceptor outer segments in the retinas of ins2-noninjected mice . to evaluate whether the waveform appearance and occasional retinal detachment might be a consequence of the cryosectioning method of preparing sections , we examined eyes that had been fixed prior to embedding and processed for jb-4 plastic embedding . in this case , the ins2-noninjected mice showed minimal detachment , although slight irregularities of the nuclear layers were still detectable ( fig . the jb-4embedded retinas of the ( + ) -ptz treated ins2 mice had a generally uniform appearance ( figs . 2j , 2k ) similar to that observed in the cryosections of retinas of these mice ( figs . preservation of retinal structure and cells in the gcl in ins2 mice administered ( + ) -ptz after diabetes onset . ( a , b ) representative h&e - stained retinal cryosections of ins2 mice that received no ( + ) -ptz for the duration of the study ; the inl is slightly disrupted , and gcl density is decreased . representative h&e - stained retinal cryosections of ( c , d ) ins2 mice that received 0.5 mg kg ( + ) -ptz beginning 4 weeks after onset of diabetes ( at age 8 weeks ) or ( e , f ) ins2 mice that received 0.5 mg kg ( + ) -ptz beginning 8 weeks after onset of diabetes ( at age 12 weeks ) . morphometric analysis revealed no significant change in total retinal thickness in any of the eyes evaluated ( g ) , but a significant difference in the number of cell bodies in the gcl per 100 m length of retina ( h ) . eyes were processed for jb-4 embedding and stained with h&e to permit visualization of retinal architecture of ( i ) ins2 mice that received no ( + ) -ptz ; ( j ) ins2 mice that received 0.5 mg kg ( + ) -ptz beginning 4 weeks after onset of diabetes ( at age 8 weeks ) ; or ( k ) ins2 mice that received 0.5 mg kg ( + ) -ptz beginning 8 weeks after onset of diabetes ( at age 12 weeks ) . gcl , ganglion cell layer ; inl , inner nuclear layer ; onl , outer nuclear layer ; is / os , inner / outer segments . we examined the expression levels of gfap , which is up - regulated during retinal gliosis . we observed labeling in the wt retina limited to astrocytes , which typically express this protein ( fig . 3a ) . in the ins2-noninjected mice , the level of gfap increased , and the distribution of labeling this is an indication of gliosis characteristic of this diabetic retinopathy model . in the ins2 ( + ) the observation of minimal detection of gfap ( except the normal detection in astrocytes ) was true for mice treated beginning at 8 weeks ( 4 weeks after onset of diabetes ; fig . 3c ) and in mice treated beginning at 12 weeks ( 8 weeks after onset of diabetes ; fig . immunofluorescent detection of gfap , a marker of gliosis in ins2 mice administered ( + ) -ptz after diabetes onset . representative photomicrographs of immunofluorescent detection of gfap in retinal cryosections from ( a ) ins2 ( wt ) , ( b ) ins2 [ no ( + ) -ptz treatment ] , ( c ) ins2 mice that received 0.5 mg kg ( + ) -ptz beginning 4 weeks after onset of diabetes ( at age 8 weeks ) , and ( d ) ins2 mice that received 0.5 mg kg ( + ) -ptz beginning 8 weeks after onset of diabetes ( at age 12 weeks ) . immunofluorescence analysis used anti - gfap followed by incubation with alexa fluor 488 ( green - labeled secondary antibody ) , showing marked increase in gfap in radially oriented fibers of mller glial cells ( radial labeling ) in ins2 [ no ( + ) -ptz treatment ] , but minimal labeling in mller cells of the wt or ( + ) -ptz treated mice . ( e ) quantification of gfap fluorescence intensity data obtained from metamorphic analysis ( significant difference : * p < 0.05 , * * p < 0.01 ) . regarding the monitoring of blood glucose in this study ( table 1 ) , wt mice had blood glucose values below 240 mg / dl and were not hyperglycemic . the ( + ) -ptz treated ins2 mice remained hyperglycemic throughout the study ; blood glucose levels were approximately 400 mg / dl ( similar to untreated ins2 mice ) and were significantly higher than wt mice ( 116197 mg / dl ) , suggesting that hyperglycemia per se may not be sufficient to trigger neuronal loss in diabetes . in addition to investigating the effects of delayed administration of ( + ) -ptz on the retinal structure in the ins2diabetic mice described above , we examined the consequences on retinal phenotype when sigma1r was absent by generating ins2/sig1rmice . we performed sd - oct in wt ( ins2 ) , ins2 mice , and ins2/sig1rmice that were either 12 or 16 weeks of age . volume image projections and the accompanying b - scans showed generally normal retinal architecture and no evidence of gross disruption in the retinal nuclear or plexiform layers among the three groups ( figs . postimage analysis of retinal layers used invivovue 2.4 diver software ( bioptigen , inc . , durham , nc , usa ) and confirmed that the thickness of most retinal layers was comparable among the three groups of mice , with the exception of the measurement of the nerve fiber layer ( nfl ) . in this case , the nfl was thicker in retinas of ins2/sig1rmice compared with wt ( ins2 ) or ins2 mice ( fig . measurement of other retinal layers did not differ significantly among the three mouse groups ( data not shown ) . wild - type ( ins2 ) , ins2 , and ins2/sig1r mice were anesthetized and subjected to sd - oct to obtain images of the retina in situ . representative vip for wt ( ins2 ) mice : age 12 ( a ) and 16 weeks ( b ) ; ins2 mice : age 12 ( c ) and 16 weeks ( d ) ; and ins2/sig1rmice : age 12 ( e ) and 16 weeks ( f ) . the panel to the right of each vip is the b - scans of the same animal . autosegmentation analysis was performed to determine whether there were significant differences in thickness of retinal layers among the three mouse groups . there was a significant increase in thickness of the nerve fiber layer ( g ) ( * significantly different at p < 0.05 ) . mice were subjected to fundoscopy wt ( ins2 ) at 12 ( h ) and 16 weeks ( j ) ; ins2 at 12 ( l ) and 16 weeks ( n ) ; ins2/sig1r at 12 ( p ) and 16 weeks ( r ) . fundus images were normal for wt and ins2 , whereas vitreal opacities ( denoted by long arrows ) were detected in the ins2/sig1r mice ( p , r ) . wild - type ( ins2 ) showed no alterations at either 12 ( i ) or 16 weeks ( k ) . ins2 mice also had normal vascularization patterns at 12 ( m ) and 16 weeks ( o ) , whereas ins2/sig1r mice had beading at 12 ( q ) and at 16 weeks ( s ) . the long arrows in q and s point to areas of vitreal opacity ; the indented arrowhead points to a vessel with beading ( s ) . ( t ) enlarged image of image shown in s. ( u , v ) fa images of two additional mice at 16 weeks showing vitreal opacity ( long arrow ) and occasional beading of vessels ( indented arrowhead ) . we then examined wt ( ins2 ) , ins2 , and ins2/sig1r mice by retinal fundoscopy and fa at 12 and 16 weeks of age . fluorescein dye was injected , and images were acquired 30 seconds after injection and every minute thereafter for 5 minutes . images were arranged according to capture time , and data were compared among the three mouse groups at ages 12 and 16 weeks . fluorescein angiography performed in wt mice showed normal vessel filling , no leakage , and uniform capillary network around blood vessels at both ages studied ( figs . 4i , 4k ) . similarly , vessel filling in ins2 mice appeared normal , and leakage was not observed at either 12 ( fig . 4o ) . in ins2/sig1rmice , however , there was evidence of vessel beading at 12 weeks ( fig . 4s , indented arrowhead ) . a larger image of figure 4s is provided ( fig . the long arrow in figure 4 t points to a region of vitreal opacity , not an area of leakiness . , there appears to be an area of capillary dropout ( denoted by an asterisk ) in the retina of a 16-week ins2/sig1rmouse . it is noteworthy that all of the 16-week ins2/sig1rmice examined showed some evidence of beading and of vitreal opacities . the data suggest that in the absence of sigma1r subtle retinal vascular abnormalities develop that are not normally observed by fa in the ins2 diabetic mouse retina . eyes from wt ( ins2 ) , ins2 , and ins2/sig1r mice were processed in jb-4 embedding compound for light microscopic evaluation and retinal morphometric analysis at 12 and 16 weeks . although the retinal architecture becomes mildly disrupted by 25 weeks in the ins2 mice as reported previously and figure 2 of this study , the retinal architecture of ins2 mice at the younger ages ( 12 and 16 weeks ) analyzed in this study ( fig . a notable feature that distinguished the ins2/sig1r mice from the wt and ins2 mice was the fragmented appearance and disruption of the nfl in these mice compared with the wt ( ins2 ) and ins2 mice ( fig . although the disrupted appearance of the nfl may reflect increased sensitivity to the fixative used in processing the tissue , there appears to be involvement of nfl in the ins2/sig1r mice when examined in vivo by sd - oct imaging ( fig . the nfl is comprised of the axons of the ganglion cells ; thus , the decrease in the numbers of cells observed in the ins2/sig1r mice could account for disruption of their axons . eyes of ( a ) wt ( ins2 ) , ( b ) ins2 , and ( c ) ins2/sig1rmice were processed for jb-4 embedding and stained with h&e to permit visualization of retinal architecture . panels shown are from 16-week - old mice . in wt ( ins2 ) mice , the retinas are well organized and layers are of normal thickness . the retinas of the ins2 and ins2/sig1rmice are similar in appearance , with the exception of the nerve fiber layer in the ins2/sig1rmice ( arrow ) , which is markedly disrupted . retinas were subjected to morphometric analysis ; there was a significant decrease in the number of cells in the gcl in the ins2/sig1rmice at 12 ( d ) and 16 weeks ( e ) . retinal cryosections of the same mice were subjected to immunofluorescent detection of tunel - positive cells ( green labeling ) , cell nuclei were blue ( dapi ) in ( f ) wt ( ins2 ) , ( g ) ins2 , and ( h ) ins2/sig1rmice . additional images of tunel staining were obtained using a lower - powered objective focused on the inl for ( i ) wt ( ins2 ) , ( j ) ins2 , and ( k ) ins2/sig1rmice . gcl , ganglion cell layer ; ipl , inner plexiform layer ; inl , inner nuclear layer ; opl , outer plexiform layer ; onl , outer nuclear layer ; is , inner segment ; os , outer segment ; rpe , retinal pigment epithelium . statistical significance : * * p < 0.01 , * * * p < 0.001 . in the mouse retina , the number of cell bodies in the gcl is typically 1214 per 100 m length of retina . this was observed in the wt ( ins2 ) mouse retina at both 12 and 16 weeks ( figs . , there was minimal difference at 12 and 16 weeks in the number of cells bodies in the gcl compared with wt mice ( figs . 5d , 5e ) . in ins2/sig1r mice , however , there was a significant decrease in the number of cells in the gcl at 12 ( 11 cells/100 m retinal length ) and 16 weeks ( 8 cells/100 m retinal length ) . the data suggest that , in the absence of sigma1r , the loss of ganglion cells observed at 25 weeks in the ins2 is markedly accelerated . we subjected retinal cryosections from the three mouse groups to immunofluorescence to detect dying cells using the apoptag fluorescein in situ apoptosis detection kit ( tunel staining ) . we did not detect dying cells in gcl of wt ( ins2 ) mice ( fig . we observed some tunel - positive cells in the gcl of the ins2 retina ( fig . 5 g ) and many more tunel - positive cells in the gcl of ins2/sig1r mice ( fig . the findings confirm the reduced numbers of cells in the gcl determined by morphometric analysis . we also examined the inl for tunel - positive cells and observed very few tunel - positive cells in retinas of wt ( ins2 ) ( fig . 5j ) , but a significant increase in tunel - positive cells in the inl of retinas of ins2/sig1r mice ( fig . increased expression of gfap has been reported in mller cells at 6 months ( and fig . 3 of this study ) . in the current study , we investigated the levels of gfap in retinal cryosections of wt ( ins2 ) , ins2 , and ins2/sig1r mice by immunofluorescence at 12 and 16 weeks . photomicrographs shown in figure 6 are from 16-week mice . aside from gfap labeling typically observed in retinal astrocytes , we detected minimal gfap in retinas of wt ( ins2 ) mice ( fig . there was a slight increase in gfap levels in retinas of ins2 mice ( fig . 6b ) and a substantial increase in gfap detected in retinas of ins2/sig1r mice ( fig . the data suggest that , in younger ins2 mice lacking sigma1r , there is increased retinal gliosis compared with age - matched diabetic mice that have sigma1r . immunofluorescent detection of gfap , a marker of gliosis in wt ( ins2 ) , ins2 , and ins2/sig1rmice at 16 weeks . representative photomicrographs of immunofluorescent detection of gfap in retinal cryosections from ( a ) wt ( ins2 ) , ( b ) ins2 , and ( c ) ins2/sig1rmice at age 16 weeks . immunofluorescence analysis used anti - gfap followed by incubation with alexa fluor 488 ( green - labeled secondary antibody ) , showing marked increase in gfap in radially oriented fibers of mller glial cells ( radial labeling ) in ins2/sig1rmice but much less gfap labeling in mller cells of the wt or ins2 mice at this age . gcl , ganglion cell layer ; inl , inner nuclear layer ; onl , outer nuclear layer . the blood glucose levels measured in wt ( ins2 ) were within normal limits , whereas levels were hyperglycemic in both ins2 and ins2/sig1r mice ( table 2 ) . earlier studies demonstrated that twice weekly administration of ( + ) -ptz at the 0.5-mg kg dosage reduced apoptotic neuron death characteristic of ins2mice and preserved retinal architecture , when treatment started at diabetes onset . we used this same ( + ) -ptz dosage and treatment regimen in the present study , but initiated injections either 4 or 8 weeks after diabetes onset , which meant that injections began at age 8 or 12 weeks in ins2mice . representative photomicrographs of retinas of wt mice receiving either no ( + ) -ptz injection or receiving ( + ) -ptz twice weekly beginning at age 8 weeks are shown ( fig . 1 ) . there was no deleterious effect on retinal architecture when wt mice were administered the sigma1r ligand ( fig . 1 ) and no differences in morphometric analysis of retinal layers ( data not shown ) representative photomicrographs of h&e - stained retinas of control ( c57bl/6 ) mice ( wt ) that received ( a ) no ( + ) -ptz injection or ( b ) 0.5 mg kg ( + ) -ptz injection twice weekly beginning at 8 weeks . gcl , ganglion cell layer ; ipl , inner plexiform layer ; inl , inner nuclear layer ; onl , outer nuclear layer ; rpe , retinal pigment epithelial layer . in the case of the ins2 mice that received no ( + ) -ptz injections , the retinas showed mild disruption . figures 2a and 2b show representative photomicrographs of retinas from two of the mice in the nontreated group . the alterations in the inl by 25 weeks in the ins2 mice have been reported . the nuclear layers did not have their characteristic uniform stratified appearance , but this was not attributable to presence of the rd8 mutation . mice that had been injected intraperitoneally beginning at 4 or 8 weeks after onset of diabetes onset with 0.5 mg kg ( + ) -ptz twice weekly showed preservation of retinal architecture ( figs . the retinas were subjected to morphometric analysis , and the total retinal thickness did not differ between the wt mice , ins2-noninjected mice , and ins2-injected mice beginning at 4 or 8 weeks ( fig . 2 g ) . nor did the thickness of the individual layers ( ipl , inl , opl , and onl ) differ significantly between groups ( data not shown ) . however , when the numbers of cells in the gcl were quantified , there was a significant difference between the wt mice and the ins2-noninjected mice ( fig . wild - type mice had approximately 13 cells per 100 m length of retina , whereas the ins2-noninjected mice had approximately 9 cells for the same retinal length ( fig . for the mice treated with sigma1r ligand at either 4 or 8 weeks after onset of diabetes , there were approximately 11 cells per 100 m retinal length , which was significantly greater than the ins2-noninjected mice . although the numbers of cells in the gcl of ( + ) -ptz - treated ins2 mice were not equal to that of the wt mice , there was clear preservation of these cells compared with diabetic mice not treated with the sigma1r ligand . we also noted that there was waveform irregularity of the onl accompanied by shallow retinal detachment and irregularly elongated photoreceptor outer segments in the retinas of ins2-noninjected mice . to evaluate whether the waveform appearance and occasional retinal detachment might be a consequence of the cryosectioning method of preparing sections , we examined eyes that had been fixed prior to embedding and processed for jb-4 plastic embedding . in this case , the ins2-noninjected mice showed minimal detachment , although slight irregularities of the nuclear layers were still detectable ( fig . the jb-4embedded retinas of the ( + ) -ptz treated ins2 mice had a generally uniform appearance ( figs . 2j , 2k ) similar to that observed in the cryosections of retinas of these mice ( figs . preservation of retinal structure and cells in the gcl in ins2 mice administered ( + ) -ptz after diabetes onset . ( a , b ) representative h&e - stained retinal cryosections of ins2 mice that received no ( + ) -ptz for the duration of the study ; the inl is slightly disrupted , and gcl density is decreased . representative h&e - stained retinal cryosections of ( c , d ) ins2 mice that received 0.5 mg kg ( + ) -ptz beginning 4 weeks after onset of diabetes ( at age 8 weeks ) or ( e , f ) ins2 mice that received 0.5 mg kg ( + ) -ptz beginning 8 weeks after onset of diabetes ( at age 12 weeks ) . morphometric analysis revealed no significant change in total retinal thickness in any of the eyes evaluated ( g ) , but a significant difference in the number of cell bodies in the gcl per 100 m length of retina ( h ) . eyes were processed for jb-4 embedding and stained with h&e to permit visualization of retinal architecture of ( i ) ins2 mice that received no ( + ) -ptz ; ( j ) ins2 mice that received 0.5 mg kg ( + ) -ptz beginning 4 weeks after onset of diabetes ( at age 8 weeks ) ; or ( k ) ins2 mice that received 0.5 mg kg ( + ) -ptz beginning 8 weeks after onset of diabetes ( at age 12 weeks ) . gcl , ganglion cell layer ; inl , inner nuclear layer ; onl , outer nuclear layer ; is / os , inner / outer segments . we examined the expression levels of gfap , which is up - regulated during retinal gliosis . we observed labeling in the wt retina limited to astrocytes , which typically express this protein ( fig . 3a ) . in the ins2-noninjected mice , the level of gfap increased , and the distribution of labeling this is an indication of gliosis characteristic of this diabetic retinopathy model . in the ins2 ( + ) the observation of minimal detection of gfap ( except the normal detection in astrocytes ) was true for mice treated beginning at 8 weeks ( 4 weeks after onset of diabetes ; fig . 3c ) and in mice treated beginning at 12 weeks ( 8 weeks after onset of diabetes ; fig . immunofluorescent detection of gfap , a marker of gliosis in ins2 mice administered ( + ) -ptz after diabetes onset . representative photomicrographs of immunofluorescent detection of gfap in retinal cryosections from ( a ) ins2 ( wt ) , ( b ) ins2 [ no ( + ) -ptz treatment ] , ( c ) ins2 mice that received 0.5 mg kg ( + ) -ptz beginning 4 weeks after onset of diabetes ( at age 8 weeks ) , and ( d ) ins2 mice that received 0.5 mg kg ( + ) -ptz beginning 8 weeks after onset of diabetes ( at age 12 weeks ) . immunofluorescence analysis used anti - gfap followed by incubation with alexa fluor 488 ( green - labeled secondary antibody ) , showing marked increase in gfap in radially oriented fibers of mller glial cells ( radial labeling ) in ins2 [ no ( + ) -ptz treatment ] , but minimal labeling in mller cells of the wt or ( + ) -ptz treated mice . ( e ) quantification of gfap fluorescence intensity data obtained from metamorphic analysis ( significant difference : * p < 0.05 , * * p < 0.01 ) . regarding the monitoring of blood glucose in this study ( table 1 ) , wt mice had blood glucose values below 240 mg / dl and were not hyperglycemic . the ( + ) -ptz treated ins2 mice remained hyperglycemic throughout the study ; blood glucose levels were approximately 400 mg / dl ( similar to untreated ins2 mice ) and were significantly higher than wt mice ( 116197 mg / dl ) , suggesting that hyperglycemia per se may not be sufficient to trigger neuronal loss in diabetes . in addition to investigating the effects of delayed administration of ( + ) -ptz on the retinal structure in the ins2diabetic mice described above , we examined the consequences on retinal phenotype when sigma1r was absent by generating ins2/sig1rmice . we performed sd - oct in wt ( ins2 ) , ins2 mice , and ins2/sig1rmice that were either 12 or 16 weeks of age . volume image projections and the accompanying b - scans showed generally normal retinal architecture and no evidence of gross disruption in the retinal nuclear or plexiform layers among the three groups ( figs . postimage analysis of retinal layers used invivovue 2.4 diver software ( bioptigen , inc . , durham , nc , usa ) and confirmed that the thickness of most retinal layers was comparable among the three groups of mice , with the exception of the measurement of the nerve fiber layer ( nfl ) . in this case , the nfl was thicker in retinas of ins2/sig1rmice compared with wt ( ins2 ) or ins2 mice ( fig . measurement of other retinal layers did not differ significantly among the three mouse groups ( data not shown ) . wild - type ( ins2 ) , ins2 , and ins2/sig1r mice were anesthetized and subjected to sd - oct to obtain images of the retina in situ . representative vip for wt ( ins2 ) mice : age 12 ( a ) and 16 weeks ( b ) ; ins2 mice : age 12 ( c ) and 16 weeks ( d ) ; and ins2/sig1rmice : age 12 ( e ) and 16 weeks ( f ) . the panel to the right of each vip is the b - scans of the same animal . autosegmentation analysis was performed to determine whether there were significant differences in thickness of retinal layers among the three mouse groups . there was a significant increase in thickness of the nerve fiber layer ( g ) ( * significantly different at p < 0.05 ) . mice were subjected to fundoscopy wt ( ins2 ) at 12 ( h ) and 16 weeks ( j ) ; ins2 at 12 ( l ) and 16 weeks ( n ) ; ins2/sig1r at 12 ( p ) and 16 weeks ( r ) . fundus images were normal for wt and ins2 , whereas vitreal opacities ( denoted by long arrows ) were detected in the ins2/sig1r mice ( p , r ) . wild - type ( ins2 ) showed no alterations at either 12 ( i ) or 16 weeks ( k ) . ins2 mice also had normal vascularization patterns at 12 ( m ) and 16 weeks ( o ) , whereas ins2/sig1r mice had beading at 12 ( q ) and at 16 weeks ( s ) . the long arrows in q and s point to areas of vitreal opacity ; the indented arrowhead points to a vessel with beading ( s ) . ( t ) enlarged image of image shown in s. ( u , v ) fa images of two additional mice at 16 weeks showing vitreal opacity ( long arrow ) and occasional beading of vessels ( indented arrowhead ) . we then examined wt ( ins2 ) , ins2 , and ins2/sig1r mice by retinal fundoscopy and fa at 12 and 16 weeks of age . fluorescein dye was injected , and images were acquired 30 seconds after injection and every minute thereafter for 5 minutes . images were arranged according to capture time , and data were compared among the three mouse groups at ages 12 and 16 weeks . fluorescein angiography performed in wt mice showed normal vessel filling , no leakage , and uniform capillary network around blood vessels at both ages studied ( figs . similarly , vessel filling in ins2 mice appeared normal , and leakage was not observed at either 12 ( fig . 4o ) . in ins2/sig1rmice , however , there was evidence of vessel beading at 12 weeks ( fig . the long arrow in figure 4 t points to a region of vitreal opacity , not an area of leakiness . , there appears to be an area of capillary dropout ( denoted by an asterisk ) in the retina of a 16-week ins2/sig1rmouse . it is noteworthy that all of the 16-week ins2/sig1rmice examined showed some evidence of beading and of vitreal opacities . the data suggest that in the absence of sigma1r subtle retinal vascular abnormalities develop that are not normally observed by fa in the ins2 diabetic mouse retina . eyes from wt ( ins2 ) , ins2 , and ins2/sig1r mice were processed in jb-4 embedding compound for light microscopic evaluation and retinal morphometric analysis at 12 and 16 weeks . although the retinal architecture becomes mildly disrupted by 25 weeks in the ins2 mice as reported previously and figure 2 of this study , the retinal architecture of ins2 mice at the younger ages ( 12 and 16 weeks ) analyzed in this study ( fig . a notable feature that distinguished the ins2/sig1r mice from the wt and ins2 mice was the fragmented appearance and disruption of the nfl in these mice compared with the wt ( ins2 ) and ins2 mice ( fig . although the disrupted appearance of the nfl may reflect increased sensitivity to the fixative used in processing the tissue , there appears to be involvement of nfl in the ins2/sig1r mice when examined in vivo by sd - oct imaging ( fig . the nfl is comprised of the axons of the ganglion cells ; thus , the decrease in the numbers of cells observed in the ins2/sig1r mice could account for disruption of their axons . the images in figures 5a5c are from 16-week mice . retinal structure of wt ( ins2 ) , ins2 , and ins2/sig1rmice . eyes of ( a ) wt ( ins2 ) , ( b ) ins2 , and ( c ) ins2/sig1rmice were processed for jb-4 embedding and stained with h&e to permit visualization of retinal architecture . panels shown are from 16-week - old mice . in wt ( ins2 ) mice , the retinas are well organized and layers are of normal thickness . the retinas of the ins2 and ins2/sig1rmice are similar in appearance , with the exception of the nerve fiber layer in the ins2/sig1rmice ( arrow ) , which is markedly disrupted . retinas were subjected to morphometric analysis ; there was a significant decrease in the number of cells in the gcl in the ins2/sig1rmice at 12 ( d ) and 16 weeks ( e ) . retinal cryosections of the same mice were subjected to immunofluorescent detection of tunel - positive cells ( green labeling ) , cell nuclei were blue ( dapi ) in ( f ) wt ( ins2 ) , ( g ) ins2 , and ( h ) ins2/sig1rmice . additional images of tunel staining were obtained using a lower - powered objective focused on the inl for ( i ) wt ( ins2 ) , ( j ) ins2 , and ( k ) ins2/sig1rmice . gcl , ganglion cell layer ; ipl , inner plexiform layer ; inl , inner nuclear layer ; opl , outer plexiform layer ; onl , outer nuclear layer ; is , inner segment ; os , outer segment ; rpe , retinal pigment epithelium . statistical significance : * * p < 0.01 , * * * p < 0.001 . in the mouse retina , the number of cell bodies in the gcl is typically 1214 per 100 m length of retina . this was observed in the wt ( ins2 ) mouse retina at both 12 and 16 weeks ( figs . , there was minimal difference at 12 and 16 weeks in the number of cells bodies in the gcl compared with wt mice ( figs . 5d , 5e ) . in ins2/sig1r mice , however , there was a significant decrease in the number of cells in the gcl at 12 ( 11 cells/100 m retinal length ) and 16 weeks ( 8 cells/100 m retinal length ) . the data suggest that , in the absence of sigma1r , the loss of ganglion cells observed at 25 weeks in the ins2 is markedly accelerated . we subjected retinal cryosections from the three mouse groups to immunofluorescence to detect dying cells using the apoptag fluorescein in situ apoptosis detection kit ( tunel staining ) . we did not detect dying cells in gcl of wt ( ins2 ) mice ( fig . we observed some tunel - positive cells in the gcl of the ins2 retina ( fig . 5 g ) and many more tunel - positive cells in the gcl of ins2/sig1r mice ( fig . the findings confirm the reduced numbers of cells in the gcl determined by morphometric analysis . we also examined the inl for tunel - positive cells and observed very few tunel - positive cells in retinas of wt ( ins2 ) ( fig . 5j ) , but a significant increase in tunel - positive cells in the inl of retinas of ins2/sig1r mice ( fig . in the ins2 retina , increased expression of gfap has been reported in mller cells at 6 months ( and fig . 3 of this study ) . in the current study , we investigated the levels of gfap in retinal cryosections of wt ( ins2 ) , ins2 , and ins2/sig1r mice by immunofluorescence at 12 and 16 weeks . typically observed in retinal astrocytes , we detected minimal gfap in retinas of wt ( ins2 ) mice ( fig . there was a slight increase in gfap levels in retinas of ins2 mice ( fig . 6b ) and a substantial increase in gfap detected in retinas of ins2/sig1r mice ( fig . the data suggest that , in younger ins2 mice lacking sigma1r , there is increased retinal gliosis compared with age - matched diabetic mice that have sigma1r . immunofluorescent detection of gfap , a marker of gliosis in wt ( ins2 ) , ins2 , and ins2/sig1rmice at 16 weeks . representative photomicrographs of immunofluorescent detection of gfap in retinal cryosections from ( a ) wt ( ins2 ) , ( b ) ins2 , and ( c ) ins2/sig1rmice at age 16 weeks . immunofluorescence analysis used anti - gfap followed by incubation with alexa fluor 488 ( green - labeled secondary antibody ) , showing marked increase in gfap in radially oriented fibers of mller glial cells ( radial labeling ) in ins2/sig1rmice but much less gfap labeling in mller cells of the wt or ins2 mice at this age . gcl , ganglion cell layer ; inl , inner nuclear layer ; onl , outer nuclear layer . the blood glucose levels measured in wt ( ins2 ) were within normal limits , whereas levels were hyperglycemic in both ins2 and ins2/sig1r mice ( table 2 ) . one was to investigate whether delaying administration of a high - affinity sigma1r ligand after onset of diabetes could afford neuroprotection in a model of diabetic retinopathy and the second was to evaluate whether absence of sigma1r would accelerate neuron loss in this model . although diabetic retinopathy has long been recognized as a retinal vascular disease , significant neuronal loss is also a feature of this disease . we chose to use the diabetic ins2 mouse as an endogenous diabetic model with modest progressive loss of cells in the gcl . in our study examining whether administration of ( + ) -ptz after onset of diabetes would be neuroprotective , we investigated four mouse groups . we used wt mice , ins2 mice that received no injection , and two groups of ins2 mice that were injected beginning at 8 or 12 weeks ( which was either 4 or 8 weeks after diabetes onset ) . earlier studies had shown that by the time ins2 mice were 25 weeks , there was an approximate 20% loss of cells in the gcl . we used that same time point to investigate whether ( + ) -ptz could attenuate cell loss and found that , in both the 8- and 12-week injection groups , there was attenuation of cell loss . that is , even though ( + ) -ptz was not administered at the onset of diabetes , there still appeared to be significant neuroprotection when the retinas were examined at 25 weeks . the retinas of ins2 mice that were in the delayed injection groups retained significantly more cells in the gcl than noninjected ins2 mice . in addition , examination of retinal architecture showed that retinas of ( + ) -ptz - injected mice were well organized . most intervention studies ( including our own study with the ins2 mouse several years ago ) start therapeutic intervention as early as possible , sometimes prior to onset of disease , to achieve the greatest likelihood of a beneficial outcome . a recent example of this involving a sigma1r ligand was a report from shimazawa et al . , in which the effects of cutamesine dihydrochloride ( sa4503 ) were investigated in a light - induced mouse model of photoreceptor degeneration . in this case , the mice were pretreated with the sigma1r ligand ( intravitreal injection ) 1 hour prior to exposure to intense light . the effects of cutamesine on the photoreceptor cell loss were impressive , but it is not known whether administering the ligand after the exposure to intense light would be protective . similarly , sun et al . induced ganglion cell death in rats using episcleral vein cauterization ( evc ) and tested whether the sigma1r ligand pregnenolone sulfate would lower evc - induced increase in intraocular pressure ( iop ) . they observed normalization of iop in this treatment regimen , but again it is not known whether the treatment could lower iop if administered after the evc insult . our findings that administration of ( + ) -ptz after onset of diabetes can afford some degree of neuroprotection is important because most human patients would not receive treatment at the onset of disease . our findings that gliosis was attenuated as shown by decreased gfap expression in the ins2 mouse retina , even though ( + ) -ptz administration was delayed until after diabetes onset , is noteworthy given the recent report that activation of sigma1r using pre-084 inhibits osmotic swelling of retinal mller glial cells and may contribute to the neuroprotective properties of sigma1r ligands . it binds sigmar1 with a very high affinity and is presumed to be an agonist for the receptor ; however , because the actual signaling events associated with sigmar1 activation have not been characterized , it is difficult to ascribe definitively an agonist / antagonist function to this ligand . there is convincing evidence that ( + ) -ptz is specific for sigmar1 because in experiments where the gene is absent , ( + ) -ptz does not afford protection . the preservation of retinal structure and attenuated neuronal death do not appear to be a direct function of decreased hyperglycemia because ( + ) -ptz injected ins2 mice continue to have elevated blood glucose throughout the study . retinal neuronal loss associated with diabetes may not involve hyperglycemia directly ; rather , it may be due to complications secondary to hyperglycemia such as oxidative stress . the beneficial effects of ( + ) -ptz administered at the onset of diabetes , as well as after onset of diabetes reported herein , suggest that activation of the sigma1r may be a promising treatment strategy for retinal degenerative diseases . the precise physiologic role of sigma1r in retina is not known ; however , findings from many studies support the notion that sigma1r is a molecular chaperone and modulates cellular stress . sigma1r has been shown to modulate oxidative stress via nrf2 signaling , to activate extracellular signal regulated kinases1/2 , to suppress inflammatory responses in microglial cells , to attenuate calcium influx in ganglion cells , to modulate voltage - gated calcium channels , to decrease retinal glial cytokine release and promote cytosolic - to - nuclear nf-b translocation , and to modulate er stress . the multiple protective functions of sigma1r prompted the present study to investigate whether absence of sigma1r would accelerate the retinal phenotype in ins2 mice . sig1r mice have a late - onset retinal degeneration characterized by optic nerve axonal degeneration at approximately 6 months and progressive loss of ganglion cells by 1 year ; the retina is normal in appearance in the first few months of life . no retinal vasculopathy has been reported in sig1r mice . in the present study , we evaluated retinas of ins2/sig1r mice and compared them with ins2 mice at 12 and 16 weeks , time points earlier than the reported ganglion cell loss ( 25 weeks ) . we found that ganglion cell loss was minimal in ins2 mice at 12 and 16 weeks but was significantly worse in ins2/sig1r mice , particularly at 16 weeks . the data suggest that in the absence of sigma1r , retinal neuronal loss is accelerated . the data were confirmed by detection of significantly more tunel - positive cells ( indicative of apoptosis ) , in retinas of ins2/sig1r mice compared with wt or ins2 mice . in addition , the nfl examined in retinal sections was fragmented and disrupted in ins2/sig1r mice . this was not due simply to fixation artifacts because oct analysis showed that , although the total retinal thickness and most of the individual retinal layers did not differ from wt , there was a significant increase in the nfl thickness in the ins2/sig1r mice . in addition to oct analysis , imaging of the retinal vessels of ins2/sig1r mice showed subtle vascular changes that are not typically observed in ins2 mice . especially at 16 weeks , fa revealed vessel beading and occasional capillary dropout . the findings suggest that despite the minimal vascular involvement of retina in ins2 mice normally , lack of sigma1r increases slightly the incidence of vascular alterations . in addition to neuronal and vascular features , the ins2/sig1r mice also demonstrate significant increase in gfap compared with ins2 mice , suggesting that retinal gliosis , which has been reported in the ins2 mice , is more pronounced when sigma1r is absent . the findings suggest a role for sigma1r as a modulator of stress ; in the absence of sigma1r , there appears to be heightened sensitivity to stress . taken collectively , the data from the present study provide insights into the role of sigma1r in retinal disease . the findings that ( 1 ) administration of a high affinity sigma1r ligand after onset of diabetes affords a degree of retinal neuroprotection and ( 2 ) absence of the receptor in diabetic retinopathy worsens the phenotype suggest that sigma1r may have an important role in preservation of retinal structure . it remains to be determined whether other types of retinal degeneration , for example , glaucoma , will respond positively to targeting of sigma1r . in addition it will be important to explore targeting sigma1r in treatment of other more severe retinopathies including photoreceptor cell degeneration . there is an additional aspect of the present study that warrants discussion , which concerns the retinal phenotype of the ins2 diabetic mouse . several years ago , our group published in investigative ophthalmology and visual science observations about the protective effects of ( + ) -ptz in ins2 diabetic mice . in that study , we observed and reported a very severe retinal phenotype in ins2 mice affecting the inl and the gcl . barber and others had initially reported the ganglion cell loss and decreased thickness of inl in this mouse , although they did not report a pronounced disruption as we had observed . we do not know what factors caused the ins2 mice to have such a disrupted retinal architecture in our colony at that time . for several years thereafter , we did not use the ins2 mice in our studies . when we reestablished our colony more recently , we again purchased the mice from the jackson laboratories ( the source of the original mice ) , but the animals derived from establishing the new colony showed a much milder phenotype , which is reported here . the ganglion cell loss , reported initially by barber and others and by our group , is still a characteristic feature of this mouse model , but the fulminant retinopathy we observed is not present . we can not account for the apparent phenotypic drift that appears to have occurred between our initial studies and the current work . we do not know whether our original colony harbored the rd8 mutation ; however , we do not think this was the case because the characteristic retinal rosette pattern was not observed . at any rate , the ins2 mice phenotype observed and reported in the present work is very similar to that observed by other investigators , and the severe phenotype is no longer detectable in our animals .

purposesigma receptor 1 ( sigma1r ) , a nonopioid putative molecular chaperone , has neuroprotective properties in retina . this study sought to determine whether delaying administration of ( + ) -pentazocine , a high - affinity sigma1r ligand after onset of diabetes in ins2akita/+ diabetic mice would afford retinal neuroprotection and to determine consequences on retinal phenotype in ins2akita/+ diabetic mice in the absence of sigma1r.methodsins2akita/+ diabetic and wt mice received intraperitoneal injections of ( + ) -pentazocine beginning 4 or 8 weeks after onset of diabetes ; eyes were harvested at 25 weeks . retinal histologic sections were analyzed to determine thicknesses of retinal layers , number of ganglion cells , and evidence of gliosis ( increased glial fibrillary acidic protein levels ) . ins2akita/+/sig1r/mice were generated and subjected to in vivo assessment of retinal architecture ( optical coherence tomography [ oct ] ) and retinal vasculature using fluorescein angiography ( fa ) at 12 and 16 weeks compared with age - matched ins2akita/+ mice . eyes were then harvested for retinal morphometric assessment and gliosis assessment.resultswild-type mice had 13 0.06 cells/100 m retinal length ; cell bodies in ins2akita/+ mice injected 4 and 8 weeks after onset of diabetes with ( + ) -pentazocine retained significantly more ganglion cells compared with ins2akita/+ mice ( 9 0.04 ) and demonstrated significant attenuation of gliosis . ins2akita/+/sig1r/mouse retinas , analyzed to determine whether the ins2akita/+ phenotype was accelerated when lacking sigma1r , revealed increased nerve fiber layer thickness ( oct ) , evidence of vitreal opacities , and vessel beading ( fa ) compared with ins2akita/+ mice . morphometric analysis revealed significantly fewer ganglion cells in ins2akita/+/sig1r/mice compared with ins2akita/+ mice.conclusionssigma1r may be a novel retinal stress modulator , and targeting it even after disease onset may afford retinal neuroprotection .

Materials and Methods Animals Administration of (+)-PTZ After Onset of Diabetes Spectral-Domain OCT Imaging, Fundoscopy, and FA Tissue Processing and Morphometric Analysis Assessment of Gliosis and Cell Death Statistical Analysis Results Effects of Administration of (+)-PTZ After Diabetes Onset on Neuronal Death in Diabetic Retinopathy SD-OCT Imaging, Fundoscopy, and FA in Retinal Morphometric Analysis in Assessment of Gliosis in Effects of Sigma1R on Blood Glucose Levels in Discussion

in the first study , we examined the effects of delaying ( + ) -ptz injection ( after onset of diabetes ) on retinal neuronal death in diabetic retinopathy . , we determined whether the retinal neurodegeneration of ins2 mice would be accelerated if the mice lacked sigma1r , and we asked whether the retinal phenotype would involve vasculopathy when sigma1r was absent . mice were administered ( + ) -ptz either 4 or 8 weeks after onset of diabetes ( thus , one group of ins2 mice was injected beginning at age 8 weeks and a second was injected beginning at age 12 weeks ) . mice were euthanized at 25 weeks , as was the case in the previous study in which ins2 mice were administered ( + ) -ptz at diabetes onset . glial fibrillary acidic protein ( gfap ) is a 54-kda intermediate filament protein that is a major constituent of retinal astrocytes ; under retinal stress conditions , gfap expression is up - regulated in retinal mller glial cells in a process termed gliosis . analysis of variance was used to determine whether there were significant differences in morphologic measurements in mice administered ( + ) -ptz at the two time intervals compared with noninjected animals . in the first study , we examined the effects of delaying ( + ) -ptz injection ( after onset of diabetes ) on retinal neuronal death in diabetic retinopathy . , we determined whether the retinal neurodegeneration of ins2 mice would be accelerated if the mice lacked sigma1r , and we asked whether the retinal phenotype would involve vasculopathy when sigma1r was absent . mice were administered ( + ) -ptz either 4 or 8 weeks after onset of diabetes ( thus , one group of ins2 mice was injected beginning at age 8 weeks and a second was injected beginning at age 12 weeks ) . mice were euthanized at 25 weeks , as was the case in the previous study in which ins2 mice were administered ( + ) -ptz at diabetes onset . per mouse , glial fibrillary acidic protein ( gfap ) is a 54-kda intermediate filament protein that is a major constituent of retinal astrocytes ; under retinal stress conditions , gfap expression is up - regulated in retinal mller glial cells in a process termed gliosis . earlier studies demonstrated that twice weekly administration of ( + ) -ptz at the 0.5-mg kg dosage reduced apoptotic neuron death characteristic of ins2mice and preserved retinal architecture , when treatment started at diabetes onset . we used this same ( + ) -ptz dosage and treatment regimen in the present study , but initiated injections either 4 or 8 weeks after diabetes onset , which meant that injections began at age 8 or 12 weeks in ins2mice . 1 ) and no differences in morphometric analysis of retinal layers ( data not shown ) representative photomicrographs of h&e - stained retinas of control ( c57bl/6 ) mice ( wt ) that received ( a ) no ( + ) -ptz injection or ( b ) 0.5 mg kg ( + ) -ptz injection twice weekly beginning at 8 weeks . mice that had been injected intraperitoneally beginning at 4 or 8 weeks after onset of diabetes onset with 0.5 mg kg ( + ) -ptz twice weekly showed preservation of retinal architecture ( figs . the retinas were subjected to morphometric analysis , and the total retinal thickness did not differ between the wt mice , ins2-noninjected mice , and ins2-injected mice beginning at 4 or 8 weeks ( fig . for the mice treated with sigma1r ligand at either 4 or 8 weeks after onset of diabetes , there were approximately 11 cells per 100 m retinal length , which was significantly greater than the ins2-noninjected mice . although the numbers of cells in the gcl of ( + ) -ptz - treated ins2 mice were not equal to that of the wt mice , there was clear preservation of these cells compared with diabetic mice not treated with the sigma1r ligand . preservation of retinal structure and cells in the gcl in ins2 mice administered ( + ) -ptz after diabetes onset . representative h&e - stained retinal cryosections of ( c , d ) ins2 mice that received 0.5 mg kg ( + ) -ptz beginning 4 weeks after onset of diabetes ( at age 8 weeks ) or ( e , f ) ins2 mice that received 0.5 mg kg ( + ) -ptz beginning 8 weeks after onset of diabetes ( at age 12 weeks ) . morphometric analysis revealed no significant change in total retinal thickness in any of the eyes evaluated ( g ) , but a significant difference in the number of cell bodies in the gcl per 100 m length of retina ( h ) . eyes were processed for jb-4 embedding and stained with h&e to permit visualization of retinal architecture of ( i ) ins2 mice that received no ( + ) -ptz ; ( j ) ins2 mice that received 0.5 mg kg ( + ) -ptz beginning 4 weeks after onset of diabetes ( at age 8 weeks ) ; or ( k ) ins2 mice that received 0.5 mg kg ( + ) -ptz beginning 8 weeks after onset of diabetes ( at age 12 weeks ) . in the ins2 ( + ) the observation of minimal detection of gfap ( except the normal detection in astrocytes ) was true for mice treated beginning at 8 weeks ( 4 weeks after onset of diabetes ; fig . 3c ) and in mice treated beginning at 12 weeks ( 8 weeks after onset of diabetes ; fig . representative photomicrographs of immunofluorescent detection of gfap in retinal cryosections from ( a ) ins2 ( wt ) , ( b ) ins2 [ no ( + ) -ptz treatment ] , ( c ) ins2 mice that received 0.5 mg kg ( + ) -ptz beginning 4 weeks after onset of diabetes ( at age 8 weeks ) , and ( d ) ins2 mice that received 0.5 mg kg ( + ) -ptz beginning 8 weeks after onset of diabetes ( at age 12 weeks ) . the ( + ) -ptz treated ins2 mice remained hyperglycemic throughout the study ; blood glucose levels were approximately 400 mg / dl ( similar to untreated ins2 mice ) and were significantly higher than wt mice ( 116197 mg / dl ) , suggesting that hyperglycemia per se may not be sufficient to trigger neuronal loss in diabetes . in addition to investigating the effects of delayed administration of ( + ) -ptz on the retinal structure in the ins2diabetic mice described above , we examined the consequences on retinal phenotype when sigma1r was absent by generating ins2/sig1rmice . mice were subjected to fundoscopy wt ( ins2 ) at 12 ( h ) and 16 weeks ( j ) ; ins2 at 12 ( l ) and 16 weeks ( n ) ; ins2/sig1r at 12 ( p ) and 16 weeks ( r ) . ins2 mice also had normal vascularization patterns at 12 ( m ) and 16 weeks ( o ) , whereas ins2/sig1r mice had beading at 12 ( q ) and at 16 weeks ( s ) . we then examined wt ( ins2 ) , ins2 , and ins2/sig1r mice by retinal fundoscopy and fa at 12 and 16 weeks of age . eyes from wt ( ins2 ) , ins2 , and ins2/sig1r mice were processed in jb-4 embedding compound for light microscopic evaluation and retinal morphometric analysis at 12 and 16 weeks . although the retinal architecture becomes mildly disrupted by 25 weeks in the ins2 mice as reported previously and figure 2 of this study , the retinal architecture of ins2 mice at the younger ages ( 12 and 16 weeks ) analyzed in this study ( fig . eyes of ( a ) wt ( ins2 ) , ( b ) ins2 , and ( c ) ins2/sig1rmice were processed for jb-4 embedding and stained with h&e to permit visualization of retinal architecture . retinas were subjected to morphometric analysis ; there was a significant decrease in the number of cells in the gcl in the ins2/sig1rmice at 12 ( d ) and 16 weeks ( e ) . , there was minimal difference at 12 and 16 weeks in the number of cells bodies in the gcl compared with wt mice ( figs . in ins2/sig1r mice , however , there was a significant decrease in the number of cells in the gcl at 12 ( 11 cells/100 m retinal length ) and 16 weeks ( 8 cells/100 m retinal length ) . the data suggest that , in the absence of sigma1r , the loss of ganglion cells observed at 25 weeks in the ins2 is markedly accelerated . in the current study , we investigated the levels of gfap in retinal cryosections of wt ( ins2 ) , ins2 , and ins2/sig1r mice by immunofluorescence at 12 and 16 weeks . the data suggest that , in younger ins2 mice lacking sigma1r , there is increased retinal gliosis compared with age - matched diabetic mice that have sigma1r . immunofluorescent detection of gfap , a marker of gliosis in wt ( ins2 ) , ins2 , and ins2/sig1rmice at 16 weeks . earlier studies demonstrated that twice weekly administration of ( + ) -ptz at the 0.5-mg kg dosage reduced apoptotic neuron death characteristic of ins2mice and preserved retinal architecture , when treatment started at diabetes onset . we used this same ( + ) -ptz dosage and treatment regimen in the present study , but initiated injections either 4 or 8 weeks after diabetes onset , which meant that injections began at age 8 or 12 weeks in ins2mice . 1 ) and no differences in morphometric analysis of retinal layers ( data not shown ) representative photomicrographs of h&e - stained retinas of control ( c57bl/6 ) mice ( wt ) that received ( a ) no ( + ) -ptz injection or ( b ) 0.5 mg kg ( + ) -ptz injection twice weekly beginning at 8 weeks . mice that had been injected intraperitoneally beginning at 4 or 8 weeks after onset of diabetes onset with 0.5 mg kg ( + ) -ptz twice weekly showed preservation of retinal architecture ( figs . the retinas were subjected to morphometric analysis , and the total retinal thickness did not differ between the wt mice , ins2-noninjected mice , and ins2-injected mice beginning at 4 or 8 weeks ( fig . for the mice treated with sigma1r ligand at either 4 or 8 weeks after onset of diabetes , there were approximately 11 cells per 100 m retinal length , which was significantly greater than the ins2-noninjected mice . although the numbers of cells in the gcl of ( + ) -ptz - treated ins2 mice were not equal to that of the wt mice , there was clear preservation of these cells compared with diabetic mice not treated with the sigma1r ligand . preservation of retinal structure and cells in the gcl in ins2 mice administered ( + ) -ptz after diabetes onset . representative h&e - stained retinal cryosections of ( c , d ) ins2 mice that received 0.5 mg kg ( + ) -ptz beginning 4 weeks after onset of diabetes ( at age 8 weeks ) or ( e , f ) ins2 mice that received 0.5 mg kg ( + ) -ptz beginning 8 weeks after onset of diabetes ( at age 12 weeks ) . morphometric analysis revealed no significant change in total retinal thickness in any of the eyes evaluated ( g ) , but a significant difference in the number of cell bodies in the gcl per 100 m length of retina ( h ) . eyes were processed for jb-4 embedding and stained with h&e to permit visualization of retinal architecture of ( i ) ins2 mice that received no ( + ) -ptz ; ( j ) ins2 mice that received 0.5 mg kg ( + ) -ptz beginning 4 weeks after onset of diabetes ( at age 8 weeks ) ; or ( k ) ins2 mice that received 0.5 mg kg ( + ) -ptz beginning 8 weeks after onset of diabetes ( at age 12 weeks ) . in the ins2 ( + ) the observation of minimal detection of gfap ( except the normal detection in astrocytes ) was true for mice treated beginning at 8 weeks ( 4 weeks after onset of diabetes ; fig . 3c ) and in mice treated beginning at 12 weeks ( 8 weeks after onset of diabetes ; fig . representative photomicrographs of immunofluorescent detection of gfap in retinal cryosections from ( a ) ins2 ( wt ) , ( b ) ins2 [ no ( + ) -ptz treatment ] , ( c ) ins2 mice that received 0.5 mg kg ( + ) -ptz beginning 4 weeks after onset of diabetes ( at age 8 weeks ) , and ( d ) ins2 mice that received 0.5 mg kg ( + ) -ptz beginning 8 weeks after onset of diabetes ( at age 12 weeks ) . the ( + ) -ptz treated ins2 mice remained hyperglycemic throughout the study ; blood glucose levels were approximately 400 mg / dl ( similar to untreated ins2 mice ) and were significantly higher than wt mice ( 116197 mg / dl ) , suggesting that hyperglycemia per se may not be sufficient to trigger neuronal loss in diabetes . in addition to investigating the effects of delayed administration of ( + ) -ptz on the retinal structure in the ins2diabetic mice described above , we examined the consequences on retinal phenotype when sigma1r was absent by generating ins2/sig1rmice . mice were subjected to fundoscopy wt ( ins2 ) at 12 ( h ) and 16 weeks ( j ) ; ins2 at 12 ( l ) and 16 weeks ( n ) ; ins2/sig1r at 12 ( p ) and 16 weeks ( r ) . ins2 mice also had normal vascularization patterns at 12 ( m ) and 16 weeks ( o ) , whereas ins2/sig1r mice had beading at 12 ( q ) and at 16 weeks ( s ) . we then examined wt ( ins2 ) , ins2 , and ins2/sig1r mice by retinal fundoscopy and fa at 12 and 16 weeks of age . eyes from wt ( ins2 ) , ins2 , and ins2/sig1r mice were processed in jb-4 embedding compound for light microscopic evaluation and retinal morphometric analysis at 12 and 16 weeks . although the retinal architecture becomes mildly disrupted by 25 weeks in the ins2 mice as reported previously and figure 2 of this study , the retinal architecture of ins2 mice at the younger ages ( 12 and 16 weeks ) analyzed in this study ( fig . eyes of ( a ) wt ( ins2 ) , ( b ) ins2 , and ( c ) ins2/sig1rmice were processed for jb-4 embedding and stained with h&e to permit visualization of retinal architecture . retinas were subjected to morphometric analysis ; there was a significant decrease in the number of cells in the gcl in the ins2/sig1rmice at 12 ( d ) and 16 weeks ( e ) . , there was minimal difference at 12 and 16 weeks in the number of cells bodies in the gcl compared with wt mice ( figs . in ins2/sig1r mice , however , there was a significant decrease in the number of cells in the gcl at 12 ( 11 cells/100 m retinal length ) and 16 weeks ( 8 cells/100 m retinal length ) . the data suggest that , in the absence of sigma1r , the loss of ganglion cells observed at 25 weeks in the ins2 is markedly accelerated . in the current study , we investigated the levels of gfap in retinal cryosections of wt ( ins2 ) , ins2 , and ins2/sig1r mice by immunofluorescence at 12 and 16 weeks . the data suggest that , in younger ins2 mice lacking sigma1r , there is increased retinal gliosis compared with age - matched diabetic mice that have sigma1r . immunofluorescent detection of gfap , a marker of gliosis in wt ( ins2 ) , ins2 , and ins2/sig1rmice at 16 weeks . one was to investigate whether delaying administration of a high - affinity sigma1r ligand after onset of diabetes could afford neuroprotection in a model of diabetic retinopathy and the second was to evaluate whether absence of sigma1r would accelerate neuron loss in this model . in our study examining whether administration of ( + ) -ptz after onset of diabetes would be neuroprotective , we investigated four mouse groups . we used wt mice , ins2 mice that received no injection , and two groups of ins2 mice that were injected beginning at 8 or 12 weeks ( which was either 4 or 8 weeks after diabetes onset ) . that is , even though ( + ) -ptz was not administered at the onset of diabetes , there still appeared to be significant neuroprotection when the retinas were examined at 25 weeks . in addition , examination of retinal architecture showed that retinas of ( + ) -ptz - injected mice were well organized . our findings that administration of ( + ) -ptz after onset of diabetes can afford some degree of neuroprotection is important because most human patients would not receive treatment at the onset of disease . our findings that gliosis was attenuated as shown by decreased gfap expression in the ins2 mouse retina , even though ( + ) -ptz administration was delayed until after diabetes onset , is noteworthy given the recent report that activation of sigma1r using pre-084 inhibits osmotic swelling of retinal mller glial cells and may contribute to the neuroprotective properties of sigma1r ligands . the beneficial effects of ( + ) -ptz administered at the onset of diabetes , as well as after onset of diabetes reported herein , suggest that activation of the sigma1r may be a promising treatment strategy for retinal degenerative diseases . in the present study , we evaluated retinas of ins2/sig1r mice and compared them with ins2 mice at 12 and 16 weeks , time points earlier than the reported ganglion cell loss ( 25 weeks ) . the data suggest that in the absence of sigma1r , retinal neuronal loss is accelerated . the findings suggest a role for sigma1r as a modulator of stress ; in the absence of sigma1r , there appears to be heightened sensitivity to stress . the findings that ( 1 ) administration of a high affinity sigma1r ligand after onset of diabetes affords a degree of retinal neuroprotection and ( 2 ) absence of the receptor in diabetic retinopathy worsens the phenotype suggest that sigma1r may have an important role in preservation of retinal structure .

it performs iterative profile searching similar to psi - blast ( 1 ) , but with full dynamic programing on each cycle and on - the - fly estimation of significance . the scanps web server has been developed to simplify the running and analysis of scanps searches . an innovative aspect of the server is its novel tree - based presentation of results for searches against the scop domain database ( 2 ) . a comparison of a protein domain to the domains in scop can be of considerable value in elucidating its structure and function . facilities for comparing a query sequence to scop sequences are also provided by fps ( http://fps.sdsc.edu/ ) , gtop ( http://spock.genes.nig.ac.jp/~genome/grpsblt.html ) and cascadeblast ( http://crick.mbu.iisc.ernet.in/~cascade/cascadeblast.html ) , all of which use psi - blast as the search algorithm . however , interpreting the results of scop searches in the context of the classification is hampered by the fact that search methods typically produce a linear table of hits ; understanding the relationships between hits to the scop database usually requires a manual mapping of the results table onto the scop hierarchy . this procedure is tedious and error prone and therefore a browser interface has been developed that maps hits onto the scop hierarchy for viewing using a tree - based framework . the new interface allows the user to perform a search of the complete non - redundant scop sequence database and view results in a form that allows the information inherent in the scop classification to be exploited in interpreting those results . although scanps has been available for over 15 years and has been accessible as a service at the european bioinformatics institute ( ebi ) for 10 years , it has not previously been described in the literature . accordingly , as background to the new web server , a brief overview of the motivation , novel features and performance of the program is given here . waterman algorithm ( 3 ) comparison of a protein sequence to a protein sequence database with either length dependent or affine gap penalties ( 4 ) . the program is written in c and effort was put into coding for performance on conventional workstation hardware . this enabled the program to be used routinely for searching large databases on modest computer hardware in contrast to the belief in the early 1990s that smith parallel processing by dynamically splitting the database across multiple processors was demonstrated on a network of five loosely coupled workstations ( 6 ) , then refined to exploit symmetric multi processing ( smp ) hardware via openmp ( 7 ) . the smp implementation gave near linear speedup on a 24 processor silicon graphics challenge . with the move to commodity pc hardware in the late 1990s , fourway onchip parallelism was implemented on intel and amd chips with a speedup over linear code of at least 3 depending on the query and database size . in addition to the onchip parallelism , multiprocessor parallelism was implemented through mpi ( 8,9 ) . the mpi implementation gave parallel efficiency of over 90% on 16 processors on an intel piii cluster connected by 100 mb network . the speed obtained by parallel processing , coupled with the on - the - fly statistics described below , permitted the implementation of iterative searching . in this mode , a multiple sequence alignment is constructed for sequences that score above a preset significance threshold in the initial search . the multiple alignment is built up by aligning to the query sequence as a template , but using a position specific scoring matrix ( pssm ) as appropriate at each iteration . alignment columns which contain gaps in the query sequence are deleted ; as a result , the alignment is always the same length as the query sequence . a pssm ( 10,11 ) is derived from this alignment and used to re - search the database . since sequences that are very similar to the query sequence contribute little information to the pssm , a percentage identity threshold ( pidt ) is employed which excludes all sequences from the alignment whose similarity to the query sequence exceeds the threshold . the contributions of each sequence to the pssm are weighted according to the method of henikoff and henikoff ( 12 ) . the scoring matrix is then constructed at each alignment position similarly to standard log odds matrices ( 12,13 ) . the process of constructing a pssm and searching the database is repeated until convergence , or until a preset number of iterations has been completed . in each database search , the statistical significance of a score between the query and any sequence in the database is assessed by on - the - fly modeling the distribution of query database sequence pair scores . scores are binned according to the log of the product of the query and database sequence lengths ( lpl ) . within each lpl bin the extreme - value location and scale parameters are then fitted to exponential and linear equations respectively with respect to the lpl . the resulting extreme - value equations are applied back to all query sequence pairs and the resulting probabilities converted to e - values for ranking and display . this method of estimating significance is similar to those implemented in fasta / ssearch ( 14 ) ; for a full discussion of the similarities and differences between the different fitting schemes and the effect on performance in benchmarks see ( 15 ) . the search performance of scanps was compared with that of psi - blast using a benchmark based on scop ( 2 ) . the benchmark dataset comprises 1113 sequences taken from the pdb40d - b dataset constructed by brenner et al . single - segment domains whose structure had been determined by x - ray crystallography were selected , representing 479 scop superfamilies across 343 scop folds . true positives were defined as pairs of sequences belonging to the same scop superfamily ; true negatives were defined as those pairs in which the sequences belong to different scop folds . the resulting set of sequence pairs contain 2528 true positives and 616 923 true negatives , resulting in a total of 618 821 pairs . benchmarking was performed by searching the benchmark set with each of the benchmark sequences in turn . since scanps and psi - blast use sequence profiles to enhance search sensitivity , it is necessary to embed the benchmark set in a larger sequence database in order to ensure that there is an adequate set of related sequences to construct the search profile for each benchmark sequence . accordingly , the benchmark set was embedded in swall ( 17 ) to create the search database . both scanps version 2.3.9 and psi - blast version 2.2 - 17 were run for a maximum of 10 iterations . search parameters were chosen to reflect the typical use case where a low rate of false positives is acceptable in return for a high rate of true positives found . scanps was run with a profile e - value inclusion value of 0.015 , a pidt of 97% and using the blosum50 scoring matrix . gap penalties for opening and extending gaps were set to 12 and 2 , respectively . these parameters have previously been established to provide the optimal combination of good sequence coverage and low error rate ( 15 ) . to allow direct comparison with psi - blast defaults , psi - blast scans were performed with the default blosum62 scoring matrix and profile e - value cutoff of 0.002 . the e - values for the pairs in the benchmark were collected and ranked in order from lowest to highest and used to calculate coverage versus error - per - query plots . figure 1 shows plots of percentage true positive pairs versus percentage errors - per - query ( epq ) for scanps and psi - blast . scanps offers significantly increased coverage versus psi - blast for a given rate of epq . for example , at 1% epq , scanps using the blosum 62 scoring matrix finds 27% of true positives , while psi - blast finds 24.4% . scanps when run with the optimal blosum 50 scoring matrix finds 28.6% of true pairs at 1% epq . the vertical axis ( coverage ) represents the number of true positives found divided by the total number of true positives in the benchmark , expressed as a percentage . true positives are those pairs in which the domains belong to the same scop superfamily . the horizontal axis ( epq ) represents the number of false positives found divided by the total number of true positives , expressed as a percentage . the black line show results for scanps run with the blosum50 scoring matrix ; profile inclusion e - value = 0.015 , gap opening penalty = 12 , gap extension penalty = 2 and profile identity threshold = 97% . the green line show results for scanps run with the blosum62 scoring matrix ; profile inclusion e - value = 0.015 , gap opening penalty = 12 , gap extension penalty = 2 and profile identity threshold = 97% . the blue line show results for psi - blast run with the blosum62 matrix ; profile inclusion value = 0.002 , gap opening penalty = 11 and gap extension penalty = 1 . all runs were for a maximum of 10 iterations and pairs were collected from the final iteration of each search . the vertical axis ( coverage ) represents the number of true positives found divided by the total number of true positives in the benchmark , expressed as a percentage . true positives are those pairs in which the domains belong to the same scop superfamily . the horizontal axis ( epq ) represents the number of false positives found divided by the total number of true positives , expressed as a percentage . the black line show results for scanps run with the blosum50 scoring matrix ; profile inclusion e - value = 0.015 , gap opening penalty = 12 , gap extension penalty = 2 and profile identity threshold = 97% . the green line show results for scanps run with the blosum62 scoring matrix ; profile inclusion e - value = 0.015 , gap opening penalty = 12 , gap extension penalty = 2 and profile identity threshold = 97% . the blue line show results for psi - blast run with the blosum62 matrix ; profile inclusion value = 0.002 , gap opening penalty = 11 and gap extension penalty = 1 . all runs were for a maximum of 10 iterations and pairs were collected from the final iteration of each search . the output from scanps consists of tables of hits for each iteration , together with associated pairwise alignments and multiple alignments for all of the hits in a given iteration . this output is usually voluminous , so a major advantage of transforming the raw output into a browsable format is that it becomes much easier to navigate through the results . the web interface also permits crossreferencing to sequence databases and integration of the jalview viewer ( 18 ) for viewing alignments and further analysis . the web interface allows the possibility of more sophisticated representations for scop search results as described below . the server input page ( figure 2 ) allows the user either to upload a sequence file or paste a sequence directly into a text box . the search database is selected from a pulldown menu , with the current options of uniref100 , uniref90 , uniref50 ( 19 ) , pdb ( 20 ) and scop ( 2 ) . the uniref and pdb databases are updated automatically on biweekly and weekly schedules , respectively . the scop sequences used are generated by astral ( http://astral.berkeley.edu ) ( 21 ) from the seqres records of the corresponding pdb entries and are non - redundant at the level of 100% sequence identity . the user can set all of the search parameters or use the defaults , which , with the exception of iteration number , are those found to be optimum in benchmarking on scop superfamily data . each parameter is documented by a link to the corresponding section of the help documentation . the search is run non - interactively and an email message provides the user with the url where the results can be viewed . the search time required depends on the inputs , parameters and the load on the server . searches of scop and pdb are typically returned in within 5 min ; uniref searches may require several hours . the server does not at present use the mpi implementation of scanps but it is planned to do so to take full advantage of the large computing cluster that is available to the server . the default output format is a linear presentation of the hits and corresponding pairwise and multiple alignments for each iteration . hits for each iteration are listed in a table which displays the hit rank , the identifier assigned to the hit in the source database , a descriptive string , the e - value , the raw score and a button to display the pairwise alignment of the query and the hit in jalview ( 18 ) . this is followed by a multiple alignment of the query with all of the hits found in the iteration and then the pairwise alignments . for each pairwise and multiple alignment , when searching against the pdb and scop , jalview uses secondary structure assignments from dssp ( 22 ) to display the secondary structure elements in the database sequences . if a scop search has been performed , the results can also be viewed in a hierarchical viewer that maps the results onto the scop hierarchy ( figure 3 ) . a separate mapping is generated for each search iteration and can be displayed by clicking a button in the linear interface . the tree frame displays a tree - like representation of the scop hierarchy , the branches of which may be expanded or collapsed to display particular branches at more fine - grained levels of classification . the nodes in the tree frame are annotated with the number of scanps hits at that node and the lowest e - value found for a hit at that node , allowing the viewer to quickly identify those parts of the scop hierarchy in which hits are clustered . clicking on a scop node in the tree frame displays the corresponding node page in the node frame . node frame displays , for a given scop node , a page ( the node page ) , which contains a table of the domains in the node and summary information about the node . for each domain , the table lists the domain 's scop classification and the data returned by the search method . figure 3.result of a scanps search of the scop database displayed in the tree - based scop results interface , with a pairwise alignment displayed in jalview in the foreground . the tree frame on the left displays the scop hierarchy ; the node frame on the right on displays the page for a particular node . this is followed by summary data from scanps and then the domain table . for each domain , the table lists its scop classification below the current node , followed by the data for the domain returned by scanps . pairwise alignments for each hit are listed below the domain table ; these can also be displayed in jalview by clicking the appropriate button . the alignment in jalview is shaded to indicate the positions of strands ( blue ) and helices ( red ) in the database structure . result of a scanps search of the scop database displayed in the tree - based scop results interface , with a pairwise alignment displayed in jalview in the foreground . the tree frame on the left displays the scop hierarchy ; the node frame on the right on displays the page for a particular node . this is followed by summary data from scanps and then the domain table . for each domain , the table lists its scop classification below the current node , followed by the data for the domain returned by scanps . pairwise alignments for each hit are listed below the domain table ; these can also be displayed in jalview by clicking the appropriate button . the alignment in jalview is shaded to indicate the positions of strands ( blue ) and helices ( red ) in the database structure . although scanps has been available for over 15 years and has been accessible as a service at the european bioinformatics institute ( ebi ) for 10 years , it has not previously been described in the literature . accordingly , as background to the new web server , a brief overview of the motivation , novel features and performance of the program is given here . waterman algorithm ( 3 ) comparison of a protein sequence to a protein sequence database with either length dependent or affine gap penalties ( 4 ) . the program is written in c and effort was put into coding for performance on conventional workstation hardware . this enabled the program to be used routinely for searching large databases on modest computer hardware in contrast to the belief in the early 1990s that smith parallel processing by dynamically splitting the database across multiple processors was demonstrated on a network of five loosely coupled workstations ( 6 ) , then refined to exploit symmetric multi processing ( smp ) hardware via openmp ( 7 ) . the smp implementation gave near linear speedup on a 24 processor silicon graphics challenge . with the move to commodity pc hardware in the late 1990s , fourway onchip parallelism was implemented on intel and amd chips with a speedup over linear code of at least 3 depending on the query and database size . in addition to the onchip parallelism , multiprocessor parallelism was implemented through mpi ( 8,9 ) . the mpi implementation gave parallel efficiency of over 90% on 16 processors on an intel piii cluster connected by 100 mb network . the speed obtained by parallel processing , coupled with the on - the - fly statistics described below , permitted the implementation of iterative searching . in this mode , a multiple sequence alignment is constructed for sequences that score above a preset significance threshold in the initial search . the multiple alignment is built up by aligning to the query sequence as a template , but using a position specific scoring matrix ( pssm ) as appropriate at each iteration . alignment columns which contain gaps in the query sequence are deleted ; as a result , the alignment is always the same length as the query sequence . a pssm ( 10,11 ) is derived from this alignment and used to re - search the database . since sequences that are very similar to the query sequence contribute little information to the pssm , a percentage identity threshold ( pidt ) is employed which excludes all sequences from the alignment whose similarity to the query sequence exceeds the threshold . the contributions of each sequence to the pssm are weighted according to the method of henikoff and henikoff ( 12 ) . the scoring matrix is then constructed at each alignment position similarly to standard log odds matrices ( 12,13 ) . the process of constructing a pssm and searching the database is repeated until convergence , or until a preset number of iterations has been completed . in each database search , the statistical significance of a score between the query and any sequence in the database is assessed by on - the - fly modeling the distribution of query database sequence pair scores . scores are binned according to the log of the product of the query and database sequence lengths ( lpl ) . within each lpl bin the extreme - value location and scale parameters are then fitted to exponential and linear equations respectively with respect to the lpl . the resulting extreme - value equations are applied back to all query sequence pairs and the resulting probabilities converted to e - values for ranking and display . this method of estimating significance is similar to those implemented in fasta / ssearch ( 14 ) ; for a full discussion of the similarities and differences between the different fitting schemes and the effect on performance in benchmarks see ( 15 ) . the search performance of scanps was compared with that of psi - blast using a benchmark based on scop ( 2 ) . the benchmark dataset comprises 1113 sequences taken from the pdb40d - b dataset constructed by brenner et al . single - segment domains whose structure had been determined by x - ray crystallography were selected , representing 479 scop superfamilies across 343 scop folds . true positives were defined as pairs of sequences belonging to the same scop superfamily ; true negatives were defined as those pairs in which the sequences belong to different scop folds . the resulting set of sequence pairs contain 2528 true positives and 616 923 true negatives , resulting in a total of 618 821 pairs . benchmarking was performed by searching the benchmark set with each of the benchmark sequences in turn . since scanps and psi - blast use sequence profiles to enhance search sensitivity , it is necessary to embed the benchmark set in a larger sequence database in order to ensure that there is an adequate set of related sequences to construct the search profile for each benchmark sequence . accordingly , the benchmark set was embedded in swall ( 17 ) to create the search database . both scanps version 2.3.9 and psi - blast version 2.2 - 17 were run for a maximum of 10 iterations . search parameters were chosen to reflect the typical use case where a low rate of false positives is acceptable in return for a high rate of true positives found . scanps was run with a profile e - value inclusion value of 0.015 , a pidt of 97% and using the blosum50 scoring matrix . gap penalties for opening and extending gaps were set to 12 and 2 , respectively . these parameters have previously been established to provide the optimal combination of good sequence coverage and low error rate ( 15 ) . to allow direct comparison with psi - blast defaults , psi - blast scans were performed with the default blosum62 scoring matrix and profile e - value cutoff of 0.002 . the gap opening and extension penalties were set to 11 and 1 , respectively . the e - values for the pairs in the benchmark were collected and ranked in order from lowest to highest and used to calculate coverage versus error - per - query plots . figure 1 shows plots of percentage true positive pairs versus percentage errors - per - query ( epq ) for scanps and psi - blast . scanps offers significantly increased coverage versus psi - blast for a given rate of epq . for example , at 1% epq , scanps using the blosum 62 scoring matrix finds 27% of true positives , while psi - blast finds 24.4% . scanps when run with the optimal blosum 50 scoring matrix finds 28.6% of true pairs at 1% epq . the vertical axis ( coverage ) represents the number of true positives found divided by the total number of true positives in the benchmark , expressed as a percentage . true positives are those pairs in which the domains belong to the same scop superfamily . the horizontal axis ( epq ) represents the number of false positives found divided by the total number of true positives , expressed as a percentage . the black line show results for scanps run with the blosum50 scoring matrix ; profile inclusion e - value = 0.015 , gap opening penalty = 12 , gap extension penalty = 2 and profile identity threshold = 97% . the green line show results for scanps run with the blosum62 scoring matrix ; profile inclusion e - value = 0.015 , gap opening penalty = 12 , gap extension penalty = 2 and profile identity threshold = 97% . the blue line show results for psi - blast run with the blosum62 matrix ; profile inclusion value = 0.002 , gap opening penalty = 11 and gap extension penalty = 1 . all runs were for a maximum of 10 iterations and pairs were collected from the final iteration of each search . the vertical axis ( coverage ) represents the number of true positives found divided by the total number of true positives in the benchmark , expressed as a percentage . true positives are those pairs in which the domains belong to the same scop superfamily . the horizontal axis ( epq ) represents the number of false positives found divided by the total number of true positives , expressed as a percentage . the black line show results for scanps run with the blosum50 scoring matrix ; profile inclusion e - value = 0.015 , gap opening penalty = 12 , gap extension penalty = 2 and profile identity threshold = 97% . the green line show results for scanps run with the blosum62 scoring matrix ; profile inclusion e - value = 0.015 , gap opening penalty = 12 , gap extension penalty = 2 and profile identity threshold = 97% . the blue line show results for psi - blast run with the blosum62 matrix ; profile inclusion value = 0.002 , gap opening penalty = 11 and gap extension penalty = 1 . all runs were for a maximum of 10 iterations and pairs were collected from the final iteration of each search . the output from scanps consists of tables of hits for each iteration , together with associated pairwise alignments and multiple alignments for all of the hits in a given iteration . this output is usually voluminous , so a major advantage of transforming the raw output into a browsable format is that it becomes much easier to navigate through the results . the web interface also permits crossreferencing to sequence databases and integration of the jalview viewer ( 18 ) for viewing alignments and further analysis . the web interface allows the possibility of more sophisticated representations for scop search results as described below . the server input page ( figure 2 ) allows the user either to upload a sequence file or paste a sequence directly into a text box . the search database is selected from a pulldown menu , with the current options of uniref100 , uniref90 , uniref50 ( 19 ) , pdb ( 20 ) and scop ( 2 ) . the uniref and pdb databases are updated automatically on biweekly and weekly schedules , respectively . the scop sequences used are generated by astral ( http://astral.berkeley.edu ) ( 21 ) from the seqres records of the corresponding pdb entries and are non - redundant at the level of 100% sequence identity . the user can set all of the search parameters or use the defaults , which , with the exception of iteration number , are those found to be optimum in benchmarking on scop superfamily data . each parameter is documented by a link to the corresponding section of the help documentation . the search is run non - interactively and an email message provides the user with the url where the results can be viewed . the search time required depends on the inputs , parameters and the load on the server . searches of scop and pdb are typically returned in within 5 min ; uniref searches may require several hours . the server does not at present use the mpi implementation of scanps but it is planned to do so to take full advantage of the large computing cluster that is available to the server the default output format is a linear presentation of the hits and corresponding pairwise and multiple alignments for each iteration . hits for each iteration are listed in a table which displays the hit rank , the identifier assigned to the hit in the source database , a descriptive string , the e - value , the raw score and a button to display the pairwise alignment of the query and the hit in jalview ( 18 ) . this is followed by a multiple alignment of the query with all of the hits found in the iteration and then the pairwise alignments . for each pairwise and multiple alignment , a button is provided to view the alignment in the jalview alignment tool . when searching against the pdb and scop , jalview uses secondary structure assignments from dssp ( 22 ) to display the secondary structure elements in the database sequences . if a scop search has been performed , the results can also be viewed in a hierarchical viewer that maps the results onto the scop hierarchy ( figure 3 ) . a separate mapping is generated for each search iteration and can be displayed by clicking a button in the linear interface . the tree frame displays a tree - like representation of the scop hierarchy , the branches of which may be expanded or collapsed to display particular branches at more fine - grained levels of classification . the nodes in the tree frame are annotated with the number of scanps hits at that node and the lowest e - value found for a hit at that node , allowing the viewer to quickly identify those parts of the scop hierarchy in which hits are clustered . clicking on a scop node in the tree frame displays the corresponding node page in the node frame . node frame displays , for a given scop node , a page ( the node page ) , which contains a table of the domains in the node and summary information about the node . for each domain , the table lists the domain 's scop classification and the data returned by the search method . figure 3.result of a scanps search of the scop database displayed in the tree - based scop results interface , with a pairwise alignment displayed in jalview in the foreground . the tree frame on the left displays the scop hierarchy ; the node frame on the right on displays the page for a particular node . this is followed by summary data from scanps and then the domain table . for each domain , the table lists its scop classification below the current node , followed by the data for the domain returned by scanps . pairwise alignments for each hit are listed below the domain table ; these can also be displayed in jalview by clicking the appropriate button . the alignment in jalview is shaded to indicate the positions of strands ( blue ) and helices ( red ) in the database structure . result of a scanps search of the scop database displayed in the tree - based scop results interface , with a pairwise alignment displayed in jalview in the foreground . the tree frame on the left displays the scop hierarchy ; the node frame on the right on displays the page for a particular node . this is followed by summary data from scanps and then the domain table . for each domain , the table lists its scop classification below the current node , followed by the data for the domain returned by scanps . pairwise alignments for each hit are listed below the domain table ; these can also be displayed in jalview by clicking the appropriate button . the alignment in jalview is shaded to indicate the positions of strands ( blue ) and helices ( red ) in the database structure . the server is implemented as a set of perl cgi scripts but most of the functionality is contained in a set of common perl modules used by all the scripts . the scop interface is built using an object - oriented perl library that is designed to facilitate building interfaces for any program that searches scop . the scanps web server ( http://www.compbio.dundee.ac.uk/www-scanps ) provides access to scanps in the form of a user - friendly web interface with regularly updated databases and postprocessing of results to present them in a form that facilitates analysis and interpretation . the server provides the facility to search the complete scop database with a query sequence and display the results in a tree view . this maps hits directly onto the scop hierarchy with links to the scop database website . it also integrates the jalview alignment editor for viewing alignments between the hits and the query sequence . planned enhancements to the server include deploying the mpi implementation of scanps to reduce search times further , as well as allowing search of scop embedded in uniref to improve sensitivity for iterative searches .

scanps performs iterative profile searching similar to psi - blast but with full dynamic programing on each cycle and on - the - fly estimation of significance . this combination gives good sensitivity and selectivity that outperforms psi - blast in domain - searching benchmarks . although computationally expensive , scanps exploits onchip parallelism ( mmx and sse2 instructions on intel chips ) as well as mpi parallelism to give acceptable turnround times even for large databases . a web server developed to run scanps searches is now available at http://www.compbio.dundee.ac.uk/www-scanps . the server interface allows a range of different protein sequence databases to be searched including the scop database of protein domains . the server provides the user with regularly updated versions of the main protein sequence databases and is backed up by significant computing resources which ensure that searches are performed rapidly . for scop searches , the results may be viewed in a new tree - based representation that reflects the structure of the scop hierarchy ; this aids the user in placing each hit in the context of its scop classification and understanding its relationship to other domains in scop .

INTRODUCTION MATERIALS AND METHODS Overview of SCANPS Benchmarking of SCANPS The SCANPS web interface IMPLEMENTATION CONCLUSION

it performs iterative profile searching similar to psi - blast ( 1 ) , but with full dynamic programing on each cycle and on - the - fly estimation of significance . the scanps web server has been developed to simplify the running and analysis of scanps searches . an innovative aspect of the server is its novel tree - based presentation of results for searches against the scop domain database ( 2 ) . a comparison of a protein domain to the domains in scop can be of considerable value in elucidating its structure and function . facilities for comparing a query sequence to scop sequences are also provided by fps ( http://fps.sdsc.edu/ ) , gtop ( http://spock.genes.nig.ac.jp/~genome/grpsblt.html ) and cascadeblast ( http://crick.mbu.iisc.ernet.in/~cascade/cascadeblast.html ) , all of which use psi - blast as the search algorithm . however , interpreting the results of scop searches in the context of the classification is hampered by the fact that search methods typically produce a linear table of hits ; understanding the relationships between hits to the scop database usually requires a manual mapping of the results table onto the scop hierarchy . this procedure is tedious and error prone and therefore a browser interface has been developed that maps hits onto the scop hierarchy for viewing using a tree - based framework . the new interface allows the user to perform a search of the complete non - redundant scop sequence database and view results in a form that allows the information inherent in the scop classification to be exploited in interpreting those results . although scanps has been available for over 15 years and has been accessible as a service at the european bioinformatics institute ( ebi ) for 10 years , it has not previously been described in the literature . accordingly , as background to the new web server , a brief overview of the motivation , novel features and performance of the program is given here . waterman algorithm ( 3 ) comparison of a protein sequence to a protein sequence database with either length dependent or affine gap penalties ( 4 ) . this enabled the program to be used routinely for searching large databases on modest computer hardware in contrast to the belief in the early 1990s that smith parallel processing by dynamically splitting the database across multiple processors was demonstrated on a network of five loosely coupled workstations ( 6 ) , then refined to exploit symmetric multi processing ( smp ) hardware via openmp ( 7 ) . with the move to commodity pc hardware in the late 1990s , fourway onchip parallelism was implemented on intel and amd chips with a speedup over linear code of at least 3 depending on the query and database size . in addition to the onchip parallelism , multiprocessor parallelism was implemented through mpi ( 8,9 ) . the speed obtained by parallel processing , coupled with the on - the - fly statistics described below , permitted the implementation of iterative searching . in this mode , a multiple sequence alignment is constructed for sequences that score above a preset significance threshold in the initial search . the multiple alignment is built up by aligning to the query sequence as a template , but using a position specific scoring matrix ( pssm ) as appropriate at each iteration . alignment columns which contain gaps in the query sequence are deleted ; as a result , the alignment is always the same length as the query sequence . since sequences that are very similar to the query sequence contribute little information to the pssm , a percentage identity threshold ( pidt ) is employed which excludes all sequences from the alignment whose similarity to the query sequence exceeds the threshold . in each database search , the statistical significance of a score between the query and any sequence in the database is assessed by on - the - fly modeling the distribution of query database sequence pair scores . this method of estimating significance is similar to those implemented in fasta / ssearch ( 14 ) ; for a full discussion of the similarities and differences between the different fitting schemes and the effect on performance in benchmarks see ( 15 ) . the search performance of scanps was compared with that of psi - blast using a benchmark based on scop ( 2 ) . since scanps and psi - blast use sequence profiles to enhance search sensitivity , it is necessary to embed the benchmark set in a larger sequence database in order to ensure that there is an adequate set of related sequences to construct the search profile for each benchmark sequence . accordingly , the benchmark set was embedded in swall ( 17 ) to create the search database . both scanps version 2.3.9 and psi - blast version 2.2 - 17 were run for a maximum of 10 iterations . to allow direct comparison with psi - blast defaults , psi - blast scans were performed with the default blosum62 scoring matrix and profile e - value cutoff of 0.002 . the e - values for the pairs in the benchmark were collected and ranked in order from lowest to highest and used to calculate coverage versus error - per - query plots . figure 1 shows plots of percentage true positive pairs versus percentage errors - per - query ( epq ) for scanps and psi - blast . scanps offers significantly increased coverage versus psi - blast for a given rate of epq . for example , at 1% epq , scanps using the blosum 62 scoring matrix finds 27% of true positives , while psi - blast finds 24.4% . the blue line show results for psi - blast run with the blosum62 matrix ; profile inclusion value = 0.002 , gap opening penalty = 11 and gap extension penalty = 1 . the vertical axis ( coverage ) represents the number of true positives found divided by the total number of true positives in the benchmark , expressed as a percentage . the blue line show results for psi - blast run with the blosum62 matrix ; profile inclusion value = 0.002 , gap opening penalty = 11 and gap extension penalty = 1 . the output from scanps consists of tables of hits for each iteration , together with associated pairwise alignments and multiple alignments for all of the hits in a given iteration . this output is usually voluminous , so a major advantage of transforming the raw output into a browsable format is that it becomes much easier to navigate through the results . the web interface also permits crossreferencing to sequence databases and integration of the jalview viewer ( 18 ) for viewing alignments and further analysis . the web interface allows the possibility of more sophisticated representations for scop search results as described below . the server input page ( figure 2 ) allows the user either to upload a sequence file or paste a sequence directly into a text box . the scop sequences used are generated by astral ( http://astral.berkeley.edu ) ( 21 ) from the seqres records of the corresponding pdb entries and are non - redundant at the level of 100% sequence identity . the user can set all of the search parameters or use the defaults , which , with the exception of iteration number , are those found to be optimum in benchmarking on scop superfamily data . the search is run non - interactively and an email message provides the user with the url where the results can be viewed . the search time required depends on the inputs , parameters and the load on the server . the server does not at present use the mpi implementation of scanps but it is planned to do so to take full advantage of the large computing cluster that is available to the server . hits for each iteration are listed in a table which displays the hit rank , the identifier assigned to the hit in the source database , a descriptive string , the e - value , the raw score and a button to display the pairwise alignment of the query and the hit in jalview ( 18 ) . this is followed by a multiple alignment of the query with all of the hits found in the iteration and then the pairwise alignments . for each pairwise and multiple alignment , when searching against the pdb and scop , jalview uses secondary structure assignments from dssp ( 22 ) to display the secondary structure elements in the database sequences . if a scop search has been performed , the results can also be viewed in a hierarchical viewer that maps the results onto the scop hierarchy ( figure 3 ) . a separate mapping is generated for each search iteration and can be displayed by clicking a button in the linear interface . the tree frame displays a tree - like representation of the scop hierarchy , the branches of which may be expanded or collapsed to display particular branches at more fine - grained levels of classification . the nodes in the tree frame are annotated with the number of scanps hits at that node and the lowest e - value found for a hit at that node , allowing the viewer to quickly identify those parts of the scop hierarchy in which hits are clustered . node frame displays , for a given scop node , a page ( the node page ) , which contains a table of the domains in the node and summary information about the node . for each domain , the table lists the domain 's scop classification and the data returned by the search method . figure 3.result of a scanps search of the scop database displayed in the tree - based scop results interface , with a pairwise alignment displayed in jalview in the foreground . the tree frame on the left displays the scop hierarchy ; the node frame on the right on displays the page for a particular node . for each domain , the table lists its scop classification below the current node , followed by the data for the domain returned by scanps . the alignment in jalview is shaded to indicate the positions of strands ( blue ) and helices ( red ) in the database structure . result of a scanps search of the scop database displayed in the tree - based scop results interface , with a pairwise alignment displayed in jalview in the foreground . the tree frame on the left displays the scop hierarchy ; the node frame on the right on displays the page for a particular node . for each domain , the table lists its scop classification below the current node , followed by the data for the domain returned by scanps . although scanps has been available for over 15 years and has been accessible as a service at the european bioinformatics institute ( ebi ) for 10 years , it has not previously been described in the literature . accordingly , as background to the new web server , a brief overview of the motivation , novel features and performance of the program is given here . waterman algorithm ( 3 ) comparison of a protein sequence to a protein sequence database with either length dependent or affine gap penalties ( 4 ) . this enabled the program to be used routinely for searching large databases on modest computer hardware in contrast to the belief in the early 1990s that smith parallel processing by dynamically splitting the database across multiple processors was demonstrated on a network of five loosely coupled workstations ( 6 ) , then refined to exploit symmetric multi processing ( smp ) hardware via openmp ( 7 ) . with the move to commodity pc hardware in the late 1990s , fourway onchip parallelism was implemented on intel and amd chips with a speedup over linear code of at least 3 depending on the query and database size . in addition to the onchip parallelism , multiprocessor parallelism was implemented through mpi ( 8,9 ) . the speed obtained by parallel processing , coupled with the on - the - fly statistics described below , permitted the implementation of iterative searching . the multiple alignment is built up by aligning to the query sequence as a template , but using a position specific scoring matrix ( pssm ) as appropriate at each iteration . alignment columns which contain gaps in the query sequence are deleted ; as a result , the alignment is always the same length as the query sequence . since sequences that are very similar to the query sequence contribute little information to the pssm , a percentage identity threshold ( pidt ) is employed which excludes all sequences from the alignment whose similarity to the query sequence exceeds the threshold . in each database search , the statistical significance of a score between the query and any sequence in the database is assessed by on - the - fly modeling the distribution of query database sequence pair scores . scores are binned according to the log of the product of the query and database sequence lengths ( lpl ) . this method of estimating significance is similar to those implemented in fasta / ssearch ( 14 ) ; for a full discussion of the similarities and differences between the different fitting schemes and the effect on performance in benchmarks see ( 15 ) . the search performance of scanps was compared with that of psi - blast using a benchmark based on scop ( 2 ) . the resulting set of sequence pairs contain 2528 true positives and 616 923 true negatives , resulting in a total of 618 821 pairs . benchmarking was performed by searching the benchmark set with each of the benchmark sequences in turn . since scanps and psi - blast use sequence profiles to enhance search sensitivity , it is necessary to embed the benchmark set in a larger sequence database in order to ensure that there is an adequate set of related sequences to construct the search profile for each benchmark sequence . both scanps version 2.3.9 and psi - blast version 2.2 - 17 were run for a maximum of 10 iterations . to allow direct comparison with psi - blast defaults , psi - blast scans were performed with the default blosum62 scoring matrix and profile e - value cutoff of 0.002 . figure 1 shows plots of percentage true positive pairs versus percentage errors - per - query ( epq ) for scanps and psi - blast . scanps offers significantly increased coverage versus psi - blast for a given rate of epq . for example , at 1% epq , scanps using the blosum 62 scoring matrix finds 27% of true positives , while psi - blast finds 24.4% . the blue line show results for psi - blast run with the blosum62 matrix ; profile inclusion value = 0.002 , gap opening penalty = 11 and gap extension penalty = 1 . the vertical axis ( coverage ) represents the number of true positives found divided by the total number of true positives in the benchmark , expressed as a percentage . the blue line show results for psi - blast run with the blosum62 matrix ; profile inclusion value = 0.002 , gap opening penalty = 11 and gap extension penalty = 1 . the output from scanps consists of tables of hits for each iteration , together with associated pairwise alignments and multiple alignments for all of the hits in a given iteration . this output is usually voluminous , so a major advantage of transforming the raw output into a browsable format is that it becomes much easier to navigate through the results . the web interface also permits crossreferencing to sequence databases and integration of the jalview viewer ( 18 ) for viewing alignments and further analysis . the web interface allows the possibility of more sophisticated representations for scop search results as described below . the server input page ( figure 2 ) allows the user either to upload a sequence file or paste a sequence directly into a text box . the scop sequences used are generated by astral ( http://astral.berkeley.edu ) ( 21 ) from the seqres records of the corresponding pdb entries and are non - redundant at the level of 100% sequence identity . the user can set all of the search parameters or use the defaults , which , with the exception of iteration number , are those found to be optimum in benchmarking on scop superfamily data . each parameter is documented by a link to the corresponding section of the help documentation . the search is run non - interactively and an email message provides the user with the url where the results can be viewed . the search time required depends on the inputs , parameters and the load on the server . the server does not at present use the mpi implementation of scanps but it is planned to do so to take full advantage of the large computing cluster that is available to the server the default output format is a linear presentation of the hits and corresponding pairwise and multiple alignments for each iteration . hits for each iteration are listed in a table which displays the hit rank , the identifier assigned to the hit in the source database , a descriptive string , the e - value , the raw score and a button to display the pairwise alignment of the query and the hit in jalview ( 18 ) . this is followed by a multiple alignment of the query with all of the hits found in the iteration and then the pairwise alignments . for each pairwise and multiple alignment , a button is provided to view the alignment in the jalview alignment tool . if a scop search has been performed , the results can also be viewed in a hierarchical viewer that maps the results onto the scop hierarchy ( figure 3 ) . the tree frame displays a tree - like representation of the scop hierarchy , the branches of which may be expanded or collapsed to display particular branches at more fine - grained levels of classification . the nodes in the tree frame are annotated with the number of scanps hits at that node and the lowest e - value found for a hit at that node , allowing the viewer to quickly identify those parts of the scop hierarchy in which hits are clustered . node frame displays , for a given scop node , a page ( the node page ) , which contains a table of the domains in the node and summary information about the node . for each domain , the table lists the domain 's scop classification and the data returned by the search method . figure 3.result of a scanps search of the scop database displayed in the tree - based scop results interface , with a pairwise alignment displayed in jalview in the foreground . the tree frame on the left displays the scop hierarchy ; the node frame on the right on displays the page for a particular node . for each domain , the table lists its scop classification below the current node , followed by the data for the domain returned by scanps . pairwise alignments for each hit are listed below the domain table ; these can also be displayed in jalview by clicking the appropriate button . the alignment in jalview is shaded to indicate the positions of strands ( blue ) and helices ( red ) in the database structure . result of a scanps search of the scop database displayed in the tree - based scop results interface , with a pairwise alignment displayed in jalview in the foreground . the tree frame on the left displays the scop hierarchy ; the node frame on the right on displays the page for a particular node . for each domain , the table lists its scop classification below the current node , followed by the data for the domain returned by scanps . the alignment in jalview is shaded to indicate the positions of strands ( blue ) and helices ( red ) in the database structure . the server is implemented as a set of perl cgi scripts but most of the functionality is contained in a set of common perl modules used by all the scripts . the scop interface is built using an object - oriented perl library that is designed to facilitate building interfaces for any program that searches scop . the scanps web server ( http://www.compbio.dundee.ac.uk/www-scanps ) provides access to scanps in the form of a user - friendly web interface with regularly updated databases and postprocessing of results to present them in a form that facilitates analysis and interpretation . the server provides the facility to search the complete scop database with a query sequence and display the results in a tree view . this maps hits directly onto the scop hierarchy with links to the scop database website . planned enhancements to the server include deploying the mpi implementation of scanps to reduce search times further , as well as allowing search of scop embedded in uniref to improve sensitivity for iterative searches .

from january 1999 to december 2004 , we identified 43 patients with a definitive diagnosis of nsip and 115 patients with a definitive diagnosis of uip by searching the patient records at our hospital and at another teaching hospital . over the same period , 252 individuals were identified who had no abnormal lesion on high - resolution ct ( hrct ) and they had normal pulmonary function tests , so they were selected as controls . to avoid any bias due to age or gender , the uip , nsip and control subjects were individually matched for age and gender . finally , 26 patients with nsip , 26 patients with uip and 26 controls were selected . for each of the three groups , the ages were matched within two years ( age range : 40 - 77 years , mean age : 56.3 ) . nonspecific interstitial pneumonia was diagnosed in 26 patients from lung wedge resections by consensus between a panel of experienced pathologists who worked in conference , and the diagnosis was based on the criteria developed by the american thoracic society and european respiratory society ( ats / ers ) ( 6 ) . the histologic features of nsip included a cellular pattern ( n = 3 ) , a fibrosing pattern ( n = 10 ) , and both patterns ( n = 13 ) . uip was also diagnosed from lung resections in five cases or from a combination of the typical hrct findings ( as described by the ats ) and from the clinical information in 21 cases ( 6 ) . two authors ( c.h.l . and k.r.s . ) reviewed the clinical histories and the underlying medical conditions . to exclude other factors that might change the amount and shape of the anterior mediastinal fat , patients with a history of tuberculosis , empyema , connective tissue diseases , exposure to organic or inorganic dust or toxic fumes , cushing disease , asthma or any other diseases requiring steroid medication were excluded from the study population . patients who had been treated with steroid before ct were excluded for the same reason . our institutional review board did not require approval of our study or informed consents for the patients ' records or image reviews . the scans were 1 - 1.5 mm thick / section , and they were reconstructed using a high spatial frequency algorithm . the hrct scans were obtained with a variety of scanners ( somatom plus-4 scanner , siemens medical systems , erlangen , germany ; somatom plus - s scanner , siemens medical systems , erlangen , germany ; and hi - speed advantage , ge medical systems , milwaukee , wi ) . the ct scan digital imaging communications in medicine ( dicom ) format was used for the quantitative measurements . the amount of anterior mediastinal fat was determined from scans at the level of the aortic arch , and it was quantified using rapidia software ( 3dmed , seoul , korea ) ( fig . this software automatically calculates the areas of anterior mediastinal fat with the attenuation range of -190 to -50 hus . this attenuation threshold for the anterior mediastinal fat measurement was modified from the value used for the standardized abdominal fat measurement on ct by yoshizumi et al . the shape of the anterior mediastinal fat was categorized as concave , flat or convex at the level of the main pulmonary trunk rather than determining the shape of the anterior mediastinal fat at the level of aortic arch because the amount of anterior mediastinal fat at the level of aortic arch was insufficient for the purposes of classifying it in some cases . the anteroposterior ( ap ) ( from the posterior wall of the sternum to the anterior wall of the ascending aorta ) and transverse dimensions ( the width of the posterior wall of the sternum in contact with the anterior mediastinal fat ) of the anterior mediastinal fat at the level of the main pulmonary trunk were also measured ( fig . all the analyses were performed with sas system software ( version 9.0 , sas institute , cary , nc ) . the 26 1:1:1 matched sets were analyzed , and the amounts of anterior mediastinal fat , the retrosternal ap and transverse dimensions of the anterior mediastinum , and the weights and body mass indexes ( bmis ) were compared using oneway analysis of variance ( anova ) . the shapes of the anterior mediastinal fat were compared using the chi - square test . the conditional exact logistic regression model was used to assess whether convexity of the anterior mediastinum , as determined by ct , was a risk factor for pulmonary fibrosis . the potential confounding factors including weight , bmi , the ap and transverse dimensions and the amount of fat in the anterior mediastinum , were categorized ( the weight was divided for 10 kg intervals and the other factors were divided into four groups in reference to the control ) . because weight was the most important confounding factor , the crude odds ratios and adjusted odds ratios for weight with the corresponding 95% confidence intervals ( ci ) were calculated . a polychotomous logistic regression test model was used to calculate the overall p - values for the uip , nsip and control groups . from january 1999 to december 2004 , we identified 43 patients with a definitive diagnosis of nsip and 115 patients with a definitive diagnosis of uip by searching the patient records at our hospital and at another teaching hospital . over the same period , 252 individuals were identified who had no abnormal lesion on high - resolution ct ( hrct ) and they had normal pulmonary function tests , so they were selected as controls . to avoid any bias due to age or gender , the uip , nsip and control subjects were individually matched for age and gender . finally , 26 patients with nsip , 26 patients with uip and 26 controls were selected . for each of the three groups , the ages were matched within two years ( age range : 40 - 77 years , mean age : 56.3 ) . nonspecific interstitial pneumonia was diagnosed in 26 patients from lung wedge resections by consensus between a panel of experienced pathologists who worked in conference , and the diagnosis was based on the criteria developed by the american thoracic society and european respiratory society ( ats / ers ) ( 6 ) . the histologic features of nsip included a cellular pattern ( n = 3 ) , a fibrosing pattern ( n = 10 ) , and both patterns ( n = 13 ) . uip was also diagnosed from lung resections in five cases or from a combination of the typical hrct findings ( as described by the ats ) and from the clinical information in 21 cases ( 6 ) . two authors ( c.h.l . and k.r.s . ) reviewed the clinical histories and the underlying medical conditions . to exclude other factors that might change the amount and shape of the anterior mediastinal fat , patients with a history of tuberculosis , empyema , connective tissue diseases , exposure to organic or inorganic dust or toxic fumes , cushing disease , asthma or any other diseases requiring steroid medication were excluded from the study population . patients who had been treated with steroid before ct were excluded for the same reason . our institutional review board did not require approval of our study or informed consents for the patients ' records or image reviews . the scans were 1 - 1.5 mm thick / section , and they were reconstructed using a high spatial frequency algorithm . the hrct scans were obtained with a variety of scanners ( somatom plus-4 scanner , siemens medical systems , erlangen , germany ; somatom plus - s scanner , siemens medical systems , erlangen , germany ; and hi - speed advantage , ge medical systems , milwaukee , wi ) . the ct scan digital imaging communications in medicine ( dicom ) format was used for the quantitative measurements . the amount of anterior mediastinal fat was determined from scans at the level of the aortic arch , and it was quantified using rapidia software ( 3dmed , seoul , korea ) ( fig . this software automatically calculates the areas of anterior mediastinal fat with the attenuation range of -190 to -50 hus . this attenuation threshold for the anterior mediastinal fat measurement was modified from the value used for the standardized abdominal fat measurement on ct by yoshizumi et al . the shape of the anterior mediastinal fat was categorized as concave , flat or convex at the level of the main pulmonary trunk rather than determining the shape of the anterior mediastinal fat at the level of aortic arch because the amount of anterior mediastinal fat at the level of aortic arch was insufficient for the purposes of classifying it in some cases . the anteroposterior ( ap ) ( from the posterior wall of the sternum to the anterior wall of the ascending aorta ) and transverse dimensions ( the width of the posterior wall of the sternum in contact with the anterior mediastinal fat ) of the anterior mediastinal fat at the level of the main pulmonary trunk were also measured ( fig . all the analyses were performed with sas system software ( version 9.0 , sas institute , cary , nc ) . the 26 1:1:1 matched sets were analyzed , and the amounts of anterior mediastinal fat , the retrosternal ap and transverse dimensions of the anterior mediastinum , and the weights and body mass indexes ( bmis ) were compared using oneway analysis of variance ( anova ) . the shapes of the anterior mediastinal fat were compared using the chi - square test . the conditional exact logistic regression model was used to assess whether convexity of the anterior mediastinum , as determined by ct , was a risk factor for pulmonary fibrosis . the potential confounding factors including weight , bmi , the ap and transverse dimensions and the amount of fat in the anterior mediastinum , were categorized ( the weight was divided for 10 kg intervals and the other factors were divided into four groups in reference to the control ) . because weight was the most important confounding factor , the crude odds ratios and adjusted odds ratios for weight with the corresponding 95% confidence intervals ( ci ) were calculated . a polychotomous logistic regression test model was used to calculate the overall p - values for the uip , nsip and control groups . no significant differences in age , weight and bmi were observed among the three groups . the amount of anterior mediastinal fat was not different among the three groups ( p = 0.175 ) . the retrosternal ap dimension ( p = 0.037 ) and transverse dimension ( p < 0.001 ) of the anterior mediastinal fat were significantly different among the three groups . bonferroni post hoc test showed that the uip retrosternal ap dimension was shorter ( p = 0.037 ) and the transverse dimension was longer ( p = 0.001 ) than that in the normal control group . for the nsip group , only the transverse dimension was significantly longer than that in the normal control group ( p < 0.001 ) . however , no significant difference in the ap and transverse dimensions of the anterior mediastinal fat was observed between the nsip and uip groups . the shapes of anterior mediastinum were significantly different among the three groups ( p < 0.001 ) ( table 2 ) . the convex shape of the anterior mediastinum was predictive of nsip ( or = 19.7 , ci 3.32- , p < 0.001 ) and uip ( or = 24.42 , ci 4.06- , p < 0.001 ) . when the data were adjusted for weight , a convex shape was also predictive for nsip ( adjusted or = 17.16 , ci 2.89- , p < 0.002 ) and uip ( adjusted or = 32.64 , ci 5.71- , p < 0.002 ) . the uip patients were also found to have a higher likelihood of a convex shape of the anterior mediastinum than were those patients with nsip ( or = 3.96 , ci 0.74 - 39.79 , p < 0.001 ; adjusted or = 6.11 , ci although mediastinal widening in idiopathic pulmonary fibrosis have been previously described , no objective ct analysis has been reported ( 4 ) . thus , this is the first study to investigate the use of ct images to quantitatively and qualitatively analyze the mediastinal morphologies in pulmonary fibrosis . therefore , the shape of the mediastinum readily adapts to the changes in lung pathology ( 1 ) . mediastinal widening can be seen in the setting of idiopathic pulmonary fibrosis ( 4 ) . according to the results of our study , idiopathic interstitial pneumonias such as uip and nsip can change the shape of the anterior mediastinal fat . mediastinal lipomatosis refers to the accumulation of excess fat , and this is usually associated with corticosteroid administration ( 8 , 9 ) . for those patients with uip , the first line treatment is corticosteroid ; thus , mediastinal widening can be the result of steroid medication or fibrous scarring . however , because we excluded the patients who had previously received corticosteroid medications , its effect could be ignored in the present study . in patients with a treated neoplasm , however , if the patient has been irradiated , then mediastinal widening can be due to adjacent lung scarring , which retracts the mediastinal pleura laterally and draws in fat . for example , localized lung or pleural scarring caused by lung infection can tether the pleura and thicken the overlying fat in the chest wall or mediastinum ( 2 , 3 ) . proto ( 1 ) have also reported that a retrosternal band is a common finding on the lateral radiographs of patients with an abnormally low lung volume ( congenital or acquired ) on one side . retrosternal soft tissues develop because the diminished lung volume pulls the mediastinum toward the affected side and this draws mediastinal fat anterolaterally in front of the lung . before the advent of ct , this retrosternal band was attributed to excessive areolar tissue or to an accessory hemidiaphragm ( 10 ) , but ct has shown that the actual cause of this retrosternal band is mediastinal rotation and mediastinal fat displacement ( 11 ) . we measured the amount of mediastinal fat at the level of the aortic arch , which is where most of the fat tissue can be consistently observed on hrct . however , for classifying the shape of anterior mediastinum into three types , we selected the level of the main pulmonary trunk because the shape of the anterior mediastinum could be more objectively and consistently determined . in some cases , the retrosternal space was so narrowed that the shape of the anterior mediastinum could not be determined at the level of aortic arch . our results showed that uip tends to be associated with a reduced ap dimension and an increased transverse dimension with the resultant convex shape of the anterior mediastinum . in uip patients , we can speculate that the bilateral tensile force exerted by the subpleural fibrosis of the lung adjacent to the mediastinum could result in widening of the transverse dimension and shortening of the ap dimension of the anterior mediastinum ( fig . ( 5 ) analyzed the changes in the amounts and distributions of the anterior mediastinal fat after left upper lobectomy , as determined by ct . the postoperative anterior mediastinal fat distribution was distinctly changed with showing a marked increase from the aortic arch to the main pulmonary arterial level , with a converse decrease at the upper and lower slices , but no significant postoperative change was noted in the total anterior mediastinal fat volume . likewise , in our study , the amounts of anterior mediastinal fat in the cases of nsip and uip were not significantly different from the controls . this indicates that lung fibrosis did not induce a quantitative increase of the anterior mediastinal fat , but rather that it induces a redistribution of the mediastinal fat . the loss of volume of both lower basal lungs due to the fibrosis in uip might cause a redistribution of fat tissues and the anterior mediastinal shape changes . several studies have found that considerable overlap exists between the thin section ct patterns of nsip and uip patients , i.e. , up to one - third of nsip patients were found to have ct patterns that were similar to those of uip patients ( 12 - 14 ) . however , these radiologic findings were solely concerned with the intrapulmonary manifestations . even the pathologic differential diagnosis of uip and fibrotic nsip is not straightforward ; therefore , the current gold standard involves a clinicopathologic work - up by the physicians and radiologists , with a subsequent review of the final clinicopathologic diagnosis ( 6 ) . the prognosis of the nsip subgroups differs ; cellular nsip responds rather well to corticosteroid treatment and it has a better prognosis than fibrotic nsip ( 15 - 17 ) . patients with uip show a more favorable prognosis than those without honeycombing ( 18 ) . therefore , we can also hypothesize that the prognosis of idiopathic pulmonary fibrosis correlates well with the progression of pulmonary fibrosis . however , measuring the extent of fibrosis and the volume loss of the lung parenchyma in uip patients is not so easy , and there is no widely accepted standardized scoring system ( 19 ) . honeycombing represents end - stage fibrosis , but the ground - glass opacity associated with honeycombing can also be caused by interstitial fibrosis ( 20 - 22 ) . moreover , some ground - glass opacities disappear on the follow up studies for the nsip patients . thus , there are problems for how to measure the extent of overall fibrosis in idiopathic pulmonary fibrosis . ct analysis of a dynamic mediastinum might reflect the overall amount of fibrosis in lungs because the mediastinum tends to change its shape and adapt to various thoracic conditions . our results showed that a convex shape of the anterior mediastinum was due to a reduced ap dimension , and an increased transverse dimension tends to indicate a risk of nsip and uip , that is , the progression of fibrosis . though the evaluation of the mediastinum shape is not presented as a critical method or as a definitive tool for diagnosing any specific disease entity that causes pulmonary fibrosis , it could provide a straightforward and convenient way of observing the overall fibrosis in the lung parenchyma , and it could be an indirect means of determining whether the radiologic findings are transient or not . whether the convexity of the anterior mediastinum correlates well with the prognosis of idiopathic pulmonary fibrosis is not known , but this could be elucidated in a larger study . the confidence interval of our logistic regression model was excessive and this may have been caused by the limited sample size . another limitation was that other conditions such as emphysema , chest wall movement and the phase of respiration , but not age and gender , might have affected the measurement of the anterior mediastinal fat , although inspiratory hrct scans were performed . in summary , the retrosternal ap and transverse dimensions of uip patients differed from those of the normal individuals , whereas the amounts of anterior mediastinal fat were similar . uip and nsip patients have a tendency to have a convex shape of their anterior mediastinal fat . therefore , the shape of the mediastinum readily adapts to the changes in lung pathology ( 1 ) . mediastinal widening can be seen in the setting of idiopathic pulmonary fibrosis ( 4 ) . according to the results of our study , idiopathic interstitial pneumonias such as uip and nsip can change the shape of the anterior mediastinal fat . mediastinal lipomatosis refers to the accumulation of excess fat , and this is usually associated with corticosteroid administration ( 8 , 9 ) . for those patients with uip , the first line treatment is corticosteroid ; thus , mediastinal widening can be the result of steroid medication or fibrous scarring . however , because we excluded the patients who had previously received corticosteroid medications , its effect could be ignored in the present study . in patients with a treated neoplasm , . however , if the patient has been irradiated , then mediastinal widening can be due to adjacent lung scarring , which retracts the mediastinal pleura laterally and draws in fat . for example , localized lung or pleural scarring caused by lung infection can tether the pleura and thicken the overlying fat in the chest wall or mediastinum ( 2 , 3 ) . proto ( 1 ) have also reported that a retrosternal band is a common finding on the lateral radiographs of patients with an abnormally low lung volume ( congenital or acquired ) on one side . retrosternal soft tissues develop because the diminished lung volume pulls the mediastinum toward the affected side and this draws mediastinal fat anterolaterally in front of the lung . before the advent of ct , this retrosternal band was attributed to excessive areolar tissue or to an accessory hemidiaphragm ( 10 ) , but ct has shown that the actual cause of this retrosternal band is mediastinal rotation and mediastinal fat displacement ( 11 ) . we measured the amount of mediastinal fat at the level of the aortic arch , which is where most of the fat tissue can be consistently observed on hrct . however , for classifying the shape of anterior mediastinum into three types , we selected the level of the main pulmonary trunk because the shape of the anterior mediastinum could be more objectively and consistently determined . in some cases , the retrosternal space was so narrowed that the shape of the anterior mediastinum could not be determined at the level of aortic arch . our results showed that uip tends to be associated with a reduced ap dimension and an increased transverse dimension with the resultant convex shape of the anterior mediastinum . in uip patients , honeycombing change with volume loss usually occurs at the periphery of the lung base . we can speculate that the bilateral tensile force exerted by the subpleural fibrosis of the lung adjacent to the mediastinum could result in widening of the transverse dimension and shortening of the ap dimension of the anterior mediastinum ( fig . ( 5 ) analyzed the changes in the amounts and distributions of the anterior mediastinal fat after left upper lobectomy , as determined by ct . the postoperative anterior mediastinal fat distribution was distinctly changed with showing a marked increase from the aortic arch to the main pulmonary arterial level , with a converse decrease at the upper and lower slices , but no significant postoperative change was noted in the total anterior mediastinal fat volume . likewise , in our study , the amounts of anterior mediastinal fat in the cases of nsip and uip were not significantly different from the controls . this indicates that lung fibrosis did not induce a quantitative increase of the anterior mediastinal fat , but rather that it induces a redistribution of the mediastinal fat . the loss of volume of both lower basal lungs due to the fibrosis in uip might cause a redistribution of fat tissues and the anterior mediastinal shape changes . several studies have found that considerable overlap exists between the thin section ct patterns of nsip and uip patients , i.e. , up to one - third of nsip patients were found to have ct patterns that were similar to those of uip patients ( 12 - 14 ) . however , these radiologic findings were solely concerned with the intrapulmonary manifestations . even the pathologic differential diagnosis of uip and fibrotic nsip is not straightforward ; therefore , the current gold standard involves a clinicopathologic work - up by the physicians and radiologists , with a subsequent review of the final clinicopathologic diagnosis ( 6 ) . the prognosis of the nsip subgroups differs ; cellular nsip responds rather well to corticosteroid treatment and it has a better prognosis than fibrotic nsip ( 15 - 17 ) . patients with uip show a more favorable prognosis than those without honeycombing ( 18 ) . therefore , we can also hypothesize that the prognosis of idiopathic pulmonary fibrosis correlates well with the progression of pulmonary fibrosis . however , measuring the extent of fibrosis and the volume loss of the lung parenchyma in uip patients is not so easy , and there is no widely accepted standardized scoring system ( 19 ) . honeycombing represents end - stage fibrosis , but the ground - glass opacity associated with honeycombing can also be caused by interstitial fibrosis ( 20 - 22 ) . moreover , some ground - glass opacities disappear on the follow up studies for the nsip patients . thus , there are problems for how to measure the extent of overall fibrosis in idiopathic pulmonary fibrosis . ct analysis of a dynamic mediastinum might reflect the overall amount of fibrosis in lungs because the mediastinum tends to change its shape and adapt to various thoracic conditions . our results showed that a convex shape of the anterior mediastinum was due to a reduced ap dimension , and an increased transverse dimension tends to indicate a risk of nsip and uip , that is , the progression of fibrosis . though the evaluation of the mediastinum shape is not presented as a critical method or as a definitive tool for diagnosing any specific disease entity that causes pulmonary fibrosis , it could provide a straightforward and convenient way of observing the overall fibrosis in the lung parenchyma , and it could be an indirect means of determining whether the radiologic findings are transient or not . whether the convexity of the anterior mediastinum correlates well with the prognosis of idiopathic pulmonary fibrosis is not known , but this could be elucidated in a larger study . the confidence interval of our logistic regression model was excessive and this may have been caused by the limited sample size . another limitation was that other conditions such as emphysema , chest wall movement and the phase of respiration , but not age and gender , might have affected the measurement of the anterior mediastinal fat , although inspiratory hrct scans were performed . in summary , the retrosternal ap and transverse dimensions of uip patients differed from those of the normal individuals , whereas the amounts of anterior mediastinal fat were similar . uip and nsip patients have a tendency to have a convex shape of their anterior mediastinal fat .

objectivewe wanted to determine whether the amount and shape of the anterior mediastinal fat in the patients suffering with usual interstitial pneumonia ( uip ) or nonspecific interstitial pneumonia ( nsip ) was different from those of the normal control group.materials and methodswe selected patients who suffered with uip ( n = 26 ) and nsip ( n = 26 ) who had undergone ct scans . twenty - six controls were selected from individuals with normal ct findings and normal pulmonary function tests . all three groups ( n = 78 ) were individually matched for age and gender . the amounts of anterior mediastinal fat , and the retrosternal anteroposterior ( ap ) and transverse dimensions of the anterior mediastinal fat were compared by one - way analysis of variance and bonferroni 's test . the shapes of the anterior mediastinum were compared using the chi - square test . exact logistic regression analysis and polychotomous logistic regression analysis were employed to assess whether the patients with nsip or uip had a tendency to show a convex shape of their anterior mediastinal fat.resultsthe amount of anterior mediastinal fat was not different among the three groups ( p = 0.175 ) . for the uip patients , the retrosternal ap dimension of the anterior mediastinal fat was shorter ( p = 0.037 ) and the transverse dimension of the anterior mediastinal fat was longer ( p = 0.001 ) than those of the normal control group . for the nsip patients , only the transverse dimension was significantly longer than those of the normal control group ( p < 0.001 ) . the convex shape of the anterior mediastinum was predictive of nsip ( or = 19.7 , ci 3.32 - , p < 0.001 ) and uip ( or = 24.42 , ci 4.06 - , p < 0.001).conclusionfor uip patients , the retrosternal ap and transverse dimensions are different from those of normal individuals , whereas the amounts of anterior mediastinal fat are similar . uip and nsip patients have a tendency to have a convex shape of their anterior mediastinal fat .

MATERIALS AND METHODS Patients High-Resolution CT CT Image Analysis Statistical Analysis RESULTS DISCUSSION Changes in the Shape of the Anterior Mediastinum in Various Pathologic Conditions Mediastinal Changes and its Mechanism Differential Diagnosis of Nonspecific Interstitial Pneumonia and Uusual Interstitial Pneumonia by CT

over the same period , 252 individuals were identified who had no abnormal lesion on high - resolution ct ( hrct ) and they had normal pulmonary function tests , so they were selected as controls . to avoid any bias due to age or gender , the uip , nsip and control subjects were individually matched for age and gender . finally , 26 patients with nsip , 26 patients with uip and 26 controls were selected . for each of the three groups , the ages were matched within two years ( age range : 40 - 77 years , mean age : 56.3 ) . nonspecific interstitial pneumonia was diagnosed in 26 patients from lung wedge resections by consensus between a panel of experienced pathologists who worked in conference , and the diagnosis was based on the criteria developed by the american thoracic society and european respiratory society ( ats / ers ) ( 6 ) . the histologic features of nsip included a cellular pattern ( n = 3 ) , a fibrosing pattern ( n = 10 ) , and both patterns ( n = 13 ) . to exclude other factors that might change the amount and shape of the anterior mediastinal fat , patients with a history of tuberculosis , empyema , connective tissue diseases , exposure to organic or inorganic dust or toxic fumes , cushing disease , asthma or any other diseases requiring steroid medication were excluded from the study population . patients who had been treated with steroid before ct were excluded for the same reason . the amount of anterior mediastinal fat was determined from scans at the level of the aortic arch , and it was quantified using rapidia software ( 3dmed , seoul , korea ) ( fig . this software automatically calculates the areas of anterior mediastinal fat with the attenuation range of -190 to -50 hus . this attenuation threshold for the anterior mediastinal fat measurement was modified from the value used for the standardized abdominal fat measurement on ct by yoshizumi et al . the shape of the anterior mediastinal fat was categorized as concave , flat or convex at the level of the main pulmonary trunk rather than determining the shape of the anterior mediastinal fat at the level of aortic arch because the amount of anterior mediastinal fat at the level of aortic arch was insufficient for the purposes of classifying it in some cases . the anteroposterior ( ap ) ( from the posterior wall of the sternum to the anterior wall of the ascending aorta ) and transverse dimensions ( the width of the posterior wall of the sternum in contact with the anterior mediastinal fat ) of the anterior mediastinal fat at the level of the main pulmonary trunk were also measured ( fig . the 26 1:1:1 matched sets were analyzed , and the amounts of anterior mediastinal fat , the retrosternal ap and transverse dimensions of the anterior mediastinum , and the weights and body mass indexes ( bmis ) were compared using oneway analysis of variance ( anova ) . the shapes of the anterior mediastinal fat were compared using the chi - square test . the conditional exact logistic regression model was used to assess whether convexity of the anterior mediastinum , as determined by ct , was a risk factor for pulmonary fibrosis . the potential confounding factors including weight , bmi , the ap and transverse dimensions and the amount of fat in the anterior mediastinum , were categorized ( the weight was divided for 10 kg intervals and the other factors were divided into four groups in reference to the control ) . because weight was the most important confounding factor , the crude odds ratios and adjusted odds ratios for weight with the corresponding 95% confidence intervals ( ci ) were calculated . a polychotomous logistic regression test model was used to calculate the overall p - values for the uip , nsip and control groups . over the same period , 252 individuals were identified who had no abnormal lesion on high - resolution ct ( hrct ) and they had normal pulmonary function tests , so they were selected as controls . to avoid any bias due to age or gender , the uip , nsip and control subjects were individually matched for age and gender . finally , 26 patients with nsip , 26 patients with uip and 26 controls were selected . for each of the three groups , the ages were matched within two years ( age range : 40 - 77 years , mean age : 56.3 ) . nonspecific interstitial pneumonia was diagnosed in 26 patients from lung wedge resections by consensus between a panel of experienced pathologists who worked in conference , and the diagnosis was based on the criteria developed by the american thoracic society and european respiratory society ( ats / ers ) ( 6 ) . the histologic features of nsip included a cellular pattern ( n = 3 ) , a fibrosing pattern ( n = 10 ) , and both patterns ( n = 13 ) . to exclude other factors that might change the amount and shape of the anterior mediastinal fat , patients with a history of tuberculosis , empyema , connective tissue diseases , exposure to organic or inorganic dust or toxic fumes , cushing disease , asthma or any other diseases requiring steroid medication were excluded from the study population . patients who had been treated with steroid before ct were excluded for the same reason . the amount of anterior mediastinal fat was determined from scans at the level of the aortic arch , and it was quantified using rapidia software ( 3dmed , seoul , korea ) ( fig . this software automatically calculates the areas of anterior mediastinal fat with the attenuation range of -190 to -50 hus . this attenuation threshold for the anterior mediastinal fat measurement was modified from the value used for the standardized abdominal fat measurement on ct by yoshizumi et al . the shape of the anterior mediastinal fat was categorized as concave , flat or convex at the level of the main pulmonary trunk rather than determining the shape of the anterior mediastinal fat at the level of aortic arch because the amount of anterior mediastinal fat at the level of aortic arch was insufficient for the purposes of classifying it in some cases . the anteroposterior ( ap ) ( from the posterior wall of the sternum to the anterior wall of the ascending aorta ) and transverse dimensions ( the width of the posterior wall of the sternum in contact with the anterior mediastinal fat ) of the anterior mediastinal fat at the level of the main pulmonary trunk were also measured ( fig . the 26 1:1:1 matched sets were analyzed , and the amounts of anterior mediastinal fat , the retrosternal ap and transverse dimensions of the anterior mediastinum , and the weights and body mass indexes ( bmis ) were compared using oneway analysis of variance ( anova ) . the shapes of the anterior mediastinal fat were compared using the chi - square test . the conditional exact logistic regression model was used to assess whether convexity of the anterior mediastinum , as determined by ct , was a risk factor for pulmonary fibrosis . the potential confounding factors including weight , bmi , the ap and transverse dimensions and the amount of fat in the anterior mediastinum , were categorized ( the weight was divided for 10 kg intervals and the other factors were divided into four groups in reference to the control ) . a polychotomous logistic regression test model was used to calculate the overall p - values for the uip , nsip and control groups . no significant differences in age , weight and bmi were observed among the three groups . the amount of anterior mediastinal fat was not different among the three groups ( p = 0.175 ) . the retrosternal ap dimension ( p = 0.037 ) and transverse dimension ( p < 0.001 ) of the anterior mediastinal fat were significantly different among the three groups . bonferroni post hoc test showed that the uip retrosternal ap dimension was shorter ( p = 0.037 ) and the transverse dimension was longer ( p = 0.001 ) than that in the normal control group . for the nsip group , only the transverse dimension was significantly longer than that in the normal control group ( p < 0.001 ) . however , no significant difference in the ap and transverse dimensions of the anterior mediastinal fat was observed between the nsip and uip groups . the shapes of anterior mediastinum were significantly different among the three groups ( p < 0.001 ) ( table 2 ) . the convex shape of the anterior mediastinum was predictive of nsip ( or = 19.7 , ci 3.32- , p < 0.001 ) and uip ( or = 24.42 , ci 4.06- , p < 0.001 ) . when the data were adjusted for weight , a convex shape was also predictive for nsip ( adjusted or = 17.16 , ci 2.89- , p < 0.002 ) and uip ( adjusted or = 32.64 , ci 5.71- , p < 0.002 ) . the uip patients were also found to have a higher likelihood of a convex shape of the anterior mediastinum than were those patients with nsip ( or = 3.96 , ci 0.74 - 39.79 , p < 0.001 ; adjusted or = 6.11 , ci although mediastinal widening in idiopathic pulmonary fibrosis have been previously described , no objective ct analysis has been reported ( 4 ) . therefore , the shape of the mediastinum readily adapts to the changes in lung pathology ( 1 ) . according to the results of our study , idiopathic interstitial pneumonias such as uip and nsip can change the shape of the anterior mediastinal fat . for those patients with uip , the first line treatment is corticosteroid ; thus , mediastinal widening can be the result of steroid medication or fibrous scarring . however , because we excluded the patients who had previously received corticosteroid medications , its effect could be ignored in the present study . we measured the amount of mediastinal fat at the level of the aortic arch , which is where most of the fat tissue can be consistently observed on hrct . however , for classifying the shape of anterior mediastinum into three types , we selected the level of the main pulmonary trunk because the shape of the anterior mediastinum could be more objectively and consistently determined . in some cases , the retrosternal space was so narrowed that the shape of the anterior mediastinum could not be determined at the level of aortic arch . our results showed that uip tends to be associated with a reduced ap dimension and an increased transverse dimension with the resultant convex shape of the anterior mediastinum . in uip patients , we can speculate that the bilateral tensile force exerted by the subpleural fibrosis of the lung adjacent to the mediastinum could result in widening of the transverse dimension and shortening of the ap dimension of the anterior mediastinum ( fig . ( 5 ) analyzed the changes in the amounts and distributions of the anterior mediastinal fat after left upper lobectomy , as determined by ct . the postoperative anterior mediastinal fat distribution was distinctly changed with showing a marked increase from the aortic arch to the main pulmonary arterial level , with a converse decrease at the upper and lower slices , but no significant postoperative change was noted in the total anterior mediastinal fat volume . likewise , in our study , the amounts of anterior mediastinal fat in the cases of nsip and uip were not significantly different from the controls . this indicates that lung fibrosis did not induce a quantitative increase of the anterior mediastinal fat , but rather that it induces a redistribution of the mediastinal fat . the loss of volume of both lower basal lungs due to the fibrosis in uip might cause a redistribution of fat tissues and the anterior mediastinal shape changes . several studies have found that considerable overlap exists between the thin section ct patterns of nsip and uip patients , i.e. , up to one - third of nsip patients were found to have ct patterns that were similar to those of uip patients ( 12 - 14 ) . even the pathologic differential diagnosis of uip and fibrotic nsip is not straightforward ; therefore , the current gold standard involves a clinicopathologic work - up by the physicians and radiologists , with a subsequent review of the final clinicopathologic diagnosis ( 6 ) . the prognosis of the nsip subgroups differs ; cellular nsip responds rather well to corticosteroid treatment and it has a better prognosis than fibrotic nsip ( 15 - 17 ) . patients with uip show a more favorable prognosis than those without honeycombing ( 18 ) . however , measuring the extent of fibrosis and the volume loss of the lung parenchyma in uip patients is not so easy , and there is no widely accepted standardized scoring system ( 19 ) . moreover , some ground - glass opacities disappear on the follow up studies for the nsip patients . our results showed that a convex shape of the anterior mediastinum was due to a reduced ap dimension , and an increased transverse dimension tends to indicate a risk of nsip and uip , that is , the progression of fibrosis . though the evaluation of the mediastinum shape is not presented as a critical method or as a definitive tool for diagnosing any specific disease entity that causes pulmonary fibrosis , it could provide a straightforward and convenient way of observing the overall fibrosis in the lung parenchyma , and it could be an indirect means of determining whether the radiologic findings are transient or not . whether the convexity of the anterior mediastinum correlates well with the prognosis of idiopathic pulmonary fibrosis is not known , but this could be elucidated in a larger study . another limitation was that other conditions such as emphysema , chest wall movement and the phase of respiration , but not age and gender , might have affected the measurement of the anterior mediastinal fat , although inspiratory hrct scans were performed . in summary , the retrosternal ap and transverse dimensions of uip patients differed from those of the normal individuals , whereas the amounts of anterior mediastinal fat were similar . uip and nsip patients have a tendency to have a convex shape of their anterior mediastinal fat . therefore , the shape of the mediastinum readily adapts to the changes in lung pathology ( 1 ) . according to the results of our study , idiopathic interstitial pneumonias such as uip and nsip can change the shape of the anterior mediastinal fat . for those patients with uip , the first line treatment is corticosteroid ; thus , mediastinal widening can be the result of steroid medication or fibrous scarring . however , because we excluded the patients who had previously received corticosteroid medications , its effect could be ignored in the present study . we measured the amount of mediastinal fat at the level of the aortic arch , which is where most of the fat tissue can be consistently observed on hrct . however , for classifying the shape of anterior mediastinum into three types , we selected the level of the main pulmonary trunk because the shape of the anterior mediastinum could be more objectively and consistently determined . in some cases , the retrosternal space was so narrowed that the shape of the anterior mediastinum could not be determined at the level of aortic arch . our results showed that uip tends to be associated with a reduced ap dimension and an increased transverse dimension with the resultant convex shape of the anterior mediastinum . in uip patients , honeycombing change with volume loss usually occurs at the periphery of the lung base . we can speculate that the bilateral tensile force exerted by the subpleural fibrosis of the lung adjacent to the mediastinum could result in widening of the transverse dimension and shortening of the ap dimension of the anterior mediastinum ( fig . ( 5 ) analyzed the changes in the amounts and distributions of the anterior mediastinal fat after left upper lobectomy , as determined by ct . the postoperative anterior mediastinal fat distribution was distinctly changed with showing a marked increase from the aortic arch to the main pulmonary arterial level , with a converse decrease at the upper and lower slices , but no significant postoperative change was noted in the total anterior mediastinal fat volume . likewise , in our study , the amounts of anterior mediastinal fat in the cases of nsip and uip were not significantly different from the controls . this indicates that lung fibrosis did not induce a quantitative increase of the anterior mediastinal fat , but rather that it induces a redistribution of the mediastinal fat . the loss of volume of both lower basal lungs due to the fibrosis in uip might cause a redistribution of fat tissues and the anterior mediastinal shape changes . several studies have found that considerable overlap exists between the thin section ct patterns of nsip and uip patients , i.e. , up to one - third of nsip patients were found to have ct patterns that were similar to those of uip patients ( 12 - 14 ) . even the pathologic differential diagnosis of uip and fibrotic nsip is not straightforward ; therefore , the current gold standard involves a clinicopathologic work - up by the physicians and radiologists , with a subsequent review of the final clinicopathologic diagnosis ( 6 ) . the prognosis of the nsip subgroups differs ; cellular nsip responds rather well to corticosteroid treatment and it has a better prognosis than fibrotic nsip ( 15 - 17 ) . patients with uip show a more favorable prognosis than those without honeycombing ( 18 ) . however , measuring the extent of fibrosis and the volume loss of the lung parenchyma in uip patients is not so easy , and there is no widely accepted standardized scoring system ( 19 ) . moreover , some ground - glass opacities disappear on the follow up studies for the nsip patients . our results showed that a convex shape of the anterior mediastinum was due to a reduced ap dimension , and an increased transverse dimension tends to indicate a risk of nsip and uip , that is , the progression of fibrosis . though the evaluation of the mediastinum shape is not presented as a critical method or as a definitive tool for diagnosing any specific disease entity that causes pulmonary fibrosis , it could provide a straightforward and convenient way of observing the overall fibrosis in the lung parenchyma , and it could be an indirect means of determining whether the radiologic findings are transient or not . whether the convexity of the anterior mediastinum correlates well with the prognosis of idiopathic pulmonary fibrosis is not known , but this could be elucidated in a larger study . another limitation was that other conditions such as emphysema , chest wall movement and the phase of respiration , but not age and gender , might have affected the measurement of the anterior mediastinal fat , although inspiratory hrct scans were performed . in summary , the retrosternal ap and transverse dimensions of uip patients differed from those of the normal individuals , whereas the amounts of anterior mediastinal fat were similar . uip and nsip patients have a tendency to have a convex shape of their anterior mediastinal fat .

coronary heart disease ( chd ) is the major cause of death in american women as it is in men , but despite considerable scientific advances , much remains to be learned about the unique characteristics of this disease in women . young and middleaged women , in particular , have been underrepresented in research studies of cardiovascular disease . for over a decade now , studies have shown that young women have higher mortality and complication rates after an acute myocardial infarction ( mi ) compared to men of similar age , but these differences remain unexplained.1 there is also a concern that prevention efforts may have been less efficacious in young women in recent years.2 hospitalization rates for mi are on the rise in women 55 years , in contrast to other demographic groups,3 , 4 and the prehospital case fatality of mi5 and overall chd mortality6 have recently declined less in this segment of the population . the reasons for these variations are not clear but may be attributed , at least in part , to understudied or underrecognized risk factors . emotional stress is emerging as an important , albeit unappreciated , risk factor for cardiovascular disease in young women . psychosocial factors are powerful predictors of cardiovascular disease incidence and mortality in young women from the general population7 , 8 , 9 , 10 and of recurrent events and delayed recovery in young women with chd.11 , 12 this group is characterized by relatively high rates of economic disadvantage , depression , early life adversities , and perceived stress compared with men and older patients.11 , 12 , 13 , 14 as a whole , these data suggest a role of emotional factors on cardiovascular risk and outcome in young women , but objective evidence of this vulnerability is still insufficient . mental stress testing represents an objective , standardized method for assessing the cardiovascular effects of acute emotional stress exposure . mental stressinduced myocardial ischemia ( msimi ) can be provoked in the laboratory in up to approximately half of patients with chd and correlates with myocardial ischemia in daily life.15 msimi carries prognostic information similar to that of ischemia induced by conventional stress testing,16 despite not being related to severity of obstructive coronary disease.17 in a previous study of postmi patients , we reported that women 50 years , but not older women , had a doubling of the rate of msimi compared to agematched men.13 however , this study was based on a small sample of patients younger than 60 years , limiting our ability to assess whether young women are uniquely at risk for this phenomenon . in the current study , based on a large sample with broad age range , our goal was to investigate whether young women with chd are more likely to develop msimi than men and older patients . we further contrasted the results with a control condition of conventional stress testing ( a standard exercise or pharmacological stress test ) . between july 2009 and july 2014 , the mental stress ischemia mechanisms and prognosis study ( mips ) enrolled 695 patients with a diagnosis of stable chd from emory university affiliated hospitals and clinics . the main objectives of mips were to clarify mechanisms and prognosis of mental stressinduced ischemia assessed with myocardial perfusion imaging in patients with chd . criteria for chd included : ( 1 ) abnormal coronary angiography or intravascular ultrasound demonstrating atherosclerosis with at least luminal irregularities ; ( 2 ) previous percutaneous or surgical coronary revascularization ; ( 3 ) documented mi ; or ( 4 ) positive exercise or pharmacological nuclear stress test or electrocardiographic exercise stress test . patients were excluded if they were pregnant , if they had been hospitalized in the previous week for unstable angina , decompensated heart failure , or mi ; had severe psychiatric conditions , such as schizophrenia or current alcohol or substance abuse ; or had active malignancy or endstage renal disease or other severe medical problems expected to shorten life expectancy to < 5 years . study subjects underwent two 1day singlephoton emission computed tomography ( spect ) imaging studies , one with mental stress and one with exercise or pharmacological stress , within 1 week of each other . the emory university institutional review board approved the protocol , and all participants provided written informed consent . after 30 minutes of rest , mental stress was induced by a standardized public speaking task as previously described.17 , 18 patients were asked to imagine a reallife stressful situation , in which a close relative was been mistreated in a nursing home , and asked to make up a realistic story around this scenario . they were given 2 minutes to prepare a statement and then 3 minutes to present it in front of a video camera and an audience wearing white coats . we recorded blood pressure and heart rate at 5minute intervals during the resting phase and at 1minute intervals during the mental stress task , and calculated the ratepressure product . we also obtained subjective ratings of distress with the subjective units of distress scale19 on a linear scale of 0 to 100 . subjects underwent 3 spect myocardial perfusion imaging scans after injection of sestamibi radiolabeled with technetium99 m ( [ tc ] ) , at rest , during mental stress , and during conventional stress ( 1014 mci for rest imaging and 3040 mci for stress imaging , based on weight ) . testing was done in 2 separate days up to 1 week apart on a dedicated spect camera ( philips cardio md ) without attenuation correction . on the mental stress day , on the conventional stress day , the radioisotope injection was given at peak exertion or immediately after the regadenoson ( abbott , chicago , il ) injection for pharmacological stress testing . we assessed myocardial perfusion using a 17segment model both by the automated function in the emory cardiac toolbox ( ectb)20 and by visual analysis . we quantified severity of ischemia by the ectb as a total reversibility severity score ( sum of the number of sds in the reversibility map above the mean of a normal database ) and as total reversibility extent ( magnitude of stressinduced perfusion deficit as percent of the left ventricle [ lv ] ) . reversibility 2% is considered abnormal.21 , 22 quantitative analysis has excellent clinical and prognostic value , which is similar to expert visual analysis.22 , 23 two experienced readers ( p.r . and f.e . ) performed visual interpretation blinded to stress test type and clinical data ; disagreement was resolved by consensus . each myocardial segment was scored from 0 to 4 , with 0 being normal , 1 possibly normal , 2 definitely abnormal , 3 severely abnormal , and 4 no perfusion . we defined myocardial ischemia as a new perfusion abnormality ( a score of 2 in any segment ) , or worsening of preexisting abnormality by at least 2 points in a single segment , or by at least 1 point in 2 or more contiguous segments . in addition to individual segment scores , we calculated summed scores in a conventional fashion , including a summed stress score , a summed rest score , and a summed difference score ( sds ) , the latter representing a semiquantitative measure of inducible ischemia.23 we calculated percent ischemic myocardium as ( sds/68 ) 100 . the use of both automated and visual approach provided complementary information in our study . visual assessment is more accepted clinically , but it is not entirely blinded to the sex of the patient because of breast silhouette in visual images . the ectb , on the other hand , provides an operatorindependent assessment of perfusion abnormalities free of potential sexrelated bias . a research nurse obtained detailed sociodemographic and medical history information , including medication use , and measured weight and height to calculate the body mass index ( bmi ) . cad severity was quantified with the gensini scoring method , which uses a nonlinear point system for degree of luminal narrowing that weighs lesions according to specific coronary tree locations to take into account prognostic significance.24 we used validated instruments to assess behavioral , social , and health status information . angina symptoms were measured with the angina frequency subscale of the seattle angina questionnaire,25 which assesses chest pain over the past 4 weeks . depressive symptoms were assessed with the beck depression inventoryii,26 a reliable and valid selfreport measure that has been widely used in cardiac populations . in addition , we administered the structured clinical interview for dsm iv27 to derive a current and lifetime diagnosis of major depression and other psychiatric diagnoses . childhood traumatic experiences were assessed by means of the early trauma inventory , a validated measure of physical , emotional , and sexual abuse and general traumatic events before age 18 years.28 the score represents the number of traumatic exposures in each category . we also administered the cohen 's perceived stress scale,29 a 10item survey of general stress validated in multiethnic populations , and the statetrait anxiety inventory , a 40item questionnaire to measure anxiety as an emotional state or a personality trait.30 the main objective of the statistical analysis was to assess the role of age as an effect modifier of sex differences in proclivity to develop msimi , that is , whether there was a significant interaction between sex and age on quantitative and visual parameters of msimi . our main analysis used age as a continuous variable , with effects scaled per 10 years age decrements . we fitted linear and logistic regression models ( depending on the distribution of the response variable ) to assess sex differences in quantitative and visual indicators of ischemia and test for the interaction between sex and age after adjusting for a set of factors that were considered a priori as either possible confounders or mediators . because results across various indicators of ischemia were similar , multivariable analyses are shown for 2 representative parameters : the total reversibility severity score from the quantitative analysis and ischemia as a binary variable from the visual analysis . using hierarchical models , we sequentially adjusted for sociodemographic and lifestyle characteristics , clinical risk variables and cad severity , depressive symptoms , and medication use . in models predicting ischemia with mental stress finally , in order to more clearly describe the interaction effect , and because our previous research showed that women 50 years were especially at risk for msimi,13 we also reported the main results according to 4 decades of age : 50 years , 51 to 60 years , 6170 years , and > 70 years . in these analyses , we compared men and women in 4 age groups , for a total of 4 comparisons . using the bonferroni correction , all analyses were conducted using the statistical analysis system ( sas ; version 9.3 ; sas institute inc . , cary , nc ) . between july 2009 and july 2014 , the mental stress ischemia mechanisms and prognosis study ( mips ) enrolled 695 patients with a diagnosis of stable chd from emory university affiliated hospitals and clinics . the main objectives of mips were to clarify mechanisms and prognosis of mental stressinduced ischemia assessed with myocardial perfusion imaging in patients with chd . criteria for chd included : ( 1 ) abnormal coronary angiography or intravascular ultrasound demonstrating atherosclerosis with at least luminal irregularities ; ( 2 ) previous percutaneous or surgical coronary revascularization ; ( 3 ) documented mi ; or ( 4 ) positive exercise or pharmacological nuclear stress test or electrocardiographic exercise stress test . patients were excluded if they were pregnant , if they had been hospitalized in the previous week for unstable angina , decompensated heart failure , or mi ; had severe psychiatric conditions , such as schizophrenia or current alcohol or substance abuse ; or had active malignancy or endstage renal disease or other severe medical problems expected to shorten life expectancy to < 5 years . study subjects underwent two 1day singlephoton emission computed tomography ( spect ) imaging studies , one with mental stress and one with exercise or pharmacological stress , within 1 week of each other . the emory university institutional review board approved the protocol , and all participants provided written informed consent . after 30 minutes of rest , mental stress was induced by a standardized public speaking task as previously described.17 , 18 patients were asked to imagine a reallife stressful situation , in which a close relative was been mistreated in a nursing home , and asked to make up a realistic story around this scenario . they were given 2 minutes to prepare a statement and then 3 minutes to present it in front of a video camera and an audience wearing white coats . we recorded blood pressure and heart rate at 5minute intervals during the resting phase and at 1minute intervals during the mental stress task , and calculated the ratepressure product . we also obtained subjective ratings of distress with the subjective units of distress scale19 on a linear scale of 0 to 100 . subjects underwent 3 spect myocardial perfusion imaging scans after injection of sestamibi radiolabeled with technetium99 m ( [ tc ] ) , at rest , during mental stress , and during conventional stress ( 1014 mci for rest imaging and 3040 mci for stress imaging , based on weight ) . testing was done in 2 separate days up to 1 week apart on a dedicated spect camera ( philips cardio md ) without attenuation correction . on the mental stress day , [ tc]sestamibi was injected 1 minute after onset of the public speech task . on the conventional stress day , the radioisotope injection was given at peak exertion or immediately after the regadenoson ( abbott , chicago , il ) injection for pharmacological stress testing . we assessed myocardial perfusion using a 17segment model both by the automated function in the emory cardiac toolbox ( ectb)20 and by visual analysis . we quantified severity of ischemia by the ectb as a total reversibility severity score ( sum of the number of sds in the reversibility map above the mean of a normal database ) and as total reversibility extent ( magnitude of stressinduced perfusion deficit as percent of the left ventricle [ lv ] ) . reversibility 2% is considered abnormal.21 , 22 quantitative analysis has excellent clinical and prognostic value , which is similar to expert visual analysis.22 , 23 two experienced readers ( p.r . and f.e . ) performed visual interpretation blinded to stress test type and clinical data ; disagreement was resolved by consensus . each myocardial segment was scored from 0 to 4 , with 0 being normal , 1 possibly normal , 2 definitely abnormal , 3 severely abnormal , and 4 no perfusion . we defined myocardial ischemia as a new perfusion abnormality ( a score of 2 in any segment ) , or worsening of preexisting abnormality by at least 2 points in a single segment , or by at least 1 point in 2 or more contiguous segments . in addition to individual segment scores , we calculated summed scores in a conventional fashion , including a summed stress score , a summed rest score , and a summed difference score ( sds ) , the latter representing a semiquantitative measure of inducible ischemia.23 we calculated percent ischemic myocardium as ( sds/68 ) 100 . the use of both automated and visual approach provided complementary information in our study . visual assessment is more accepted clinically , but it is not entirely blinded to the sex of the patient because of breast silhouette in visual images . the ectb , on the other hand , provides an operatorindependent assessment of perfusion abnormalities free of potential sexrelated bias . a research nurse obtained detailed sociodemographic and medical history information , including medication use , and measured weight and height to calculate the body mass index ( bmi ) . cad severity was quantified with the gensini scoring method , which uses a nonlinear point system for degree of luminal narrowing that weighs lesions according to specific coronary tree locations to take into account prognostic significance.24 we used validated instruments to assess behavioral , social , and health status information . angina symptoms were measured with the angina frequency subscale of the seattle angina questionnaire,25 which assesses chest pain over the past 4 weeks . depressive symptoms were assessed with the beck depression inventoryii,26 a reliable and valid selfreport measure that has been widely used in cardiac populations . in addition , we administered the structured clinical interview for dsm iv27 to derive a current and lifetime diagnosis of major depression and other psychiatric diagnoses . childhood traumatic experiences were assessed by means of the early trauma inventory , a validated measure of physical , emotional , and sexual abuse and general traumatic events before age 18 years.28 the score represents the number of traumatic exposures in each category . we also administered the cohen 's perceived stress scale,29 a 10item survey of general stress validated in multiethnic populations , and the statetrait anxiety inventory , a 40item questionnaire to measure anxiety as an emotional state or a personality trait.30 the main objective of the statistical analysis was to assess the role of age as an effect modifier of sex differences in proclivity to develop msimi , that is , whether there was a significant interaction between sex and age on quantitative and visual parameters of msimi . our main analysis used age as a continuous variable , with effects scaled per 10 years age decrements . we fitted linear and logistic regression models ( depending on the distribution of the response variable ) to assess sex differences in quantitative and visual indicators of ischemia and test for the interaction between sex and age after adjusting for a set of factors that were considered a priori as either possible confounders or mediators . because results across various indicators of ischemia were similar , multivariable analyses are shown for 2 representative parameters : the total reversibility severity score from the quantitative analysis and ischemia as a binary variable from the visual analysis . using hierarchical models , we sequentially adjusted for sociodemographic and lifestyle characteristics , clinical risk variables and cad severity , depressive symptoms , and medication use . in models predicting ischemia with mental stress finally , in order to more clearly describe the interaction effect , and because our previous research showed that women 50 years were especially at risk for msimi,13 we also reported the main results according to 4 decades of age : 50 years , 51 to 60 years , 6170 years , and > 70 years . in these analyses , we compared men and women in 4 age groups , for a total of 4 comparisons . using the bonferroni correction , all analyses were conducted using the statistical analysis system ( sas ; version 9.3 ; sas institute inc . , cary , nc ) . in the enrollment period , 695 patients were enrolled ; of these , 686 participants ( 98.7% ) , 191 women and 495 men , had usable spect imaging data with mental stress and were included in the analysis . a flow chart of patient selection for the current study is shown in figure 1 . mean age was 63 years in both women and men , with a range of 34 to 79 years . women showed a more adverse psychosocial risk profile , but a similar or better clinical risk profile than men ( table 1 ) ; they were more often african american , unmarried , less educated , and with income below the poverty level . women also exhibited higher levels of symptoms of depression , anxiety and perceived stress , were more likely to meet clinical criteria for major depression , and had higher scores of emotional and sexual abuse in childhood , although physical abuse was more common in men . women reported more angina and lower functional status and more often had a history of diabetes mellitus , but other chd risk factors and previous cardiovascular history were similar . baseline imaging data were more favorable in women than in men , given that women showed fewer resting perfusion abnormalities , a higher ejection fraction , a lower angiographic cad severity score , and a lower rate of obstructive cad ( 50% lumen stenosis ) . sex differences in patient characteristics did not vary significantly by age with the exception of angina and history of sexual abuse , which were more common in younger women . most of the women 50 years or younger ( 62.5% ) were premenopausal by selfreport , whereas only 1 woman between 51 and 60 years of age and none older than 60 reported not to have yet reached menopause . diagram of patient selection for the main analysis ( n=686 ) and additional analyses in the study . sex differences in patient characteristics , and interaction between sex and age for each characteristic ace indicates angiotensinconverting enzyme ; bmi , body mass index ; cad , coronary artery disease ; chd , coronary heart disease ; lv , left ventricle . age as a continuous variable . as shown in table 2 , hemodynamic responses to mental stress were similar in women and men for both methods of reactivity calculation and across age . sex differences in changes in hemodynamic parameters and subjective distress with mental stress , and interaction between sex and age age as a continuous variable . of the 686 patients with spect imaging data with mental stress , 668 ( 97.4% ) also had usable data with conventional stress . of these , 471 ( 70.5% ) underwent a treadmill stress test and the rest underwent a pharmacological stress test . women were more likely to receive a pharmacological stress test than men ( 38.9% vs 25.9% ) . overall , the rate of inducible ischemia was higher with conventional stress ( 34.8% ) than with mental stress ( 15.9% ) . in the whole sample , women tended to show more ischemia , both with mental stress and conventional stress , compared to men ( table 3 ) . however , sex differences in ischemia with mental stress , but not conventional stress , varied by age : both quantitative and visual indicators of ischemia with mental stress showed larger differences between women and men in younger than in older patients . for each 10 years of decreasing age , the total reversibility severity score with mental stress was 9.6 incremental points higher in women , corresponding to 0.72% more lv myocardium with ischemia ( interaction , p<0.001 ) , and the incidence of msimi was 82.6% higher in women ( interaction , p=0.004 ) . an illustration of the interaction effect using age as a continuous variable is shown in figure 2 . to ease interpretation , women 50 years had elevated markers of ischemia with mental stress compared to men ; their percent lv with inducible ischemia was over 5fold that of men . expressed as presence or absence of msimi , 33% of women 50 years of age developed msimi compared to only 8% of men of similar age ( data not shown ) . not only had young women more ischemia with mental stress than men , but they also had significantly more ischemia with mental stress than older women ( p<0.001 for all quantitative parameters of reversibility ) . in fact , msimi was inversely associated with age in women , but not in men ( figures 2 and 3 ) . in contrast , perfusion data with conventional stress testing showed only modest differences between women and men with little variation by age ( table 3 ) . unadjusted sex differences in myocardial ischemia parameters , in all patients and per 10year decrement in age lv indicates left ventricle . inducible myocardial ischemia with mental stress according to sex and age as a continuous variable . young women ( 50 years ) showed more ischemia with mental stress than any of the other groups . ischemia was expressed as percent of ischemic myocardium and was derived with 2 separate methods : automated quantitative analysis ( a ) and visual analysis ( b ) . in both analyses , the interaction between sex and age was highly significant ( p<0.001 ) , and the comparison between women and men in the group 50 was also highly significant ( p<0.0001 ) . after adjusting for sociodemographic and lifestyle characteristics , clinical risk variables , cad severity , depressive symptoms , and medication use , the total reversibility severity score was 10 points higher , and the odds for msimi were more than doubled , in women compared to men for each 10year decrement in age . adjusting for ischemia with conventional stress did not substantially affect the results . multivariable analysis of the variation of the effect of sex on myocardial ischemia parameters with mental stress per 10year decrement in agea cad indicates coronary artery disease ; chd , coronary heart disease . the effects shown were calculated from the interaction between sex and age in each model . covariables were added sequentially , so that later models contain all the variables included in earlier models . race ( black vs nonblack ) , years of education , income below poverty level ( $20 000 ) , current smoking , and body mass index . history of myocardial infarction , heart failure , hypertension , dyslipidemia , diabetes mellitus , previous revascularization , resting left ventricular ejection fraction , and gensini angiographic score . beck depression inventory score , and use of betablockers , angiotensinconverting enzyme inhibitors , antidepressants , and anxiolytics . in the enrollment period , 695 patients were enrolled ; of these , 686 participants ( 98.7% ) , 191 women and 495 men , had usable spect imaging data with mental stress and were included in the analysis . a flow chart of patient selection for the current study is shown in figure 1 . mean age was 63 years in both women and men , with a range of 34 to 79 years . women showed a more adverse psychosocial risk profile , but a similar or better clinical risk profile than men ( table 1 ) ; they were more often african american , unmarried , less educated , and with income below the poverty level . women also exhibited higher levels of symptoms of depression , anxiety and perceived stress , were more likely to meet clinical criteria for major depression , and had higher scores of emotional and sexual abuse in childhood , although physical abuse was more common in men . women reported more angina and lower functional status and more often had a history of diabetes mellitus , but other chd risk factors and previous cardiovascular history were similar . baseline imaging data were more favorable in women than in men , given that women showed fewer resting perfusion abnormalities , a higher ejection fraction , a lower angiographic cad severity score , and a lower rate of obstructive cad ( 50% lumen stenosis ) . sex differences in patient characteristics did not vary significantly by age with the exception of angina and history of sexual abuse , which were more common in younger women . most of the women 50 years or younger ( 62.5% ) were premenopausal by selfreport , whereas only 1 woman between 51 and 60 years of age and none older than 60 reported not to have yet reached menopause . diagram of patient selection for the main analysis ( n=686 ) and additional analyses in the study . sex differences in patient characteristics , and interaction between sex and age for each characteristic ace indicates angiotensinconverting enzyme ; bmi , body mass index ; cad , coronary artery disease ; chd , coronary heart disease ; lv , left ventricle . age as a continuous variable . as shown in table 2 , hemodynamic responses to mental stress were similar in women and men for both methods of reactivity calculation and across age . sex differences in changes in hemodynamic parameters and subjective distress with mental stress , and interaction between sex and age age as a continuous variable . of the 686 patients with spect imaging data with mental stress , 668 ( 97.4% ) also had usable data with conventional stress . of these , 471 ( 70.5% ) underwent a treadmill stress test and the rest underwent a pharmacological stress test . women were more likely to receive a pharmacological stress test than men ( 38.9% vs 25.9% ) . overall , the rate of inducible ischemia was higher with conventional stress ( 34.8% ) than with mental stress ( 15.9% ) . in the whole sample , women tended to show more ischemia , both with mental stress and conventional stress , compared to men ( table 3 ) . however , sex differences in ischemia with mental stress , but not conventional stress , varied by age : both quantitative and visual indicators of ischemia with mental stress showed larger differences between women and men in younger than in older patients . for each 10 years of decreasing age , the total reversibility severity score with mental stress was 9.6 incremental points higher in women , corresponding to 0.72% more lv myocardium with ischemia ( interaction , p<0.001 ) , and the incidence of msimi was 82.6% higher in women ( interaction , p=0.004 ) . an illustration of the interaction effect using age as a continuous variable is shown in figure 2 . to ease interpretation , women 50 years had elevated markers of ischemia with mental stress compared to men ; their percent lv with inducible ischemia was over 5fold that of men . expressed as presence or absence of msimi , 33% of women 50 years of age developed msimi compared to only 8% of men of similar age ( data not shown ) . not only had young women more ischemia with mental stress than men , but they also had significantly more ischemia with mental stress than older women ( p<0.001 for all quantitative parameters of reversibility ) . in fact , msimi was inversely associated with age in women , but not in men ( figures 2 and 3 ) . in contrast , perfusion data with conventional stress testing showed only modest differences between women and men with little variation by age ( table 3 ) . unadjusted sex differences in myocardial ischemia parameters , in all patients and per 10year decrement in age lv indicates left ventricle . inducible myocardial ischemia with mental stress according to sex and age as a continuous variable . young women ( 50 years ) showed more ischemia with mental stress than any of the other groups . ischemia was expressed as percent of ischemic myocardium and was derived with 2 separate methods : automated quantitative analysis ( a ) and visual analysis ( b ) . in both analyses , the interaction between sex and age was highly significant ( p<0.001 ) , and the comparison between women and men in the group 50 was also highly significant ( p<0.0001 ) . after adjusting for sociodemographic and lifestyle characteristics , clinical risk variables , cad severity , depressive symptoms , and medication use , the total reversibility severity score was 10 points higher , and the odds for msimi were more than doubled , in women compared to men for each 10year decrement in age . adjusting for ischemia with conventional stress did not substantially affect the results . multivariable analysis of the variation of the effect of sex on myocardial ischemia parameters with mental stress per 10year decrement in agea cad indicates coronary artery disease ; chd , coronary heart disease . the effects shown were calculated from the interaction between sex and age in each model . covariables were added sequentially , so that later models contain all the variables included in earlier models . race ( black vs nonblack ) , years of education , income below poverty level ( $20 000 ) , current smoking , and body mass index . history of myocardial infarction , heart failure , hypertension , dyslipidemia , diabetes mellitus , previous revascularization , resting left ventricular ejection fraction , and gensini angiographic score . beck depression inventory score , and use of betablockers , angiotensinconverting enzyme inhibitors , antidepressants , and anxiolytics . in a large sample of patients with stable chd , we demonstrated large variations in the likelihood of developing msimi based on sex and age . using both automated and visual image analysis , younger women , especially those 50 years , exhibited more evidence of ischemia with mental stress than men of similar age . the incidence of msimi in this group was over 3fold higher than their male counterparts and was also higher than older women and men . notably , young women showed an elevated rate of ischemia also with conventional stress testing , but the difference with men was less marked . these results suggest a vulnerability toward msimi in women with earlyonset chd and are consistent with our previous findings in a sample of young survivors of an acute mi.13 given that most previous studies of msimi have included predominantly men,15 , 16 until recently little was known about msimi in women . in the remit trial of stable chd patients , which assessed msimi with echocardiography , women had a 39% higher rate of msimi than men.31 on the other hand , york et al.32 did not find sex differences in msimi assessed with a protocol similar to ours . ours is the first investigation with a sufficiently large sample to examine this question in a rigorous way . the precise mechanisms for the sex difference in msimi require further investigation . in our study , as in previous ones,11 , 13 , 31 women had a higher burden of psychosocial risk factors than men , which may signal a heightened physiological susceptibility to stress . hemodynamic responses to stress are not likely to play a role , however , given that women in our study did not show larger changes in blood pressure and heart rate with stress than men , similarly to previous work.31 , 32 women also did not report higher subjective distress with mental stress . others , however , have described a tendency for women to express morenegative emotions and lesspositive emotions , together with a higher platelet reactivity with mental stress , compared to men.31 a recent study reported a greater increase in depressed mood and feelings of social disconnection in healthy young women compared to men after exposure to an inflammatory challenge with endotoxin , even though the inflammatory response was similar.33 similarly , in a previous study , we found that women 50 years with a recent mi had a similar inflammatory response to stress than men , but the level of interleukin6 was 2 times higher in women than men before , during , and after the stress challenge.34 it is possible that the high absolute levels of inflammation reached during stressful events pose young women above a threshold of risk for abnormal emotional and vascular responses to stress . most women 50 years in our sample were premenopausal , whereas virtually all older women reported having reached menopause . indeed , brain regions involved in the processing of emotional stimuli , such as the amygdala , hippocampus , and prefrontal cortex , are sensitive to changes in the hormonal milieu , such as during the menstrual cycle , a process that may be affected by earlylife stress exposure.35 , 36 these same brain areas have direct or indirect outputs to neurohormonal systems involved in the stress response that , in turn , may affect vascular function and msimi . furthermore , ovarian hormones may be the basis for the higher levels of inflammatory biomarkers that young women show compared to similarly aged men starting at age 16 in the community37 and among mi patients.34 this higher baseline inflammation may affect women 's vascular responses to stress , as discussed above . moresevere disease in women with premature chd is not a likely explanation for our findings . in this study , as in our previous work,13 women showed similar or lesspronounced cad severity indicators than men and a similar level of traditional chd risk factors except for diabetes mellitus . in addition , there were no significant sex differences in ischemia provoked by a conventional stress test . what may be at play , however , is a propensity toward microvascular dysfunction.38 according to a prevailing model , msimi results primarily from insufficient dilation and/or constriction of the coronary microcirculation during stress.39 coronary microvascular dysfunction is common in women in the setting of chest pain without significant coronary obstruction,40 , 41 and young women show more endothelial dysfunction than men in response to mental stress.42 furthermore , emotional stress and mood disturbances in daily life , which are more common in women and have been linked to ambulatory ischemia and msimi,43 , 44 , 45 may lead to a chronic form of microvascular diastolic dysfunction.46 cumulative stressinduced sympathetic nervous system activation may be implicated , as suggested by the fact that patients with chd who develop msimi have increased peripheral vasoconstriction.17 these vascular effects may be accentuated in young women with chd , given their higher baseline levels of inflammation.34 our results are in tune with a recent statement from the american heart association highlighting the importance of stressful exposures and mood disorders as predisposing factors for premature chd in young populations.47 according to emerging data , young women may be especially vulnerable.7 , 8 , 9 given that msimi is associated with higher mortality in patients with chd,16 it could represent an important mechanism of risk and a novel prognostic indicator for young women with chd . because msimi correlates with myocardial ischemia and angina in daily life,15 , 48 it may contribute to the triggering of acute coronary syndromes in young women and may play a role in their higher mortality postmi compared to men.1 a limitation of our study is the relatively small number of young women , especially those below age 50 years ; yet , this remains the largest investigation ever conducted of msimi in women . the extent of inducible ischemia with mental stress was overall relatively mild ; however , women 50 years had on average around 3% of their myocardium with inducible ischemia with mental stress , a level that has clinical significance.21 , 22 furthermore , one third of these young women were classified as having ischemia according to accepted clinical criteria . nonetheless , future outcomes studies are needed to confirm the clinical significance of msimi in women , given that previous studies have primarily included men.16 only 1 stress task is feasible with [ tc]sestamibi spect perfusion imaging , and this could be seen as an additional limitation . however , multiple mental stress tasks with short resting periods in between may not be more informative than just 1 task , because of possible carryover effects from one condition to the next.49 , 50 the use of myocardial perfusion imaging is a strength of our study , because it remains the gold standard for ischemia assessment . furthermore , a major advantage of this technique for mental stress testing is that the radioisotope , once injected during mental stress , is trapped in the myocyte , providing a among patients with chd , we found significant differences in susceptibility to myocardial ischemia triggered by emotional stress according to sex and age , with young women showing more evidence of msimi . future investigations should examine the role of msimi in the prognosis and etiology of chd in women . these data may inform interventions specifically designed to address women 's stressors and reduce their burden of ischemic heart disease . this work was supported by the nih ( p01 hl101398 , r01 hl109413 , r01hl10941302s1 , k24hl077506 , k24 mh076955 , ul1tr000454 , and kl2tr000455 ) . dr ernest v. garcia receives royalties from the sale of the emory cardiac toolbox related to the quantitative tools used in this article . the terms of this arrangement have been reviewed and approved by emory university in accord with its conflictofinterest practice . none of the other authors have disclosures of potential conflicts of interest relevant to this article .

backgroundemerging data suggest that young women with coronary heart disease ( chd ) are disproportionally vulnerable to the adverse cardiovascular effects of psychological stress . we hypothesized that younger , but not older , women with stable chd are more likely than their male peers to develop mental stressinduced myocardial ischemia ( msimi).methods and resultswe studied 686 patients ( 191 women ) with stable coronary heart disease ( chd ) . patients underwent 99mtcsestamibi myocardial perfusion imaging at rest and with both mental ( speech task ) and conventional ( exercise / pharmacological ) stress testing . we compared quantitative ( by automated software ) and visual parameters of inducible ischemia between women and men and assessed age as an effect modifier . women had a moreadverse psychosocial profile than men whereas there were few differences in medical history and chd risk factors . both quantitative and visual indicators of ischemia with mental stress were disproportionally larger in younger women . for each 10 years of decreasing age , the total reversibility severity score with mental stress was 9.6 incremental points higher ( interaction , p<0.001 ) and the incidence of msimi was 82.6% higher ( interaction , p=0.004 ) in women than in men . incidence of msimi in women 50 years was almost 4fold higher than in men of similar age and older patients . these results persisted when adjusting for sociodemographic and medical risk factors , psychosocial factors , and medications . there were no significant sex differences in inducible ischemia with conventional stress.conclusionsyoung women with stable chd are susceptible to msimi , which could play a role in the prognosis of this group .

Introduction Methods Subjects and Design Mental Stress Procedure Myocardial Perfusion Imaging Other Measurements Statistical Analysis Results Study Sample Hemodynamic Reactivity Myocardial Perfusion Discussion Conclusion Sources of Funding Disclosures

coronary heart disease ( chd ) is the major cause of death in american women as it is in men , but despite considerable scientific advances , much remains to be learned about the unique characteristics of this disease in women . for over a decade now , studies have shown that young women have higher mortality and complication rates after an acute myocardial infarction ( mi ) compared to men of similar age , but these differences remain unexplained.1 there is also a concern that prevention efforts may have been less efficacious in young women in recent years.2 hospitalization rates for mi are on the rise in women 55 years , in contrast to other demographic groups,3 , 4 and the prehospital case fatality of mi5 and overall chd mortality6 have recently declined less in this segment of the population . psychosocial factors are powerful predictors of cardiovascular disease incidence and mortality in young women from the general population7 , 8 , 9 , 10 and of recurrent events and delayed recovery in young women with chd.11 , 12 this group is characterized by relatively high rates of economic disadvantage , depression , early life adversities , and perceived stress compared with men and older patients.11 , 12 , 13 , 14 as a whole , these data suggest a role of emotional factors on cardiovascular risk and outcome in young women , but objective evidence of this vulnerability is still insufficient . mental stressinduced myocardial ischemia ( msimi ) can be provoked in the laboratory in up to approximately half of patients with chd and correlates with myocardial ischemia in daily life.15 msimi carries prognostic information similar to that of ischemia induced by conventional stress testing,16 despite not being related to severity of obstructive coronary disease.17 in a previous study of postmi patients , we reported that women 50 years , but not older women , had a doubling of the rate of msimi compared to agematched men.13 however , this study was based on a small sample of patients younger than 60 years , limiting our ability to assess whether young women are uniquely at risk for this phenomenon . in the current study , based on a large sample with broad age range , our goal was to investigate whether young women with chd are more likely to develop msimi than men and older patients . subjects underwent 3 spect myocardial perfusion imaging scans after injection of sestamibi radiolabeled with technetium99 m ( [ tc ] ) , at rest , during mental stress , and during conventional stress ( 1014 mci for rest imaging and 3040 mci for stress imaging , based on weight ) . we quantified severity of ischemia by the ectb as a total reversibility severity score ( sum of the number of sds in the reversibility map above the mean of a normal database ) and as total reversibility extent ( magnitude of stressinduced perfusion deficit as percent of the left ventricle [ lv ] ) . we also administered the cohen 's perceived stress scale,29 a 10item survey of general stress validated in multiethnic populations , and the statetrait anxiety inventory , a 40item questionnaire to measure anxiety as an emotional state or a personality trait.30 the main objective of the statistical analysis was to assess the role of age as an effect modifier of sex differences in proclivity to develop msimi , that is , whether there was a significant interaction between sex and age on quantitative and visual parameters of msimi . we fitted linear and logistic regression models ( depending on the distribution of the response variable ) to assess sex differences in quantitative and visual indicators of ischemia and test for the interaction between sex and age after adjusting for a set of factors that were considered a priori as either possible confounders or mediators . because results across various indicators of ischemia were similar , multivariable analyses are shown for 2 representative parameters : the total reversibility severity score from the quantitative analysis and ischemia as a binary variable from the visual analysis . in models predicting ischemia with mental stress finally , in order to more clearly describe the interaction effect , and because our previous research showed that women 50 years were especially at risk for msimi,13 we also reported the main results according to 4 decades of age : 50 years , 51 to 60 years , 6170 years , and > 70 years . subjects underwent 3 spect myocardial perfusion imaging scans after injection of sestamibi radiolabeled with technetium99 m ( [ tc ] ) , at rest , during mental stress , and during conventional stress ( 1014 mci for rest imaging and 3040 mci for stress imaging , based on weight ) . we quantified severity of ischemia by the ectb as a total reversibility severity score ( sum of the number of sds in the reversibility map above the mean of a normal database ) and as total reversibility extent ( magnitude of stressinduced perfusion deficit as percent of the left ventricle [ lv ] ) . we also administered the cohen 's perceived stress scale,29 a 10item survey of general stress validated in multiethnic populations , and the statetrait anxiety inventory , a 40item questionnaire to measure anxiety as an emotional state or a personality trait.30 the main objective of the statistical analysis was to assess the role of age as an effect modifier of sex differences in proclivity to develop msimi , that is , whether there was a significant interaction between sex and age on quantitative and visual parameters of msimi . we fitted linear and logistic regression models ( depending on the distribution of the response variable ) to assess sex differences in quantitative and visual indicators of ischemia and test for the interaction between sex and age after adjusting for a set of factors that were considered a priori as either possible confounders or mediators . because results across various indicators of ischemia were similar , multivariable analyses are shown for 2 representative parameters : the total reversibility severity score from the quantitative analysis and ischemia as a binary variable from the visual analysis . in models predicting ischemia with mental stress finally , in order to more clearly describe the interaction effect , and because our previous research showed that women 50 years were especially at risk for msimi,13 we also reported the main results according to 4 decades of age : 50 years , 51 to 60 years , 6170 years , and > 70 years . in the enrollment period , 695 patients were enrolled ; of these , 686 participants ( 98.7% ) , 191 women and 495 men , had usable spect imaging data with mental stress and were included in the analysis . women showed a more adverse psychosocial risk profile , but a similar or better clinical risk profile than men ( table 1 ) ; they were more often african american , unmarried , less educated , and with income below the poverty level . baseline imaging data were more favorable in women than in men , given that women showed fewer resting perfusion abnormalities , a higher ejection fraction , a lower angiographic cad severity score , and a lower rate of obstructive cad ( 50% lumen stenosis ) . sex differences in patient characteristics did not vary significantly by age with the exception of angina and history of sexual abuse , which were more common in younger women . sex differences in patient characteristics , and interaction between sex and age for each characteristic ace indicates angiotensinconverting enzyme ; bmi , body mass index ; cad , coronary artery disease ; chd , coronary heart disease ; lv , left ventricle . sex differences in changes in hemodynamic parameters and subjective distress with mental stress , and interaction between sex and age age as a continuous variable . of the 686 patients with spect imaging data with mental stress , 668 ( 97.4% ) also had usable data with conventional stress . overall , the rate of inducible ischemia was higher with conventional stress ( 34.8% ) than with mental stress ( 15.9% ) . in the whole sample , women tended to show more ischemia , both with mental stress and conventional stress , compared to men ( table 3 ) . however , sex differences in ischemia with mental stress , but not conventional stress , varied by age : both quantitative and visual indicators of ischemia with mental stress showed larger differences between women and men in younger than in older patients . for each 10 years of decreasing age , the total reversibility severity score with mental stress was 9.6 incremental points higher in women , corresponding to 0.72% more lv myocardium with ischemia ( interaction , p<0.001 ) , and the incidence of msimi was 82.6% higher in women ( interaction , p=0.004 ) . to ease interpretation , women 50 years had elevated markers of ischemia with mental stress compared to men ; their percent lv with inducible ischemia was over 5fold that of men . expressed as presence or absence of msimi , 33% of women 50 years of age developed msimi compared to only 8% of men of similar age ( data not shown ) . not only had young women more ischemia with mental stress than men , but they also had significantly more ischemia with mental stress than older women ( p<0.001 for all quantitative parameters of reversibility ) . in fact , msimi was inversely associated with age in women , but not in men ( figures 2 and 3 ) . in contrast , perfusion data with conventional stress testing showed only modest differences between women and men with little variation by age ( table 3 ) . inducible myocardial ischemia with mental stress according to sex and age as a continuous variable . young women ( 50 years ) showed more ischemia with mental stress than any of the other groups . in both analyses , the interaction between sex and age was highly significant ( p<0.001 ) , and the comparison between women and men in the group 50 was also highly significant ( p<0.0001 ) . after adjusting for sociodemographic and lifestyle characteristics , clinical risk variables , cad severity , depressive symptoms , and medication use , the total reversibility severity score was 10 points higher , and the odds for msimi were more than doubled , in women compared to men for each 10year decrement in age . multivariable analysis of the variation of the effect of sex on myocardial ischemia parameters with mental stress per 10year decrement in agea cad indicates coronary artery disease ; chd , coronary heart disease . in the enrollment period , 695 patients were enrolled ; of these , 686 participants ( 98.7% ) , 191 women and 495 men , had usable spect imaging data with mental stress and were included in the analysis . women showed a more adverse psychosocial risk profile , but a similar or better clinical risk profile than men ( table 1 ) ; they were more often african american , unmarried , less educated , and with income below the poverty level . baseline imaging data were more favorable in women than in men , given that women showed fewer resting perfusion abnormalities , a higher ejection fraction , a lower angiographic cad severity score , and a lower rate of obstructive cad ( 50% lumen stenosis ) . sex differences in patient characteristics did not vary significantly by age with the exception of angina and history of sexual abuse , which were more common in younger women . sex differences in patient characteristics , and interaction between sex and age for each characteristic ace indicates angiotensinconverting enzyme ; bmi , body mass index ; cad , coronary artery disease ; chd , coronary heart disease ; lv , left ventricle . as shown in table 2 , hemodynamic responses to mental stress were similar in women and men for both methods of reactivity calculation and across age . sex differences in changes in hemodynamic parameters and subjective distress with mental stress , and interaction between sex and age age as a continuous variable . of the 686 patients with spect imaging data with mental stress , 668 ( 97.4% ) also had usable data with conventional stress . overall , the rate of inducible ischemia was higher with conventional stress ( 34.8% ) than with mental stress ( 15.9% ) . in the whole sample , women tended to show more ischemia , both with mental stress and conventional stress , compared to men ( table 3 ) . however , sex differences in ischemia with mental stress , but not conventional stress , varied by age : both quantitative and visual indicators of ischemia with mental stress showed larger differences between women and men in younger than in older patients . for each 10 years of decreasing age , the total reversibility severity score with mental stress was 9.6 incremental points higher in women , corresponding to 0.72% more lv myocardium with ischemia ( interaction , p<0.001 ) , and the incidence of msimi was 82.6% higher in women ( interaction , p=0.004 ) . to ease interpretation , women 50 years had elevated markers of ischemia with mental stress compared to men ; their percent lv with inducible ischemia was over 5fold that of men . expressed as presence or absence of msimi , 33% of women 50 years of age developed msimi compared to only 8% of men of similar age ( data not shown ) . not only had young women more ischemia with mental stress than men , but they also had significantly more ischemia with mental stress than older women ( p<0.001 for all quantitative parameters of reversibility ) . in fact , msimi was inversely associated with age in women , but not in men ( figures 2 and 3 ) . in contrast , perfusion data with conventional stress testing showed only modest differences between women and men with little variation by age ( table 3 ) . inducible myocardial ischemia with mental stress according to sex and age as a continuous variable . in both analyses , the interaction between sex and age was highly significant ( p<0.001 ) , and the comparison between women and men in the group 50 was also highly significant ( p<0.0001 ) . after adjusting for sociodemographic and lifestyle characteristics , clinical risk variables , cad severity , depressive symptoms , and medication use , the total reversibility severity score was 10 points higher , and the odds for msimi were more than doubled , in women compared to men for each 10year decrement in age . multivariable analysis of the variation of the effect of sex on myocardial ischemia parameters with mental stress per 10year decrement in agea cad indicates coronary artery disease ; chd , coronary heart disease . using both automated and visual image analysis , younger women , especially those 50 years , exhibited more evidence of ischemia with mental stress than men of similar age . the incidence of msimi in this group was over 3fold higher than their male counterparts and was also higher than older women and men . notably , young women showed an elevated rate of ischemia also with conventional stress testing , but the difference with men was less marked . these results suggest a vulnerability toward msimi in women with earlyonset chd and are consistent with our previous findings in a sample of young survivors of an acute mi.13 given that most previous studies of msimi have included predominantly men,15 , 16 until recently little was known about msimi in women . in the remit trial of stable chd patients , which assessed msimi with echocardiography , women had a 39% higher rate of msimi than men.31 on the other hand , york et al.32 did not find sex differences in msimi assessed with a protocol similar to ours . hemodynamic responses to stress are not likely to play a role , however , given that women in our study did not show larger changes in blood pressure and heart rate with stress than men , similarly to previous work.31 , 32 women also did not report higher subjective distress with mental stress . others , however , have described a tendency for women to express morenegative emotions and lesspositive emotions , together with a higher platelet reactivity with mental stress , compared to men.31 a recent study reported a greater increase in depressed mood and feelings of social disconnection in healthy young women compared to men after exposure to an inflammatory challenge with endotoxin , even though the inflammatory response was similar.33 similarly , in a previous study , we found that women 50 years with a recent mi had a similar inflammatory response to stress than men , but the level of interleukin6 was 2 times higher in women than men before , during , and after the stress challenge.34 it is possible that the high absolute levels of inflammation reached during stressful events pose young women above a threshold of risk for abnormal emotional and vascular responses to stress . in addition , there were no significant sex differences in ischemia provoked by a conventional stress test . what may be at play , however , is a propensity toward microvascular dysfunction.38 according to a prevailing model , msimi results primarily from insufficient dilation and/or constriction of the coronary microcirculation during stress.39 coronary microvascular dysfunction is common in women in the setting of chest pain without significant coronary obstruction,40 , 41 and young women show more endothelial dysfunction than men in response to mental stress.42 furthermore , emotional stress and mood disturbances in daily life , which are more common in women and have been linked to ambulatory ischemia and msimi,43 , 44 , 45 may lead to a chronic form of microvascular diastolic dysfunction.46 cumulative stressinduced sympathetic nervous system activation may be implicated , as suggested by the fact that patients with chd who develop msimi have increased peripheral vasoconstriction.17 these vascular effects may be accentuated in young women with chd , given their higher baseline levels of inflammation.34 our results are in tune with a recent statement from the american heart association highlighting the importance of stressful exposures and mood disorders as predisposing factors for premature chd in young populations.47 according to emerging data , young women may be especially vulnerable.7 , 8 , 9 given that msimi is associated with higher mortality in patients with chd,16 it could represent an important mechanism of risk and a novel prognostic indicator for young women with chd . because msimi correlates with myocardial ischemia and angina in daily life,15 , 48 it may contribute to the triggering of acute coronary syndromes in young women and may play a role in their higher mortality postmi compared to men.1 a limitation of our study is the relatively small number of young women , especially those below age 50 years ; yet , this remains the largest investigation ever conducted of msimi in women . the extent of inducible ischemia with mental stress was overall relatively mild ; however , women 50 years had on average around 3% of their myocardium with inducible ischemia with mental stress , a level that has clinical significance.21 , 22 furthermore , one third of these young women were classified as having ischemia according to accepted clinical criteria . nonetheless , future outcomes studies are needed to confirm the clinical significance of msimi in women , given that previous studies have primarily included men.16 only 1 stress task is feasible with [ tc]sestamibi spect perfusion imaging , and this could be seen as an additional limitation . furthermore , a major advantage of this technique for mental stress testing is that the radioisotope , once injected during mental stress , is trapped in the myocyte , providing a among patients with chd , we found significant differences in susceptibility to myocardial ischemia triggered by emotional stress according to sex and age , with young women showing more evidence of msimi . future investigations should examine the role of msimi in the prognosis and etiology of chd in women .

our cohort consisted of 50 patients below 30 years of age diagnosed with localized high - grade osteosarcoma of the extremity between january 2000 and december 2010 that were treated by members of the uthscsa sarcoma team . patients with axial primaries or metastatic disease at diagnosis were excluded from this analysis . during this time frame hospital and clinic records from university hospital , christus santa rosa children s hospital , and the cancer therapy and research center were reviewed . a retrospective analysis of patient demographics ( age at diagnosis , sex , date of diagnosis , race , and ethnicity ) , presence of predisposing factors , socioeconomic status ( based on family income obtained from institutional survey ) , and tumor characteristics ( location , histology , tumor volume , response to neoadjuvant chemotherapy , and type of primary surgery ) was performed . ethnicity was assigned based on parental report , and the national cancer institute / children s oncology group ( cog ) definitions . according to this convention , the term hispanic can include mexican americans , south americans , or cubans but our population of hispanics was exclusively mexican american . atv was defined as absolute tumor length ( atl)absolute tumor width ( atw)absolute tumor depth ( atd ) ( atv in cm = atlatwatd0.52 ) . the project was conducted after approval was received from the institutional review board at the respective institution . the chemotherapy regimens , specifically dosing , were based on body surface area and the 2 groups received equivalent treatment either on study or according to study protocol based on either aost0331 or pog9754 depending on when the patient was diagnosed . the chemotherapy included combinations of methotrexate , cisplatin , adriamycin , ifosfamide , and etoposide ; although not all agents were used in each patient . all 50 patients underwent surgery for local control and had negative surgical margins as confirmed by pathology . the type of surgery ( limb salvage , amputation , rotationplasty ) was determined by the extent of disease , involvement of neurovascular bundle , presence of pathologic fracture , and appraisal for best limb functionality after surgical resection . the resected specimens were examined for percentage of tumor necrosis in response to neoadjuvant chemotherapy and were assigned a grade of 1 to 6 as defined by salzer - kuntschik et al.4 in this grading system , grade 1 signifies no viable tumor ; grade 2 , solitary live cells or 1 islet of live cells < 0.5 cm ; grade 3 , < 10% viable cells ; grade 4 , 10% to 50% viable cells ; grade 5 , > 50% alive tumor ; and grade 6 , 100% viable tumor . this system for tumor necrosis grading was chosen to standardize the 2 different grading systems that were used in the individual pog9754 and aost0331 studies . longitudinal tumor assessment ( primary site and lungs ) was performed according to pog9754 or aost0331 recommendations . efs was calculated from the date of diagnosis until recurrence , secondary malignancy , death , or most recent follow - up examination showing absence of an event . os was calculated from the date of diagnosis to death or most recent follow - up examination . the survival curves were calculated using the kaplan - meier method and the differences of survival curves were assessed using the log - rank test . adjusted estimates were obtained from proportional hazards models with age , sex , ethnicity , family income , tumor volume , and tumor necrosis included as covariates . power analysis was completed and all statistical testing was 2-sided with a significance level of 5% . our cohort consisted of 50 patients below 30 years of age diagnosed with localized high - grade osteosarcoma of the extremity between january 2000 and december 2010 that were treated by members of the uthscsa sarcoma team . patients with axial primaries or metastatic disease at diagnosis were excluded from this analysis . during this time frame hospital and clinic records from university hospital , christus santa rosa children s hospital , and the cancer therapy and research center were reviewed . a retrospective analysis of patient demographics ( age at diagnosis , sex , date of diagnosis , race , and ethnicity ) , presence of predisposing factors , socioeconomic status ( based on family income obtained from institutional survey ) , and tumor characteristics ( location , histology , tumor volume , response to neoadjuvant chemotherapy , and type of primary surgery ) was performed . ethnicity was assigned based on parental report , and the national cancer institute / children s oncology group ( cog ) definitions . according to this convention , the term hispanic can include mexican americans , south americans , or cubans but our population of hispanics was exclusively mexican american . atv was defined as absolute tumor length ( atl)absolute tumor width ( atw)absolute tumor depth ( atd ) ( atv in cm = atlatwatd0.52 ) . the project was conducted after approval was received from the institutional review board at the respective institution . the chemotherapy regimens , specifically dosing , were based on body surface area and the 2 groups received equivalent treatment either on study or according to study protocol based on either aost0331 or pog9754 depending on when the patient was diagnosed . the chemotherapy included combinations of methotrexate , cisplatin , adriamycin , ifosfamide , and etoposide ; although not all agents were used in each patient . all 50 patients underwent surgery for local control and had negative surgical margins as confirmed by pathology . the type of surgery ( limb salvage , amputation , rotationplasty ) was determined by the extent of disease , involvement of neurovascular bundle , presence of pathologic fracture , and appraisal for best limb functionality after surgical resection . the resected specimens were examined for percentage of tumor necrosis in response to neoadjuvant chemotherapy and were assigned a grade of 1 to 6 as defined by salzer - kuntschik et al.4 in this grading system , grade 1 signifies no viable tumor ; grade 2 , solitary live cells or 1 islet of live cells < 0.5 cm ; grade 3 , < 10% viable cells ; grade 4 , 10% to 50% viable cells ; grade 5 , > 50% alive tumor ; and grade 6 , 100% viable tumor . this system for tumor necrosis grading was chosen to standardize the 2 different grading systems that were used in the individual pog9754 and aost0331 studies . longitudinal tumor assessment ( primary site and lungs ) was performed according to pog9754 or aost0331 recommendations . efs was calculated from the date of diagnosis until recurrence , secondary malignancy , death , or most recent follow - up examination showing absence of an event . os was calculated from the date of diagnosis to death or most recent follow - up examination . the survival curves were calculated using the kaplan - meier method and the differences of survival curves were assessed using the log - rank test . adjusted estimates were obtained from proportional hazards models with age , sex , ethnicity , family income , tumor volume , and tumor necrosis included as covariates . power analysis was completed and all statistical testing was 2-sided with a significance level of 5% . fifty patients with localized osteosarcoma of the extremity below 30 years of age were diagnosed between january 2000 and december 2010 at uthscsa . the cohort was composed of 35 hispanics ( 70% ) , 10 whites ( 20% ) , 4 blacks ( 8% ) , and 1 other ( 2% ) . ethnicity was assigned based on parental report , and the national cancer institute / cog definitions . therefore , although our cohort is comprised of mainly hispanics , it is not exclusively hispanics . however , as described in the methods section our hispanic population is uniquely composed of only mexican americans , whereas most of the studies that include hispanics are more heterogenous . the mean age at diagnosis was 15 years ( range , 2 to 28 y ) . there was no clinical or statistical correlation between socioeconomic status data and tumor size or extent of disease at presentation . tumor location was as follows : 41 ( 82% ) in the lower extremity and 9 ( 18% ) in the upper extremity . eighteen ( 53% ) patients had a tumor volume > 150 cm and 16 ( 47% ) patients had a tumor volume 150 cm . no significant association could be found between hispanic ethnicity and large tumor size ( p=0.73 ) . all patients had surgery for local control ; 60% had ablative surgery ( amputation , disarticulation , or rotationplasty ) , whereas 40% had limb salvage procedures . we acknowledge that this is an increased percentage of patients undergoing ablative procedures but this was due to the disproportionate number of patients with joint and neurovascular bundle involvement . seventeen ( 42% ) patients were good responders with grades 1 to 3 necrosis ( > 90% ) . twelve ( 30% ) patients had grade 4 necrosis ( between 50% and 90% ) and 11 ( 28% ) patients had a very poor response ( < 50% necrosis ) including 2 patients with 100% viable tumor after neoadjuvant chemotherapy . although the cohort of patients for which histologic response was available was 70% hispanic , 10/11 ( 91% ) of the patients with a very poor response were hispanics . the os at 3 and 5 years was 73% and 65% , respectively , for the entire cohort . eleven patients diagnosed before the age of 12 experienced a statistically significant decreased 5-year efs and os relative to those diagnosed between the ages of 12 and 29 ( 11% vs. 57% , respectively , p=<0.001 for efs and 25% vs. 76% , respectively , p=<0.001 for os ) ( table 2 ) . there was no statistically significant difference in outcomes based on ethnicity , income , or tumor volume . patient characteristics and univariate analysis although response to neoadjuvant chemotherapy has traditionally been used as a prognostic marker for both efs and os , it was of prognostic significance only for efs ( table 2 and figs . 1 , 2 ) in our population . we observed an increased percentage of patients with < 50% necrosis after neoadjuvant chemotherapy and chose to grade necrosis based on 6 categories as defined by salzer - kuntschik et al4 to further categorize the poor responders . the 5-year efs when compared between groups categorized by grades 1 to 3 , grade 4 , and grades 5 to 6 necrosis showed a statistically significant decreased outcome in patients with grades 5 to 6 necrosis ( 61% vs. 42% vs. 21% , respectively , p=0.03 ) . the 5-year os for these patients was similar across the groups suggesting that our patient population was salvageable after initial relapse . event - free survival ( efs ) of localized / extremity tumor patients based on tumor necrosis . the 5-year efs for patients with grades 5 to 6 was significantly different than the 5-year efs of patients with grades 1 to 3 and grade 4 tumor necrosis ( p=0.03 ) . overall survival ( os ) of localized / extremity tumor patients based on tumor necrosis . there was no difference in the location of relapse when comparing the preadolescent patients to the older patients ( young patients [ n=9 ] : 22% , local ; 55% , lung ; 22% , lung / local ; older patients [ n=14 ] : 21% , local ; 57% , lung ; 21% , lung / local ) . overall , 4 patients experienced a local recurrence , 5 had combined local / lung recurrence , and 1 patient had progressive disease , meaning nonresponsive to neoadjuvant chemotherapy . the mean time to relapse was 19 months ( range , 2 to 37 mo ) . salvage treatment included surgical resection for the majority of patients , with many receiving postoperative chemotherapy . eight of the 13 patients with isolated lung recurrences and 4 of the 10 with other types of recurrence / progression were successfully salvaged for an overall salvage rate of 52% for first relapses . multivariate cox regression analysis was performed to assess the association between efs or os and predictors , such as age , ethnicity , income , tumor volume , and tumor necrosis . patients below 12 years of age at diagnosis experienced a higher rate of relapse and death relative to those 12 to 29 years of age at diagnosis ( hazard ratio [ hr ] , 4.77 ; 95% ci , 2.03 - 11.17 ; p=<0.001 for efs ; hr , 5.23 ; 95% ci , 1.86 - 14.59 ; p=0.002 for os ) . grades 5 to 6 necrosis after neoadjuvant chemotherapy was significantly predictive of decreased efs ( hr , 3.76 ; 95% ci , 1.29 - 10.94 ; p=0.02 ) and there was a trend toward lower os ( hr , 3.45 ; 95% ci , 0.91 - 12.99 ; p=0.07 ) . none of ethnicity , income , or tumor volume was significantly correlated with either efs or os . fifty patients with localized osteosarcoma of the extremity below 30 years of age were diagnosed between january 2000 and december 2010 at uthscsa . the cohort was composed of 35 hispanics ( 70% ) , 10 whites ( 20% ) , 4 blacks ( 8% ) , and 1 other ( 2% ) . ethnicity was assigned based on parental report , and the national cancer institute / cog definitions . therefore , although our cohort is comprised of mainly hispanics , it is not exclusively hispanics . however , as described in the methods section our hispanic population is uniquely composed of only mexican americans , whereas most of the studies that include hispanics are more heterogenous . the mean age at diagnosis was 15 years ( range , 2 to 28 y ) . there was no clinical or statistical correlation between socioeconomic status data and tumor size or extent of disease at presentation . all patients presented with localized disease of an extremity . tumor location was as follows : 41 ( 82% ) in the lower extremity and 9 ( 18% ) in the upper extremity . eighteen ( 53% ) patients had a tumor volume > 150 cm and 16 ( 47% ) patients had a tumor volume 150 cm . no significant association could be found between hispanic ethnicity and large tumor size ( p=0.73 ) . all patients had surgery for local control ; 60% had ablative surgery ( amputation , disarticulation , or rotationplasty ) , whereas 40% had limb salvage procedures . we acknowledge that this is an increased percentage of patients undergoing ablative procedures but this was due to the disproportionate number of patients with joint and neurovascular bundle involvement . seventeen ( 42% ) patients were good responders with grades 1 to 3 necrosis ( > 90% ) . twelve ( 30% ) patients had grade 4 necrosis ( between 50% and 90% ) and 11 ( 28% ) patients had a very poor response ( < 50% necrosis ) including 2 patients with 100% viable tumor after neoadjuvant chemotherapy . although the cohort of patients for which histologic response was available was 70% hispanic , 10/11 ( 91% ) of the patients with a very poor response were hispanics . the os at 3 and 5 years was 73% and 65% , respectively , for the entire cohort . eleven patients diagnosed before the age of 12 experienced a statistically significant decreased 5-year efs and os relative to those diagnosed between the ages of 12 and 29 ( 11% vs. 57% , respectively , p=<0.001 for efs and 25% vs. 76% , respectively , p=<0.001 for os ) ( table 2 ) . there was no statistically significant difference in outcomes based on ethnicity , income , or tumor volume . patient characteristics and univariate analysis although response to neoadjuvant chemotherapy has traditionally been used as a prognostic marker for both efs and os , it was of prognostic significance only for efs ( table 2 and figs . 1 , 2 ) in our population . we observed an increased percentage of patients with < 50% necrosis after neoadjuvant chemotherapy and chose to grade necrosis based on 6 categories as defined by salzer - kuntschik et al4 to further categorize the poor responders . the 5-year efs when compared between groups categorized by grades 1 to 3 , grade 4 , and grades 5 to 6 necrosis showed a statistically significant decreased outcome in patients with grades 5 to 6 necrosis ( 61% vs. 42% vs. 21% , respectively , p=0.03 ) . the 5-year os for these patients was similar across the groups suggesting that our patient population was salvageable after initial relapse . event - free survival ( efs ) of localized / extremity tumor patients based on tumor necrosis . the 5-year efs for patients with grades 5 to 6 was significantly different than the 5-year efs of patients with grades 1 to 3 and grade 4 tumor necrosis ( p=0.03 ) . overall survival ( os ) of localized / extremity tumor patients based on tumor necrosis . there was no difference in the location of relapse when comparing the preadolescent patients to the older patients ( young patients [ n=9 ] : 22% , local ; 55% , lung ; 22% , lung / local ; older patients [ n=14 ] : 21% , local ; 57% , lung ; 21% , lung / local ) . overall , 4 patients experienced a local recurrence , 5 had combined local / lung recurrence , and 1 patient had progressive disease , meaning nonresponsive to neoadjuvant chemotherapy . the mean time to relapse was 19 months ( range , 2 to 37 mo ) . salvage treatment included surgical resection for the majority of patients , with many receiving postoperative chemotherapy . eight of the 13 patients with isolated lung recurrences and 4 of the 10 with other types of recurrence / progression were successfully salvaged for an overall salvage rate of 52% for first relapses . multivariate cox regression analysis was performed to assess the association between efs or os and predictors , such as age , ethnicity , income , tumor volume , and tumor necrosis . patients below 12 years of age at diagnosis experienced a higher rate of relapse and death relative to those 12 to 29 years of age at diagnosis ( hazard ratio [ hr ] , 4.77 ; 95% ci , 2.03 - 11.17 ; p=<0.001 for efs ; hr , 5.23 ; 95% ci , 1.86 - 14.59 ; p=0.002 for os ) . grades 5 to 6 necrosis after neoadjuvant chemotherapy was significantly predictive of decreased efs ( hr , 3.76 ; 95% ci , 1.29 - 10.94 ; p=0.02 ) and there was a trend toward lower os ( hr , 3.45 ; 95% ci , 0.91 - 12.99 ; p=0.07 ) . none of ethnicity , income , or tumor volume was significantly correlated with either efs or os . the population of localized osteosarcoma of the extremity patients treated at uthscsa was 70% hispanic ( 35/50 ) , homogenously of mexican american ancestry , giving us a unique cohort to study . of the 50 patients in our cohort , 11 were categorized as preadolescent ( below 12 y of age at diagnosis ) . nine of the 35 mexican american patients were preadolescents at diagnosis and to the best of our knowledge this is the largest series composed of such patients . in this study , the 5-year efs of 48% was similar to results from the brazilian osteosarcoma treatment group who reported a 5-year efs of only 53%.5 in this cohort , the 5-year efs was inferior to a smaller study completed on patients in low - income countries in latin america.6 these data suggest that there might be some similarities and differences in outcome among other hispanic groups . although the 5-year os of 65% was comparable with that reported by large european groups,2,7 the efs in this cohort was lower.2,812 in this regard , the data indicated that our patients had a higher risk of relapse after primary treatment , but were salvageable as denoted by os rates . notably , our predominantly mexican american preadolescent patients ( below 12 y of age at diagnosis of which 9/11 are hispanic ) had an overall poor outcome with 5-year efs of 11% and a 5-year os of 25% . in stark contrast , many published studies report the outcome of preadolescent patients to be equivalent to those diagnosed after puberty.1317 bacci et al14 reported results on a large cohort of patients treated on protocol between 1972 and 1999 , comparing the outcome of patients aged 12 years and below at diagnosis to that of patients between 13 and 40 years of age . the 5-year efs is 60% versus 58% in favor of the preadolescents.14 in our analysis , the older patients ( 12 to 30 y of age at diagnosis ) had a better 5-year efs compared with the preadolescent patients . however , there was no difference in the response to neoadjuvant chemotherapy between the 2 age groups and thus would not explain the inferior outcome of the preadolescent patients . moreover , our patients were very compliant and therefore received the recommended chemotherapy with no difference between preadolescents and older patients with regard to chemoreduction for toxicities . there is a recent report by sharib et al18 that mentioned increased toxicity in young patients and in their latino population with ewing sarcoma . we did not find an increase in toxicity in either of these groups within our cohort and thus could not use it as an explanation for the trend toward inferior outcome . traditionally , the histologic response to neoadjuvant chemotherapy has been used as a prognostic factor . in previous studies , good responders ( > 90% necrosis ) had a 5-year efs of 67% to 78% , whereas the poor responders had a 5-year efs of 49% to 51%.810 in our cohort , tumor necrosis assessment was available for 40 patients . the 5-year os was 70% and this is comparable with that reported in larger studies810 as described above . the patients who were deemed good responders to neoadjuvant chemotherapy relapsed at a higher rate than expected but were salvageable . eleven patients ( 91% hispanic ) in our cohort had a very poor response with < 50% necrosis . nine of the 11 patients relapsed , were lost to follow - up , or were censored within 3 years due to the time frame of the study . although our sample size is small ( n=50 ) , our analysis suggested that tumor necrosis in response to neoadjuvant chemotherapy regimens might not be a valid prognostic marker for efs in our study . in this study , chemotherapy was delivered according to standard protocols from the cog by pediatric or medical oncologists from different cog institutions . all patients were treated at institutions by medical teams trained in delivering treatment on osteosarcoma protocols , whether enrolled on study or not . therefore , the inferior outcome in this population might not be the result of poor adherence to treatment or a decrease in treatment because of toxicity . in our cohort , the male to female ratio was much higher than expected and a disproportionate amount of patients received ablative surgical procedures . the observation of an increase in male to female ratio may in part be due to our small sample size . the higher proportion of patients receiving ablative surgical procedures was in part because our patients presented with more invasive disease at diagnosis . in conclusion , we reviewed the clinicopathologic characteristics and disease outcome of 50 patients with localized high - grade osteosarcoma of the extremity treated by 1 members of the uthscsa sarcoma team over an 11-year period . our cohort was comprised of 70% hispanics of mexican american descent and we found a decreased efs but similar os to what is reported . more importantly , we found a strikingly increased rate of relapse in young patients diagnosed before the age of 12 . we also found that the percentage of tumor necrosis after neoadjuvant chemotherapy was not directly predictive of outcome in our population . the possibility exists that this difference in outcome is secondary to a difference in pharmacodynamics or pharmacogenomics leading to a difference in the metabolism of the various drugs used to treat osteosarcoma . it is also possible that a difference in tumor biology does exist and could be explored further in a larger study . a larger , multi - institutional study including patients with similar demographics to our study is warranted . more data related to outcomes in patients of mexican american ancestry will potentially aid in future treatment decision making and management concerning this fast - growing population .

background : osteosarcoma is the most common bone malignancy in children , adolescents , and young adults . most study cohorts have 10% to 15% hispanic patients that encompass many different hispanic backgrounds . this study characterizes the effect of mainly mexican american ethnicity on the outcome of children , adolescents , and young adults with osteosarcoma.methods:a retrospective analysis of demographics , tumor characteristics , response to treatment , and survival outcome of all localized osteosarcoma of the extremity patients below 30 years of age was performed . a kaplan - meier estimates with log - rank tests and cox proportional hazard regression models were used.results:fifty patients ( median age , 15 ; range , 2 to 28 y ) with localized high - grade osteosarcoma of the extremity were diagnosed between january 2000 and december 2010 . the cohort was 70% mexican americans . with a median follow - up of 39 months ( range , 5 to 142 mo ) , patients had a 5-year overall survival and event - free survival of 65% and 48% , respectively . we observed a significantly decreased 5-year event - free survival in patients diagnosed before age 12 relative to patients diagnosed between ages 12 and 29 ( 11% vs. 57% , p<0.001 ) . we also found that tumor necrosis was not predictive of outcome in our patients.conclusions:the preadolescent patients of predominately mexican american ethnicity had an increased rate of relapse when compared with previous studies . tumor necrosis is not directly predictive of outcome in this population .

MATERIALS AND METHODS Patient Selection and Data Elements Treatment Statistical Analysis RESULTS Demographic Data Clinicopathologic Characteristics Univariate Survival Analysis Multivariate Survival Analysis DISCUSSION

our cohort consisted of 50 patients below 30 years of age diagnosed with localized high - grade osteosarcoma of the extremity between january 2000 and december 2010 that were treated by members of the uthscsa sarcoma team . during this time frame hospital and clinic records from university hospital , christus santa rosa children s hospital , and the cancer therapy and research center were reviewed . a retrospective analysis of patient demographics ( age at diagnosis , sex , date of diagnosis , race , and ethnicity ) , presence of predisposing factors , socioeconomic status ( based on family income obtained from institutional survey ) , and tumor characteristics ( location , histology , tumor volume , response to neoadjuvant chemotherapy , and type of primary surgery ) was performed . ethnicity was assigned based on parental report , and the national cancer institute / children s oncology group ( cog ) definitions . according to this convention , the term hispanic can include mexican americans , south americans , or cubans but our population of hispanics was exclusively mexican american . the chemotherapy included combinations of methotrexate , cisplatin , adriamycin , ifosfamide , and etoposide ; although not all agents were used in each patient . the type of surgery ( limb salvage , amputation , rotationplasty ) was determined by the extent of disease , involvement of neurovascular bundle , presence of pathologic fracture , and appraisal for best limb functionality after surgical resection . the resected specimens were examined for percentage of tumor necrosis in response to neoadjuvant chemotherapy and were assigned a grade of 1 to 6 as defined by salzer - kuntschik et al.4 in this grading system , grade 1 signifies no viable tumor ; grade 2 , solitary live cells or 1 islet of live cells < 0.5 cm ; grade 3 , < 10% viable cells ; grade 4 , 10% to 50% viable cells ; grade 5 , > 50% alive tumor ; and grade 6 , 100% viable tumor . this system for tumor necrosis grading was chosen to standardize the 2 different grading systems that were used in the individual pog9754 and aost0331 studies . longitudinal tumor assessment ( primary site and lungs ) was performed according to pog9754 or aost0331 recommendations . efs was calculated from the date of diagnosis until recurrence , secondary malignancy , death , or most recent follow - up examination showing absence of an event . os was calculated from the date of diagnosis to death or most recent follow - up examination . the survival curves were calculated using the kaplan - meier method and the differences of survival curves were assessed using the log - rank test . adjusted estimates were obtained from proportional hazards models with age , sex , ethnicity , family income , tumor volume , and tumor necrosis included as covariates . power analysis was completed and all statistical testing was 2-sided with a significance level of 5% . our cohort consisted of 50 patients below 30 years of age diagnosed with localized high - grade osteosarcoma of the extremity between january 2000 and december 2010 that were treated by members of the uthscsa sarcoma team . a retrospective analysis of patient demographics ( age at diagnosis , sex , date of diagnosis , race , and ethnicity ) , presence of predisposing factors , socioeconomic status ( based on family income obtained from institutional survey ) , and tumor characteristics ( location , histology , tumor volume , response to neoadjuvant chemotherapy , and type of primary surgery ) was performed . according to this convention , the term hispanic can include mexican americans , south americans , or cubans but our population of hispanics was exclusively mexican american . the chemotherapy included combinations of methotrexate , cisplatin , adriamycin , ifosfamide , and etoposide ; although not all agents were used in each patient . the type of surgery ( limb salvage , amputation , rotationplasty ) was determined by the extent of disease , involvement of neurovascular bundle , presence of pathologic fracture , and appraisal for best limb functionality after surgical resection . the resected specimens were examined for percentage of tumor necrosis in response to neoadjuvant chemotherapy and were assigned a grade of 1 to 6 as defined by salzer - kuntschik et al.4 in this grading system , grade 1 signifies no viable tumor ; grade 2 , solitary live cells or 1 islet of live cells < 0.5 cm ; grade 3 , < 10% viable cells ; grade 4 , 10% to 50% viable cells ; grade 5 , > 50% alive tumor ; and grade 6 , 100% viable tumor . this system for tumor necrosis grading was chosen to standardize the 2 different grading systems that were used in the individual pog9754 and aost0331 studies . longitudinal tumor assessment ( primary site and lungs ) was performed according to pog9754 or aost0331 recommendations . efs was calculated from the date of diagnosis until recurrence , secondary malignancy , death , or most recent follow - up examination showing absence of an event . os was calculated from the date of diagnosis to death or most recent follow - up examination . the survival curves were calculated using the kaplan - meier method and the differences of survival curves were assessed using the log - rank test . adjusted estimates were obtained from proportional hazards models with age , sex , ethnicity , family income , tumor volume , and tumor necrosis included as covariates . fifty patients with localized osteosarcoma of the extremity below 30 years of age were diagnosed between january 2000 and december 2010 at uthscsa . the cohort was composed of 35 hispanics ( 70% ) , 10 whites ( 20% ) , 4 blacks ( 8% ) , and 1 other ( 2% ) . ethnicity was assigned based on parental report , and the national cancer institute / cog definitions . therefore , although our cohort is comprised of mainly hispanics , it is not exclusively hispanics . however , as described in the methods section our hispanic population is uniquely composed of only mexican americans , whereas most of the studies that include hispanics are more heterogenous . the mean age at diagnosis was 15 years ( range , 2 to 28 y ) . eighteen ( 53% ) patients had a tumor volume > 150 cm and 16 ( 47% ) patients had a tumor volume 150 cm . all patients had surgery for local control ; 60% had ablative surgery ( amputation , disarticulation , or rotationplasty ) , whereas 40% had limb salvage procedures . we acknowledge that this is an increased percentage of patients undergoing ablative procedures but this was due to the disproportionate number of patients with joint and neurovascular bundle involvement . twelve ( 30% ) patients had grade 4 necrosis ( between 50% and 90% ) and 11 ( 28% ) patients had a very poor response ( < 50% necrosis ) including 2 patients with 100% viable tumor after neoadjuvant chemotherapy . although the cohort of patients for which histologic response was available was 70% hispanic , 10/11 ( 91% ) of the patients with a very poor response were hispanics . the os at 3 and 5 years was 73% and 65% , respectively , for the entire cohort . eleven patients diagnosed before the age of 12 experienced a statistically significant decreased 5-year efs and os relative to those diagnosed between the ages of 12 and 29 ( 11% vs. 57% , respectively , p=<0.001 for efs and 25% vs. 76% , respectively , p=<0.001 for os ) ( table 2 ) . patient characteristics and univariate analysis although response to neoadjuvant chemotherapy has traditionally been used as a prognostic marker for both efs and os , it was of prognostic significance only for efs ( table 2 and figs . 1 , 2 ) in our population . we observed an increased percentage of patients with < 50% necrosis after neoadjuvant chemotherapy and chose to grade necrosis based on 6 categories as defined by salzer - kuntschik et al4 to further categorize the poor responders . the 5-year efs when compared between groups categorized by grades 1 to 3 , grade 4 , and grades 5 to 6 necrosis showed a statistically significant decreased outcome in patients with grades 5 to 6 necrosis ( 61% vs. 42% vs. 21% , respectively , p=0.03 ) . event - free survival ( efs ) of localized / extremity tumor patients based on tumor necrosis . the 5-year efs for patients with grades 5 to 6 was significantly different than the 5-year efs of patients with grades 1 to 3 and grade 4 tumor necrosis ( p=0.03 ) . overall survival ( os ) of localized / extremity tumor patients based on tumor necrosis . there was no difference in the location of relapse when comparing the preadolescent patients to the older patients ( young patients [ n=9 ] : 22% , local ; 55% , lung ; 22% , lung / local ; older patients [ n=14 ] : 21% , local ; 57% , lung ; 21% , lung / local ) . overall , 4 patients experienced a local recurrence , 5 had combined local / lung recurrence , and 1 patient had progressive disease , meaning nonresponsive to neoadjuvant chemotherapy . the mean time to relapse was 19 months ( range , 2 to 37 mo ) . eight of the 13 patients with isolated lung recurrences and 4 of the 10 with other types of recurrence / progression were successfully salvaged for an overall salvage rate of 52% for first relapses . multivariate cox regression analysis was performed to assess the association between efs or os and predictors , such as age , ethnicity , income , tumor volume , and tumor necrosis . patients below 12 years of age at diagnosis experienced a higher rate of relapse and death relative to those 12 to 29 years of age at diagnosis ( hazard ratio [ hr ] , 4.77 ; 95% ci , 2.03 - 11.17 ; p=<0.001 for efs ; hr , 5.23 ; 95% ci , 1.86 - 14.59 ; p=0.002 for os ) . grades 5 to 6 necrosis after neoadjuvant chemotherapy was significantly predictive of decreased efs ( hr , 3.76 ; 95% ci , 1.29 - 10.94 ; p=0.02 ) and there was a trend toward lower os ( hr , 3.45 ; 95% ci , 0.91 - 12.99 ; p=0.07 ) . fifty patients with localized osteosarcoma of the extremity below 30 years of age were diagnosed between january 2000 and december 2010 at uthscsa . the cohort was composed of 35 hispanics ( 70% ) , 10 whites ( 20% ) , 4 blacks ( 8% ) , and 1 other ( 2% ) . therefore , although our cohort is comprised of mainly hispanics , it is not exclusively hispanics . however , as described in the methods section our hispanic population is uniquely composed of only mexican americans , whereas most of the studies that include hispanics are more heterogenous . the mean age at diagnosis was 15 years ( range , 2 to 28 y ) . eighteen ( 53% ) patients had a tumor volume > 150 cm and 16 ( 47% ) patients had a tumor volume 150 cm . all patients had surgery for local control ; 60% had ablative surgery ( amputation , disarticulation , or rotationplasty ) , whereas 40% had limb salvage procedures . we acknowledge that this is an increased percentage of patients undergoing ablative procedures but this was due to the disproportionate number of patients with joint and neurovascular bundle involvement . twelve ( 30% ) patients had grade 4 necrosis ( between 50% and 90% ) and 11 ( 28% ) patients had a very poor response ( < 50% necrosis ) including 2 patients with 100% viable tumor after neoadjuvant chemotherapy . although the cohort of patients for which histologic response was available was 70% hispanic , 10/11 ( 91% ) of the patients with a very poor response were hispanics . eleven patients diagnosed before the age of 12 experienced a statistically significant decreased 5-year efs and os relative to those diagnosed between the ages of 12 and 29 ( 11% vs. 57% , respectively , p=<0.001 for efs and 25% vs. 76% , respectively , p=<0.001 for os ) ( table 2 ) . 1 , 2 ) in our population . we observed an increased percentage of patients with < 50% necrosis after neoadjuvant chemotherapy and chose to grade necrosis based on 6 categories as defined by salzer - kuntschik et al4 to further categorize the poor responders . the 5-year efs when compared between groups categorized by grades 1 to 3 , grade 4 , and grades 5 to 6 necrosis showed a statistically significant decreased outcome in patients with grades 5 to 6 necrosis ( 61% vs. 42% vs. 21% , respectively , p=0.03 ) . event - free survival ( efs ) of localized / extremity tumor patients based on tumor necrosis . the 5-year efs for patients with grades 5 to 6 was significantly different than the 5-year efs of patients with grades 1 to 3 and grade 4 tumor necrosis ( p=0.03 ) . overall survival ( os ) of localized / extremity tumor patients based on tumor necrosis . there was no difference in the location of relapse when comparing the preadolescent patients to the older patients ( young patients [ n=9 ] : 22% , local ; 55% , lung ; 22% , lung / local ; older patients [ n=14 ] : 21% , local ; 57% , lung ; 21% , lung / local ) . overall , 4 patients experienced a local recurrence , 5 had combined local / lung recurrence , and 1 patient had progressive disease , meaning nonresponsive to neoadjuvant chemotherapy . the mean time to relapse was 19 months ( range , 2 to 37 mo ) . eight of the 13 patients with isolated lung recurrences and 4 of the 10 with other types of recurrence / progression were successfully salvaged for an overall salvage rate of 52% for first relapses . multivariate cox regression analysis was performed to assess the association between efs or os and predictors , such as age , ethnicity , income , tumor volume , and tumor necrosis . patients below 12 years of age at diagnosis experienced a higher rate of relapse and death relative to those 12 to 29 years of age at diagnosis ( hazard ratio [ hr ] , 4.77 ; 95% ci , 2.03 - 11.17 ; p=<0.001 for efs ; hr , 5.23 ; 95% ci , 1.86 - 14.59 ; p=0.002 for os ) . grades 5 to 6 necrosis after neoadjuvant chemotherapy was significantly predictive of decreased efs ( hr , 3.76 ; 95% ci , 1.29 - 10.94 ; p=0.02 ) and there was a trend toward lower os ( hr , 3.45 ; 95% ci , 0.91 - 12.99 ; p=0.07 ) . the population of localized osteosarcoma of the extremity patients treated at uthscsa was 70% hispanic ( 35/50 ) , homogenously of mexican american ancestry , giving us a unique cohort to study . of the 50 patients in our cohort , 11 were categorized as preadolescent ( below 12 y of age at diagnosis ) . nine of the 35 mexican american patients were preadolescents at diagnosis and to the best of our knowledge this is the largest series composed of such patients . in this study , the 5-year efs of 48% was similar to results from the brazilian osteosarcoma treatment group who reported a 5-year efs of only 53%.5 in this cohort , the 5-year efs was inferior to a smaller study completed on patients in low - income countries in latin america.6 these data suggest that there might be some similarities and differences in outcome among other hispanic groups . although the 5-year os of 65% was comparable with that reported by large european groups,2,7 the efs in this cohort was lower.2,812 in this regard , the data indicated that our patients had a higher risk of relapse after primary treatment , but were salvageable as denoted by os rates . notably , our predominantly mexican american preadolescent patients ( below 12 y of age at diagnosis of which 9/11 are hispanic ) had an overall poor outcome with 5-year efs of 11% and a 5-year os of 25% . in stark contrast , many published studies report the outcome of preadolescent patients to be equivalent to those diagnosed after puberty.1317 bacci et al14 reported results on a large cohort of patients treated on protocol between 1972 and 1999 , comparing the outcome of patients aged 12 years and below at diagnosis to that of patients between 13 and 40 years of age . the 5-year efs is 60% versus 58% in favor of the preadolescents.14 in our analysis , the older patients ( 12 to 30 y of age at diagnosis ) had a better 5-year efs compared with the preadolescent patients . however , there was no difference in the response to neoadjuvant chemotherapy between the 2 age groups and thus would not explain the inferior outcome of the preadolescent patients . moreover , our patients were very compliant and therefore received the recommended chemotherapy with no difference between preadolescents and older patients with regard to chemoreduction for toxicities . in previous studies , good responders ( > 90% necrosis ) had a 5-year efs of 67% to 78% , whereas the poor responders had a 5-year efs of 49% to 51%.810 in our cohort , tumor necrosis assessment was available for 40 patients . eleven patients ( 91% hispanic ) in our cohort had a very poor response with < 50% necrosis . nine of the 11 patients relapsed , were lost to follow - up , or were censored within 3 years due to the time frame of the study . although our sample size is small ( n=50 ) , our analysis suggested that tumor necrosis in response to neoadjuvant chemotherapy regimens might not be a valid prognostic marker for efs in our study . in this study , chemotherapy was delivered according to standard protocols from the cog by pediatric or medical oncologists from different cog institutions . therefore , the inferior outcome in this population might not be the result of poor adherence to treatment or a decrease in treatment because of toxicity . the higher proportion of patients receiving ablative surgical procedures was in part because our patients presented with more invasive disease at diagnosis . in conclusion , we reviewed the clinicopathologic characteristics and disease outcome of 50 patients with localized high - grade osteosarcoma of the extremity treated by 1 members of the uthscsa sarcoma team over an 11-year period . our cohort was comprised of 70% hispanics of mexican american descent and we found a decreased efs but similar os to what is reported . more importantly , we found a strikingly increased rate of relapse in young patients diagnosed before the age of 12 . we also found that the percentage of tumor necrosis after neoadjuvant chemotherapy was not directly predictive of outcome in our population . more data related to outcomes in patients of mexican american ancestry will potentially aid in future treatment decision making and management concerning this fast - growing population .

chronic obstructive pulmonary disease ( copd ) , which includes both chronic bronchitis and emphysema , is characterized by airflow obstruction , and may be accompanied by airway hyperreactivity , chronic cough , sputum production , shortness of breath ( sob ) , wheezing , exercise intolerance , and poor quality of life.1,2 for 50 years , the biologic plausibility for a link between cigarette smoking and adverse respiratory system outcomes has been supported by epidemiologic and clinical evidence.3,4 risks of lung cancer , copd , and other respiratory conditions such as asthma are increased in smokers , particularly among those with extensive exposure histories.4 sex also appears to affect the risk of adverse respiratory effects from smoking . most studies show that smoking has greater adverse effects on respiratory function among women than among men.511 however , other studies have not been consistent with that finding.1215 the sex difference in many previous studies of international populations or small study populations was observed when using self - reported pack - years ( product of intensity and duration ) of smoking as a measure of tobacco exposure.68,1113,15 there has been little information about the relationship between respiratory symptoms and number of years ( duration ) of tobacco use , particularly in a population representative of a us state . this is also true with regard to the relationship between duration of tobacco use , respiratory symptoms , and sex . pack - year history requires the use of two survey questions , whereas years of tobacco use can be estimated by a single question . south carolina is a us state with a high prevalence of current smoking ( 21.0% in 2010 ) and copd ( 7.7% in 2011 ) , and a high mortality rate from copd ( 68.6 per 100,000 in 2010).16,17 the south carolina behavioral risk factor surveillance system ( brfss ) , an annual health survey , included state - added questions regarding years of tobacco use and respiratory symptoms in 2012 and provided an opportunity to assess the association of prolonged tobacco use with respiratory symptoms and copd in a contemporary state population of adult respondents with a smoking history . the brfss is an annual random - digital - dialed telephone survey conducted by state health departments in collaboration with the centers for disease control and prevention ( cdc ) in all 50 states , the district of columbia , and us territories . the state - based brfss has collected data from households with landline and cellular telephones , and has incorporated a new sample weighting method since 2011 ( http://www.cdc.gov/brfss/annual_data/annual_2011.htm ) . the brfss includes core questions about sociodemographic characteristics , risk behaviors , and chronic diseases including copd ; optional modules ( standardized questions provided by the cdc and chosen by a state based on the state s priorities and budget ) ; and questions added by the state . questions addressing respiratory symptoms and years of tobacco use were added to the 2012 south carolina brfss and were analyzed in this study . the 2012 south carolina brfss combined response rate ( the number of respondents who completed the survey as a proportion of all eligible and likely eligible persons using standards set by the american association of public opinion research response rate formula # 4 [ http://www.aapor.org/standard_definitions2.htm ] ) was 48.6% ( http://www.cdc.gov/brfss/annual_data/2012/pdf/summarydataqualityreport2012_20130712.pdf ) . this study is a secondary data analysis , which is exempt from the need for approval of an institutional review board . a history of copd was defined by the subject s affirmative responses to the question has a doctor , nurse , or other health professional ever told you that you have chronic obstructive pulmonary disease or copd , emphysema or chronic bronchitis ? the small number of persons who reported do nt know / not sure were defined as not having been diagnosed with copd . overall , 76.4% of respondents with self - reported copd also reported having had a breathing test such as spirometry . how often do you cough up mucus or phlegm ? , we defined the presence of a frequent phlegm - producing cough as a response of everyday , or most days a week to compare with respondents with a few days a month , only with occasional colds or chest infections , or never . in response to during the past 30 days , how often did you feel short of breath ? , we defined frequent sob as a response of all the time or most of the time to compare with respondents with a response of some of the time , a little of the time , or none of the time . for the third question thinking about your physical activity during the last 12 months , do you agree slightly or strongly , or disagree slightly or strongly with the following statement : i do less now than i used to because of my breathing problems , we compared those who said strongly agree with respondents who gave the other response options . these three respiratory symptom questions were selected from a validated study with a positive predictive value of 56.8% and a negative predictive value of 86.4% , which is considered to be a highly reliable questionnaire when compared with spirometry measures.18 for the analyses presented in this report , we identified respondents with a history of cigarette smoking by an affirmative response to the tobacco use question , have you smoked at least 100 cigarettes in your entire life ? years ( duration ) of tobacco use were further determined from the question over your lifetime , how many years have you smoked tobacco products ? and were divided into four duration groups ( 19 years , 1019 years , 2029 years , and 30 years ) . persons who responded not at all to the third tobacco use question , do you now smoke cigarettes , every day , some days , or not at all ? were defined as former smokers and the remainder were defined as current smokers . former smokers were also asked to identify how long it had been since they last smoked a cigarette , even one or two puffs . former smokers were then defined either by having quit smoking for 10 years ( 72.7% ) or by all other responses ( 26.8% reported quit durations < 10 years and 0.5% failed to provide information on quit duration ) . we restricted this study to adults aged 45 years because there was a low prevalence of copd ( 3.7% ) and smoking 30 years ( 1.9% ) among respondents aged 1844 years , resulting in unstable sample sizes in that age group . in the survey population of 9,386 respondents aged 45 years , 46.2% had never smoked , 33.8% were former smokers , and 18.3% were current smokers . leffondre et al has observed that inclusion of respondents who have never smoked may produce overestimations of the effects of smoking duration when that group is assigned a value of zero smoking years and is set as the referent group in an analysis;19 therefore , we restricted this study to former and current smokers ( 4,553 respondents ) aged 45 years . data from 4,135 ( 90.8% ) respondents who provided information on smoking duration , respiratory symptoms , and history of copd were analyzed in this study after those who had missing information were excluded . we estimated 95% confidence intervals ( cis ) for the percentages and used two - sided t - tests to compare characteristics between groups defined by smoking duration and to assess linear trends in characteristics by smoking duration . the distribution of smoking duration by current smoking status ( current smokers , former smokers who had quit for 10 years , and other former smokers ) was also examined . the age - adjusted prevalence and 95% ci of respiratory symptoms and copd by smoking duration and current smoking status were obtained from separate logistic regression models that included age ( 4559 years , 6069 years , and 70 years ) as the covariate . finally , we assessed the adjusted prevalence ratios and 95% cis for the likelihood of having respiratory symptoms and copd associated with smoking duration using separate multivariable logistic regression models that included sex , age , race / ethnicity ( non - hispanic white , non - hispanic black , or other / multiracial ) , education ( less than a high school diploma , a high school graduate or equivalent , or at least some college ) , and current smoking status ( current vs former ) as covariates . prior research suggesting that women might be more susceptible than men to the effects of smoking prompted testing for an interaction between sex and smoking duration in separate age - adjusted logistic regression models for each of the four dependent variables . the only sex interaction was observed for copd ( p=0.085 ) . we also assessed whether the relationships between smoking duration and the dependent variables differed by current smoking status , but failed to find a significant interaction for copd ( p=0.79 ) or for any of the three respiratory symptoms ( p>0.42 ) . there were also no significant interactions between age and sleep duration for any outcome ( p>0.30 ) . all analyses were conducted using statistical analysis system ( sas)-callable sudaan ( release 10.0.1 , research triangle institute , nc , usa ) to account for the complex sampling design . a history of copd was defined by the subject s affirmative responses to the question has a doctor , nurse , or other health professional ever told you that you have chronic obstructive pulmonary disease or copd , emphysema or chronic bronchitis ? the small number of persons who reported do nt know / not sure were defined as not having been diagnosed with copd . overall , 76.4% of respondents with self - reported copd also reported having had a breathing test such as spirometry . how often do you cough up mucus or phlegm ? , we defined the presence of a frequent phlegm - producing cough as a response of everyday , or most days a week to compare with respondents with a few days a month , only with occasional colds or chest infections , or never . in response to during the past 30 days , how often did you feel short of breath ? , we defined frequent sob as a response of all the time or most of the time to compare with respondents with a response of some of the time , a little of the time , or none of the time . for the third question thinking about your physical activity during the last 12 months , do you agree slightly or strongly , or disagree slightly or strongly with the following statement : i do less now than i used to because of my breathing problems , we compared those who said strongly agree with respondents who gave the other response options . these three respiratory symptom questions were selected from a validated study with a positive predictive value of 56.8% and a negative predictive value of 86.4% , which is considered to be a highly reliable questionnaire when compared with spirometry measures.18 for the analyses presented in this report , we identified respondents with a history of cigarette smoking by an affirmative response to the tobacco use question , have you smoked at least 100 cigarettes in your entire life ? years ( duration ) of tobacco use were further determined from the question over your lifetime , how many years have you smoked tobacco products ? and were divided into four duration groups ( 19 years , 1019 years , 2029 years , and 30 years ) . persons who responded not at all to the third tobacco use question , do you now smoke cigarettes , every day , some days , or not at all ? were defined as former smokers and the remainder were defined as current smokers . former smokers were also asked to identify how long it had been since they last smoked a cigarette , even one or two puffs . former smokers were then defined either by having quit smoking for 10 years ( 72.7% ) or by all other responses ( 26.8% reported quit durations < 10 years and 0.5% failed to provide information on quit duration ) . we restricted this study to adults aged 45 years because there was a low prevalence of copd ( 3.7% ) and smoking 30 years ( 1.9% ) among respondents aged 1844 years , resulting in unstable sample sizes in that age group . in the survey population of 9,386 respondents aged 45 years , 46.2% had never smoked , 33.8% were former smokers , and 18.3% were current smokers . leffondre et al has observed that inclusion of respondents who have never smoked may produce overestimations of the effects of smoking duration when that group is assigned a value of zero smoking years and is set as the referent group in an analysis;19 therefore , we restricted this study to former and current smokers ( 4,553 respondents ) aged 45 years . data from 4,135 ( 90.8% ) respondents who provided information on smoking duration , respiratory symptoms , and history of copd were analyzed in this study after those who had missing information were excluded . we estimated 95% confidence intervals ( cis ) for the percentages and used two - sided t - tests to compare characteristics between groups defined by smoking duration and to assess linear trends in characteristics by smoking duration . the distribution of smoking duration by current smoking status ( current smokers , former smokers who had quit for 10 years , and other former smokers ) was also examined . the age - adjusted prevalence and 95% ci of respiratory symptoms and copd by smoking duration and current smoking status were obtained from separate logistic regression models that included age ( 4559 years , 6069 years , and 70 years ) as the covariate . finally , we assessed the adjusted prevalence ratios and 95% cis for the likelihood of having respiratory symptoms and copd associated with smoking duration using separate multivariable logistic regression models that included sex , age , race / ethnicity ( non - hispanic white , non - hispanic black , or other / multiracial ) , education ( less than a high school diploma , a high school graduate or equivalent , or at least some college ) , and current smoking status ( current vs former ) as covariates . prior research suggesting that women might be more susceptible than men to the effects of smoking prompted testing for an interaction between sex and smoking duration in separate age - adjusted logistic regression models for each of the four dependent variables . we also assessed whether the relationships between smoking duration and the dependent variables differed by current smoking status , but failed to find a significant interaction for copd ( p=0.79 ) or for any of the three respiratory symptoms ( p>0.42 ) . there were also no significant interactions between age and sleep duration for any outcome ( p>0.30 ) . all analyses were conducted using statistical analysis system ( sas)-callable sudaan ( release 10.0.1 , research triangle institute , nc , usa ) to account for the complex sampling design . table 1 shows the distribution of selected characteristics by smoking duration among 4,135 adults aged 45 years with a history of smoking . the distributions of age and race / ethnicity did not differ significantly by smoking duration . respondents who had smoked for 30 years were more likely to be male ( p<0.05 ) , have less than a high school education ( p<0.05 ) , and less likely to have some college education ( p<0.05 ) than persons who had smoked for 19 years or 1019 years . the proportion of the study population who were current smokers increased with increasing smoking duration ( linear trend , p<0.05 ) such that 58.3% of those who had smoked for 30 years continued to be current smokers compared with 7.5% of those who had smoked for 19 years . the proportion of the study population comprising former smokers with quit durations of 10 years declined as smoking duration increased ( linear trend , p<0.05 ) . the distribution of smoking duration was 19.2% for 19 years , 21.6% for 1019 years , 23.0% for 2029 years , and 36.2% for 30 years . the increase in the proportions for each smoking duration was observed in men and women , all age groups , non - hispanic whites , non - hispanic blacks , and all education groups ( data not shown ) . figure 1 shows an inverse linear trend for smoking duration category in former smokers with quit durations of 10 years , while current smokers and other former smokers had a linear increase such that 60.0% of all current smokers and 43.6% of other former smokers had smoked for 30 years in contrast with 15.6% of former smokers who had quit for 10 years . we assessed the age - adjusted prevalence of copd and the three respiratory symptoms by current smoking status ( figure 2 ) . former smokers with quit durations 10 years reported a significantly lower age - adjusted percentage of frequent productive cough ( 10.6% vs 24.2% , respectively , p<0.001 ) , frequent sob ( 5.7% vs 9.9% , p=0.001 ) , agreement that sob affects physical activity ( 8.8% vs 13.8% , p<0.0001 ) , and copd ( 8.6% vs 22.7% , p<0.0001 ) compared with current smokers . in contrast , other former smokers with either no stated quit duration or quit durations < 10 years did not differ significantly from current smokers in prevalence of frequent sob , agreement that sob affects physical activity , and copd , although this former smoker group reported a significant lower age - adjusted percentage of frequent productive cough than did current smokers ( 11.5% vs 24.2% , p<0.0001 ) . data in table 2 demonstrate that the age - adjusted prevalence of each respiratory symptom and copd increased with increasing smoking duration ( linear trend , p<0.001 ) . among those who had smoked for 30 years , 25.6% reported copd , 25.0% had frequent productive cough , 11.2% had frequent sob , and 16.7% strongly agreed that sob had affected physical activity in the previous year . after controlling for age , sex , race / ethnicity , education , and current smoking status , there remained an increased likelihood for having copd and all three respiratory symptoms associated with increasing smoking duration ( linear trend , p<0.001 ) . however , after restricting the analyses to the 3,495 respondents who responded that they had not been diagnosed with copd , only one respiratory symptom remained significantly associated with smoking duration ( table 2 , p<0.001 ) . the age - adjusted percentage of respondents without copd who reported having frequent productive cough increased with increasing smoking duration ( linear trend , p<0.001 ) . this relationship persisted even after adjustment for age , sex , race / ethnicity , education , and current smoking status ( linear trend , p=0.009 ) . however , only a modest significant association was observed among respondents without copd and with a smoking duration 30 years compared with those in the 19 year duration ( prevalence ratio 1.85 ; 95% confidence interval 1.063.21 ) . there was a borderline significant sex interaction ( p=0.085 ) of the relationship between smoking duration and prevalence of copd ( figure 3 ) . the age - adjusted prevalence increased with increasing smoking duration for both men and women . however , women had a significantly higher age - adjusted prevalence of copd than men for a smoking duration of 19 years ( 10.1% vs 2.8% , p<0.001 ) , 2029 years ( 15.3% vs 9.2% , p=0.04 ) , and 30 years ( 30.9% vs 21.9% , p<0.001 ) but there was no difference at 1019 years ( 9.3% vs 8.9% , p=0.93 ) . our study is the first population - based health survey in many years that has assessed the relationship between duration of tobacco use and respiratory outcomes in adults with a smoking history in the usa . this study provides a population - based perspective of the relationship between smoking duration , three respiratory symptoms , and copd in a us state with a high prevalence of current cigarette smoking and copd . these relationships in a contemporary population in 2012 are consistent with similar dose response relationships observed in a norwegian study ( 19951997 ) for number of pack - years of smoking and chronic bronchitis , breathlessness , persistent coughing , coughing with phlegm , and reduced lung function.7,8 we observed that the significant relationship between smoking duration and frequent productive cough was also present in adults without a self - reported diagnosis of copd ; however , the relationship between smoking duration , frequent sob , and the effects of sob on physical activity were not significant in adults without copd . our results also showed that smoking cessation for 10 years in contrast with current smoking was associated with a much lower prevalence of copd and of each of the three respiratory symptoms . our results are consistent with the copd prevention strategy that smoking cessation improves respiratory function and prevents excessive decline in lung function in smokers with copd as well as in smokers without chronic symptoms.20,21 the health benefits of smoking cessation with regard to the rate of decline in forced expiratory volume in 1 second ( fev1 ) and mortality appear to be most significant for persons who quit before 4050 years of age.14,22 the sex difference in the age - adjusted relationship of smoking duration with copd demonstrated in this study is consistent with reports that women may be more susceptible to the detrimental effects of smoking at the 19 year duration than men . women were similar to men in terms of the significant relationship between smoking duration and respiratory symptoms . prior studies conducted in the late 1990s and early 2000s suggested that women smokers were particularly susceptible to the deleterious effects of smoking , with an augmented decline in fev1 compared with male smokers.6,8,9,11,20 in addition , results from the lung health study ( 19861994 ) showed that smoking cessation has a greater impact on rate of fev1 decline in women than in men.23 however , kohansal et al did not report a sex difference in the loss of lung function associated with continued or stopping smoking.14 suggested explanations for the female male difference in relationship between smoking duration and respiratory function have included sex variation in the prevalence of lifetime smoking , biologic differences such as smaller lungs and larger airways in women , hormonal factors that influence epithelial cell function , sex differences in inflammatory responses , greater awareness or recall of respiratory symptoms and diagnosed disease among women , and sex differences in the clinical presentation of copd.8,20,24 a previous study suggested that african - american women with copd had a greater loss of lung function due to cigarette smoking than other copd patients.5,25 however , the current study did not have a sample size sufficient to assess that relationship in groups defined by race , sex , and smoking duration . as would be expected , current smokers were more likely to have a frequent productive cough . smoker s cough is consistent with chronic bronchitis , and is primarily due to excessive inflammation and hypersecretion of mucus secondary to smoking tobacco products.3,26 analysis of the copdgene cohort showed that slightly more than one quarter of copd subjects reported chronic bronchitis.27 there was also a twofold increase in risk of chronic bronchitis in current smokers in the copdgene study . in contrast , more than one - third of our study respondents reported a frequent productive cough , perhaps reflecting causes of chronic cough other than copd . notably , there was less of a difference in the frequency of dyspnea between current vs former smokers ; we speculate that dyspnea may be an important reason for smoking cessation in some cases . chronic productive cough also increased with smoking duration among smokers with no evidence of copd in the current study . a study that evaluated the relationship among respiratory symptoms , copd , and airflow for over 30 years found that 40% of smokers developed chronic bronchitis and half of those developed copd.28 in the seven countries study , among continuous smokers without chronic bronchitis or any other pulmonary disease , those with a smoking history of 2040 pack - years ( p=0.001 ) or > 40 pack - years ( p=0.002 ) had lower mean fev0.75 than those with a smoking history of < 20 pack - years.28 furthermore , of smokers with no airflow obstruction in the copdgene study , those with chronic bronchitis had a greater pack - year smoking history and were more likely to be current smokers than those without chronic bronchitis.29 in that study , non - obstructive chronic bronchitis was defined by a history of cough and phlegm production for 3 consecutive months per year , and its results suggest that bronchitic symptoms such as productive cough in the absence of airflow obstruction could be an early marker of susceptibility and risk for copd.2931 unfortunately , copd in the current study was defined by having emphysema , chronic bronchitis , and/or copd , so that those subgroups can not be determined . little information has been available in us populations to address the relationship between smoking duration , copd , and respiratory symptoms , so this study is unique . however , our study had several limitations . first , the brfss is a cross - sectional study of the south carolina population , so causal relationships between smoking duration , copd , and respiratory symptoms can not be established . second , the study response rate was less than 50% and selection bias might influence our results if participation varied by smoking duration . third , the analyses relied on self - reported information from a telephone interview about current smoking , smoking duration , respiratory symptoms , and copd , and can not be validated . however , previous studies support that a self - report of provider - diagnosed copd is highly reliable,18,32 although undiagnosed copd may affect our results.3335 validity may also differ between self - reported emphysema and self - reported chronic bronchitis.36 while 76.4% of subjects with self - reported copd also reported having had a breathing test in our study , there is no information regarding test interpretation . however , having had a breathing test did not differ by smoking status among those with copd . this finding is comparable with that of a larger 2011 us survey of approximately 13,000 adults with self - reported copd , in which the percentage of subjects having had a breathing test to diagnose copd did not differ by smoking status.37 furthermore , use of smoking duration and current smoking status give only partial information about tobacco use . other useful information for such a study could include intensity ( number or packs smoked per day ) , pack - years ( a product of intensity and duration ) , age at initiation , and time since cessation , but such information can not be obtained during a time - limited interview.19 however , our results are consistent with findings for pack - years of smoking that have been applied in previous studies.57 in addition , using smoking duration rather than pack - years or additional smoking indicators that employ a time factor in the same analytic models may have helped to reduce multicollinearity and avoid a cohort effect.19 the us preventive services task force so far has found no evidence to support screening adults ( including asymptomatic smokers ) for copd using spirometry.38 however , the recommendations do suggest that current smokers should receive smoking cessation counseling and pharmacologic therapies that have been demonstrated to increase cessation rates . smoking cessation may improve respiratory symptoms and bronchial hyperresponsiveness and prevent excessive decline in lung function among smokers with copd as well as among smokers without chronic symptoms.21 smokers with respiratory symptoms , including dyspnea and productive cough , may be more susceptible to the effects of smoking on rate of decline in fev1 than those without symptoms , thus representing an important target group for smoking cessation interventions.14 the united states public health service guidelines recommend a simple intervention of 5 as , ie , ask about tobacco use , advise to quit , assess willingness to make a cessation attempt , assist in cessation attempt , and arrange follow - up to make prevention and cessation of tobacco use more successful.39 therefore , there are opportunities for physicians to provide tobacco cessation counseling to all patients who smoke , particularly those with respiratory symptoms and/or copd . such efforts will have the largest impact on decreasing copd in men and women alike . in this population - based survey of adults with a smoking history , prolonged tobacco use was associated with an increased likelihood of having copd , frequent productive cough , frequent sob , and agreement that sob affected physical activity even after controlling for current smoking behavior . former smokers who had quit smoking for 10 years had a lower prevalence of copd and respiratory symptoms than current smokers . the prevalence of copd was higher in women than in men at all levels of smoking duration . this study also demonstrates that the brfss is a useful surveillance tool to describe the association of potential risk factors for copd and respiratory symptoms in an adult state population and could be expanded to other us states for public health surveillance purposes .

backgroundthe purpose of this study was to assess the relationship of smoking duration with respiratory symptoms and history of chronic obstructive pulmonary disease ( copd ) in the south carolina behavioral risk factor surveillance system survey in 2012.methodsdata from 4,135 adults aged 45 years with a smoking history were analyzed using multivariable logistic regression that accounted for sex , age , race / ethnicity , education , and current smoking status , as well as the complex sampling design.resultsthe distribution of smoking duration ranged from 19.2% ( 19 years ) to 36.2% ( 30 years ) . among 1,454 respondents who had smoked for 30 years , 58.3% were current smokers , 25.0% had frequent productive cough , 11.2% had frequent shortness of breath , 16.7% strongly agreed that shortness of breath affected physical activity , and 25.6% had been diagnosed with copd . prevalence of copd and each respiratory symptom was lower among former smokers who quit 10 years earlier compared with current smokers . smoking duration had a linear relationship with copd ( p<0.001 ) and all three respiratory symptoms ( p<0.001 ) after adjusting for smoking status and other covariates . while copd prevalence increased with prolonged smoking duration in both men and women , women had a higher age - adjusted prevalence of copd in the 19 years , 2029 years , and 30 years duration periods.conclusionthese state population data confirm that prolonged tobacco use is associated with respiratory symptoms and copd after controlling for current smoking behavior .

Introduction Materials and methods Respiratory outcomes Smoking behaviors Statistical analysis Results Discussion Conclusion

chronic obstructive pulmonary disease ( copd ) , which includes both chronic bronchitis and emphysema , is characterized by airflow obstruction , and may be accompanied by airway hyperreactivity , chronic cough , sputum production , shortness of breath ( sob ) , wheezing , exercise intolerance , and poor quality of life.1,2 for 50 years , the biologic plausibility for a link between cigarette smoking and adverse respiratory system outcomes has been supported by epidemiologic and clinical evidence.3,4 risks of lung cancer , copd , and other respiratory conditions such as asthma are increased in smokers , particularly among those with extensive exposure histories.4 sex also appears to affect the risk of adverse respiratory effects from smoking . south carolina is a us state with a high prevalence of current smoking ( 21.0% in 2010 ) and copd ( 7.7% in 2011 ) , and a high mortality rate from copd ( 68.6 per 100,000 in 2010).16,17 the south carolina behavioral risk factor surveillance system ( brfss ) , an annual health survey , included state - added questions regarding years of tobacco use and respiratory symptoms in 2012 and provided an opportunity to assess the association of prolonged tobacco use with respiratory symptoms and copd in a contemporary state population of adult respondents with a smoking history . questions addressing respiratory symptoms and years of tobacco use were added to the 2012 south carolina brfss and were analyzed in this study . a history of copd was defined by the subject s affirmative responses to the question has a doctor , nurse , or other health professional ever told you that you have chronic obstructive pulmonary disease or copd , emphysema or chronic bronchitis ? these three respiratory symptom questions were selected from a validated study with a positive predictive value of 56.8% and a negative predictive value of 86.4% , which is considered to be a highly reliable questionnaire when compared with spirometry measures.18 for the analyses presented in this report , we identified respondents with a history of cigarette smoking by an affirmative response to the tobacco use question , have you smoked at least 100 cigarettes in your entire life ? and were divided into four duration groups ( 19 years , 1019 years , 2029 years , and 30 years ) . we restricted this study to adults aged 45 years because there was a low prevalence of copd ( 3.7% ) and smoking 30 years ( 1.9% ) among respondents aged 1844 years , resulting in unstable sample sizes in that age group . in the survey population of 9,386 respondents aged 45 years , 46.2% had never smoked , 33.8% were former smokers , and 18.3% were current smokers . leffondre et al has observed that inclusion of respondents who have never smoked may produce overestimations of the effects of smoking duration when that group is assigned a value of zero smoking years and is set as the referent group in an analysis;19 therefore , we restricted this study to former and current smokers ( 4,553 respondents ) aged 45 years . data from 4,135 ( 90.8% ) respondents who provided information on smoking duration , respiratory symptoms , and history of copd were analyzed in this study after those who had missing information were excluded . the distribution of smoking duration by current smoking status ( current smokers , former smokers who had quit for 10 years , and other former smokers ) was also examined . the age - adjusted prevalence and 95% ci of respiratory symptoms and copd by smoking duration and current smoking status were obtained from separate logistic regression models that included age ( 4559 years , 6069 years , and 70 years ) as the covariate . finally , we assessed the adjusted prevalence ratios and 95% cis for the likelihood of having respiratory symptoms and copd associated with smoking duration using separate multivariable logistic regression models that included sex , age , race / ethnicity ( non - hispanic white , non - hispanic black , or other / multiracial ) , education ( less than a high school diploma , a high school graduate or equivalent , or at least some college ) , and current smoking status ( current vs former ) as covariates . prior research suggesting that women might be more susceptible than men to the effects of smoking prompted testing for an interaction between sex and smoking duration in separate age - adjusted logistic regression models for each of the four dependent variables . we also assessed whether the relationships between smoking duration and the dependent variables differed by current smoking status , but failed to find a significant interaction for copd ( p=0.79 ) or for any of the three respiratory symptoms ( p>0.42 ) . a history of copd was defined by the subject s affirmative responses to the question has a doctor , nurse , or other health professional ever told you that you have chronic obstructive pulmonary disease or copd , emphysema or chronic bronchitis ? these three respiratory symptom questions were selected from a validated study with a positive predictive value of 56.8% and a negative predictive value of 86.4% , which is considered to be a highly reliable questionnaire when compared with spirometry measures.18 for the analyses presented in this report , we identified respondents with a history of cigarette smoking by an affirmative response to the tobacco use question , have you smoked at least 100 cigarettes in your entire life ? and were divided into four duration groups ( 19 years , 1019 years , 2029 years , and 30 years ) . we restricted this study to adults aged 45 years because there was a low prevalence of copd ( 3.7% ) and smoking 30 years ( 1.9% ) among respondents aged 1844 years , resulting in unstable sample sizes in that age group . in the survey population of 9,386 respondents aged 45 years , 46.2% had never smoked , 33.8% were former smokers , and 18.3% were current smokers . leffondre et al has observed that inclusion of respondents who have never smoked may produce overestimations of the effects of smoking duration when that group is assigned a value of zero smoking years and is set as the referent group in an analysis;19 therefore , we restricted this study to former and current smokers ( 4,553 respondents ) aged 45 years . data from 4,135 ( 90.8% ) respondents who provided information on smoking duration , respiratory symptoms , and history of copd were analyzed in this study after those who had missing information were excluded . the distribution of smoking duration by current smoking status ( current smokers , former smokers who had quit for 10 years , and other former smokers ) was also examined . the age - adjusted prevalence and 95% ci of respiratory symptoms and copd by smoking duration and current smoking status were obtained from separate logistic regression models that included age ( 4559 years , 6069 years , and 70 years ) as the covariate . finally , we assessed the adjusted prevalence ratios and 95% cis for the likelihood of having respiratory symptoms and copd associated with smoking duration using separate multivariable logistic regression models that included sex , age , race / ethnicity ( non - hispanic white , non - hispanic black , or other / multiracial ) , education ( less than a high school diploma , a high school graduate or equivalent , or at least some college ) , and current smoking status ( current vs former ) as covariates . prior research suggesting that women might be more susceptible than men to the effects of smoking prompted testing for an interaction between sex and smoking duration in separate age - adjusted logistic regression models for each of the four dependent variables . we also assessed whether the relationships between smoking duration and the dependent variables differed by current smoking status , but failed to find a significant interaction for copd ( p=0.79 ) or for any of the three respiratory symptoms ( p>0.42 ) . table 1 shows the distribution of selected characteristics by smoking duration among 4,135 adults aged 45 years with a history of smoking . respondents who had smoked for 30 years were more likely to be male ( p<0.05 ) , have less than a high school education ( p<0.05 ) , and less likely to have some college education ( p<0.05 ) than persons who had smoked for 19 years or 1019 years . the proportion of the study population who were current smokers increased with increasing smoking duration ( linear trend , p<0.05 ) such that 58.3% of those who had smoked for 30 years continued to be current smokers compared with 7.5% of those who had smoked for 19 years . the distribution of smoking duration was 19.2% for 19 years , 21.6% for 1019 years , 23.0% for 2029 years , and 36.2% for 30 years . the increase in the proportions for each smoking duration was observed in men and women , all age groups , non - hispanic whites , non - hispanic blacks , and all education groups ( data not shown ) . figure 1 shows an inverse linear trend for smoking duration category in former smokers with quit durations of 10 years , while current smokers and other former smokers had a linear increase such that 60.0% of all current smokers and 43.6% of other former smokers had smoked for 30 years in contrast with 15.6% of former smokers who had quit for 10 years . we assessed the age - adjusted prevalence of copd and the three respiratory symptoms by current smoking status ( figure 2 ) . former smokers with quit durations 10 years reported a significantly lower age - adjusted percentage of frequent productive cough ( 10.6% vs 24.2% , respectively , p<0.001 ) , frequent sob ( 5.7% vs 9.9% , p=0.001 ) , agreement that sob affects physical activity ( 8.8% vs 13.8% , p<0.0001 ) , and copd ( 8.6% vs 22.7% , p<0.0001 ) compared with current smokers . in contrast , other former smokers with either no stated quit duration or quit durations < 10 years did not differ significantly from current smokers in prevalence of frequent sob , agreement that sob affects physical activity , and copd , although this former smoker group reported a significant lower age - adjusted percentage of frequent productive cough than did current smokers ( 11.5% vs 24.2% , p<0.0001 ) . data in table 2 demonstrate that the age - adjusted prevalence of each respiratory symptom and copd increased with increasing smoking duration ( linear trend , p<0.001 ) . among those who had smoked for 30 years , 25.6% reported copd , 25.0% had frequent productive cough , 11.2% had frequent sob , and 16.7% strongly agreed that sob had affected physical activity in the previous year . after controlling for age , sex , race / ethnicity , education , and current smoking status , there remained an increased likelihood for having copd and all three respiratory symptoms associated with increasing smoking duration ( linear trend , p<0.001 ) . however , after restricting the analyses to the 3,495 respondents who responded that they had not been diagnosed with copd , only one respiratory symptom remained significantly associated with smoking duration ( table 2 , p<0.001 ) . the age - adjusted percentage of respondents without copd who reported having frequent productive cough increased with increasing smoking duration ( linear trend , p<0.001 ) . this relationship persisted even after adjustment for age , sex , race / ethnicity , education , and current smoking status ( linear trend , p=0.009 ) . however , only a modest significant association was observed among respondents without copd and with a smoking duration 30 years compared with those in the 19 year duration ( prevalence ratio 1.85 ; 95% confidence interval 1.063.21 ) . there was a borderline significant sex interaction ( p=0.085 ) of the relationship between smoking duration and prevalence of copd ( figure 3 ) . the age - adjusted prevalence increased with increasing smoking duration for both men and women . however , women had a significantly higher age - adjusted prevalence of copd than men for a smoking duration of 19 years ( 10.1% vs 2.8% , p<0.001 ) , 2029 years ( 15.3% vs 9.2% , p=0.04 ) , and 30 years ( 30.9% vs 21.9% , p<0.001 ) but there was no difference at 1019 years ( 9.3% vs 8.9% , p=0.93 ) . our study is the first population - based health survey in many years that has assessed the relationship between duration of tobacco use and respiratory outcomes in adults with a smoking history in the usa . this study provides a population - based perspective of the relationship between smoking duration , three respiratory symptoms , and copd in a us state with a high prevalence of current cigarette smoking and copd . these relationships in a contemporary population in 2012 are consistent with similar dose response relationships observed in a norwegian study ( 19951997 ) for number of pack - years of smoking and chronic bronchitis , breathlessness , persistent coughing , coughing with phlegm , and reduced lung function.7,8 we observed that the significant relationship between smoking duration and frequent productive cough was also present in adults without a self - reported diagnosis of copd ; however , the relationship between smoking duration , frequent sob , and the effects of sob on physical activity were not significant in adults without copd . our results also showed that smoking cessation for 10 years in contrast with current smoking was associated with a much lower prevalence of copd and of each of the three respiratory symptoms . our results are consistent with the copd prevention strategy that smoking cessation improves respiratory function and prevents excessive decline in lung function in smokers with copd as well as in smokers without chronic symptoms.20,21 the health benefits of smoking cessation with regard to the rate of decline in forced expiratory volume in 1 second ( fev1 ) and mortality appear to be most significant for persons who quit before 4050 years of age.14,22 the sex difference in the age - adjusted relationship of smoking duration with copd demonstrated in this study is consistent with reports that women may be more susceptible to the detrimental effects of smoking at the 19 year duration than men . prior studies conducted in the late 1990s and early 2000s suggested that women smokers were particularly susceptible to the deleterious effects of smoking , with an augmented decline in fev1 compared with male smokers.6,8,9,11,20 in addition , results from the lung health study ( 19861994 ) showed that smoking cessation has a greater impact on rate of fev1 decline in women than in men.23 however , kohansal et al did not report a sex difference in the loss of lung function associated with continued or stopping smoking.14 suggested explanations for the female male difference in relationship between smoking duration and respiratory function have included sex variation in the prevalence of lifetime smoking , biologic differences such as smaller lungs and larger airways in women , hormonal factors that influence epithelial cell function , sex differences in inflammatory responses , greater awareness or recall of respiratory symptoms and diagnosed disease among women , and sex differences in the clinical presentation of copd.8,20,24 a previous study suggested that african - american women with copd had a greater loss of lung function due to cigarette smoking than other copd patients.5,25 however , the current study did not have a sample size sufficient to assess that relationship in groups defined by race , sex , and smoking duration . smoker s cough is consistent with chronic bronchitis , and is primarily due to excessive inflammation and hypersecretion of mucus secondary to smoking tobacco products.3,26 analysis of the copdgene cohort showed that slightly more than one quarter of copd subjects reported chronic bronchitis.27 there was also a twofold increase in risk of chronic bronchitis in current smokers in the copdgene study . chronic productive cough also increased with smoking duration among smokers with no evidence of copd in the current study . a study that evaluated the relationship among respiratory symptoms , copd , and airflow for over 30 years found that 40% of smokers developed chronic bronchitis and half of those developed copd.28 in the seven countries study , among continuous smokers without chronic bronchitis or any other pulmonary disease , those with a smoking history of 2040 pack - years ( p=0.001 ) or > 40 pack - years ( p=0.002 ) had lower mean fev0.75 than those with a smoking history of < 20 pack - years.28 furthermore , of smokers with no airflow obstruction in the copdgene study , those with chronic bronchitis had a greater pack - year smoking history and were more likely to be current smokers than those without chronic bronchitis.29 in that study , non - obstructive chronic bronchitis was defined by a history of cough and phlegm production for 3 consecutive months per year , and its results suggest that bronchitic symptoms such as productive cough in the absence of airflow obstruction could be an early marker of susceptibility and risk for copd.2931 unfortunately , copd in the current study was defined by having emphysema , chronic bronchitis , and/or copd , so that those subgroups can not be determined . little information has been available in us populations to address the relationship between smoking duration , copd , and respiratory symptoms , so this study is unique . third , the analyses relied on self - reported information from a telephone interview about current smoking , smoking duration , respiratory symptoms , and copd , and can not be validated . this finding is comparable with that of a larger 2011 us survey of approximately 13,000 adults with self - reported copd , in which the percentage of subjects having had a breathing test to diagnose copd did not differ by smoking status.37 furthermore , use of smoking duration and current smoking status give only partial information about tobacco use . other useful information for such a study could include intensity ( number or packs smoked per day ) , pack - years ( a product of intensity and duration ) , age at initiation , and time since cessation , but such information can not be obtained during a time - limited interview.19 however , our results are consistent with findings for pack - years of smoking that have been applied in previous studies.57 in addition , using smoking duration rather than pack - years or additional smoking indicators that employ a time factor in the same analytic models may have helped to reduce multicollinearity and avoid a cohort effect.19 the us preventive services task force so far has found no evidence to support screening adults ( including asymptomatic smokers ) for copd using spirometry.38 however , the recommendations do suggest that current smokers should receive smoking cessation counseling and pharmacologic therapies that have been demonstrated to increase cessation rates . smoking cessation may improve respiratory symptoms and bronchial hyperresponsiveness and prevent excessive decline in lung function among smokers with copd as well as among smokers without chronic symptoms.21 smokers with respiratory symptoms , including dyspnea and productive cough , may be more susceptible to the effects of smoking on rate of decline in fev1 than those without symptoms , thus representing an important target group for smoking cessation interventions.14 the united states public health service guidelines recommend a simple intervention of 5 as , ie , ask about tobacco use , advise to quit , assess willingness to make a cessation attempt , assist in cessation attempt , and arrange follow - up to make prevention and cessation of tobacco use more successful.39 therefore , there are opportunities for physicians to provide tobacco cessation counseling to all patients who smoke , particularly those with respiratory symptoms and/or copd . in this population - based survey of adults with a smoking history , prolonged tobacco use was associated with an increased likelihood of having copd , frequent productive cough , frequent sob , and agreement that sob affected physical activity even after controlling for current smoking behavior . former smokers who had quit smoking for 10 years had a lower prevalence of copd and respiratory symptoms than current smokers .

the prospective mri part of the study was approved by the local ethics committee ( ea 1/054/09 ) . patient 1 is a 62-year old man who was admitted in 2008 because of subacute onset diplopia , dysphagia , and balance problems ( table ) . neurologic examination revealed gaze - evoked nystagmus , diplopia , limb and gait ataxia , and moderate postural tremor of the upper extremities . mri showed patchy t2 signal hyperintensities in the brainstem and cerebellar peduncles with punctate gadolinium enhancements ( figure 1 ) . three lumbar punctures during the disease course revealed mild lymphocytic pleocytosis ( 37 , 17 , and 15 leukocytes per microliter ) but no oligoclonal bands . there was no evidence of malignant cells and viral examinations of the csf for herpesviruses were negative . search for onconeural antibodies , antinuclear antibodies , antineutrophil cytoplasmic antibodies , anti - ssa , anti - ssb , anticardiolipin , elevated angiotensin - converting enzyme in sera and csf , and tuberculosis tests were normal . the patient recovered on high - dose steroid treatment , but relapsed after its withdrawal . there was no evidence of lymphoma or vessel destruction . staining for herpes simplex virus 1 and 2 were normal . the patient responded to steroid treatment , but then had a fatal brainstem hemorrhage . in 2014 , the history and initial brain biopsy specimens were re - evaluated and clippers syndrome was diagnosed . demographics , clinical symptoms , treatment , and timing of mri or neuropathologic investigations of patients in this study ( a ) mri of patient 1 shows t2-weighted hyperintensities in brainstem and cerebellar peduncles with similar pattern of gadolinium enhancement . arrows depict areas used for immunohistochemical stainings for cd45 , cd3 , cd4 , cd8 , and cd20 in brainstem ( b f ) and cerebellum ( l p ) with punctate gadolinium enhancements and parietal ( g k ) and insular cortex ( q u ) with normal mri appearance . we found prominent cd4 + t - cell infiltration in the brainstem ( d ) and cerebellum ( n ) but also in insular cortex ( s ) and parietal cortex ( i ) . the magnitude of infiltration shows a gradient with less infiltration with greater distance from the brainstem . similarly , we found less infiltration of cd8 + t cells ( e , j , o , and t ) and only a very limited number of cd20 + b cells ( f , k , p , and u ) with a similar gradient in cell numbers and degree of infiltration into the tissue . hematoxylin & eosin staining reveals inflammation also in cranial nerve roots from the brainstem ( v and w ) . magnification 5 ( b and f ) ( v and w ) , 10 ( g u ) . scale bar : 200 m ( b f ) , 100 m ( g u ) , 200 m ( v , w ) . the clinical course , mri data , and follow - up of the 3 patients have been recently described elsewhere . patient 2 is a 23-year - old man who developed subacute nystagmus and gait ataxia ( table ) . he was treated with methylprednisolone and azathioprine , but had 6 relapses in 14 years with gradual worsening of cerebellar dysarthria and gait ataxia . no systemic autoantibodies were found . at present , the patient is ambulatory ( modified rankin scale [ mrs ] 2 ) . patient 3 is a 58-year - old woman who developed subacute gait ataxia , dysarthria , and paresthesia of the face , hands , and feet ( table ) . brain biopsy indicated parenchymal and perivascular inflammatory infiltrates predominantly composed of cd3 + t cells . no systemic autoantibodies were found . at present , the patient is severely dysarthric , wheelchair - bound , and has mild involuntary movements ( mrs 4 ) . patient 4 is a 42-year - old man who developed subacute dysarthria , horizontal nystagmus , diplopia , jerky eye movements , ataxia , tetraspasticity , and paraparesis ( table ) . brain biopsy indicated parenchymal and perivascular inflammatory infiltrates predominantly composed of cd3 + t cells . mri showed multiple punctate gadolinium - enhancing lesions in the cerebellum , brainstem , and midbrain . he was treated with methotrexate and azathioprine , and had only a single relapse during the follow - up period of 19 months ( mrs 2 ) . patient 5 is a 48-year - old woman who developed subacute numbness in the left half of her body , double vision , gait instability , dysarthria , facial pain , numbness of the oral cavity , and fatigue over a period of 2 weeks in september 2013 ( table ) . during the weeks before the onset of these symptoms the patient was admitted to another hospital in october 2013 , where a cranial 1.5 t mri demonstrated multiple small t2 hyperintense lesions with punctate and curvilinear contrast enhancement in the pons and cerebellum , and a bigger right cerebellar lesion . as this case has not been published before , the brain mri findings of this patient are shown in figure e-1 at neurology.org/nn . a spinal cord mri demonstrated a small longitudinal contrast - enhancing lesion in the cervical cord . histologic analysis revealed cerebellitis with prominent cd4 + t - cellular infiltrates , compatible with clippers syndrome . antinuclear antibodies , antibodies to extractable nuclear antigens , and antineutrophil cytoplasmic antibodies as well as antibodies to aquaporin-4 were negative . she was initially treated with high - dose iv methylprednisolone ( 1 g / day for 3 days followed by 1 g / day for 5 days ) and subsequently with oral prednisolone ( starting dose 25 mg / day with a subsequent taper to 5 mg / day ) and azathioprine ( 3 50 mg ) , which was changed to methotrexate in may 2014 , when a cranial mri demonstrated constant to minimally progressive clippers lesions . at the time of the 7.0 t mri examination , she was treated with prednisolone ( 5 mg daily ) and methotrexate ( 15 mg weekly ) , and had residual facial paresthesiae but no clinical signs of new disease activity . patient 6 is a 73-year - old woman who was diagnosed with clippers in october 2010 and whose clinical and paraclinical details were previously reported in detail . the patient had mildly elevated antinuclear antibodies ( 1:320 ) and double - stranded dna antibodies ( immunoglobulin g ) . at the time of the 7.0 t mri examination , she was treated with methylprednisolone ( 4 mg daily ) and methotrexate ( 15 mg weekly ) and was in clinical remission . three - micrometer sections from formalin - fixed , paraffin - embedded tissue blocks were stained with hematoxylin & eosin ( h&e ) and for cd45 ( clone : 2b11&pd7/26 ventana - lca , ventana medical systems , tucson , az ) , cd3 ( clone : 2gv6 , ventana medical systems ) , cd4 ( sp35 , ventana medical systems ) , cd8 ( clone : c8/144b , dako , glostrup , denmark ) , cd20 ( clone : cyl26 , ventana medical systems ) , and neurofilament heavy - chain ( nf smi31 , affinity , exeter , uk ) . selected sections from brainstem and insular adjacent formalin - fixed tissue section from brainstem tissue ( autopsy ) as well as sections from 3 additional brainstem biopsies were similarly stained for light - chain restriction ( kappa - smi and lambda - smi , smlg reaction , dako ) , the zinc - finger transcription factor , b - cell lymphoma 6 protein ( bcl-6 , clone : ln22 novocastra , leica biosystems , newcastle , uk ) , the large b - cell lymphoma marker , multiple myeloma oncogene 1 ( mum-1 , irf4 , dako ) , and the precursor b lymphoma marker , cd10 ( clone : 56c6-calla , novocastra , leica biosystems ) . all immunohistochemical stainings were performed using the benchmark ultra ihc staining system ( ventana medical systems ) . for detection , digital images were obtained at a magnification of 5 or 10 ( brainstem ) or 10 ( cerebellum , parietal cortex , insular cortex / basal ganglia , nerve root , and biopsies 13 ) using a leica microscope ( leica 4000b led , leica microsystems , wetzlar , germany ) equipped with a leica digital camera ( leica dfc420 , leica microsystems ) . ultra - high - field mri were acquired using a 7.0 t whole body mri scanner ( magnetom , siemens , erlangen , germany ) applying a 24-channel receive head coil ( nova medical , wilmington , ma ) equipped with a birdcage volume coil used for transmission . the imaging protocol included 2d t2 * -weighted ( t2*w ) fast low angle shot ( echo time [ te ] = 25.0 ms , repetition time [ tr ] = 1,820 ms , spatial resolution = 0.2 0.2 2 mm , supratentorial coverage ) , pregadolinium and postgadolinium 3d t1-weighted magnetization - prepared rapid gradient echo ( t1w mprage ) ( te = 2.98 ms , tr = 2,300 ms , inversion time = 900 ms , spatial resolution = 1.0 1.0 1.0 mm , whole brain coverage ) , and 3d flow - compensated gradient echo based susceptibility - weighted imaging ( swi ) ( te = 14 ms , tr = 25 ms , flip angle = 12 , spatial resolution = 0.5 0.5 1.0 mm , supratentorial and infratentorial coverage ) . for infratentorial imaging , postgadolinium volumetric interpolated brain examination ( vibe ) sequence was used ( te = 3.3 ms , tr = 19.7 ms , spatial resolution = 0.5 0.5 1.0 mm ) . to identify vascular structures within contrast - enhancing clippers lesions , we calculated the sum of ( 1 ) coregistered t1w mprage with supratentorial t2*w images , and ( 2 ) coregistered vibe with infratentorial swi images using the image calculator function of the mipav software package ( version 7.0.1 , bethesda , md ) . the prospective mri part of the study was approved by the local ethics committee ( ea 1/054/09 ) . patient 1 is a 62-year old man who was admitted in 2008 because of subacute onset diplopia , dysphagia , and balance problems ( table ) . neurologic examination revealed gaze - evoked nystagmus , diplopia , limb and gait ataxia , and moderate postural tremor of the upper extremities . mri showed patchy t2 signal hyperintensities in the brainstem and cerebellar peduncles with punctate gadolinium enhancements ( figure 1 ) . three lumbar punctures during the disease course revealed mild lymphocytic pleocytosis ( 37 , 17 , and 15 leukocytes per microliter ) but no oligoclonal bands . there was no evidence of malignant cells and viral examinations of the csf for herpesviruses were negative . search for onconeural antibodies , antinuclear antibodies , antineutrophil cytoplasmic antibodies , anti - ssa , anti - ssb , anticardiolipin , elevated angiotensin - converting enzyme in sera and csf , and tuberculosis tests were normal . the patient recovered on high - dose steroid treatment , but relapsed after its withdrawal . there was no evidence of lymphoma or vessel destruction . staining for herpes simplex virus 1 and the patient responded to steroid treatment , but then had a fatal brainstem hemorrhage . in 2014 , the history and initial brain biopsy specimens were re - evaluated and clippers syndrome was diagnosed . demographics , clinical symptoms , treatment , and timing of mri or neuropathologic investigations of patients in this study ( a ) mri of patient 1 shows t2-weighted hyperintensities in brainstem and cerebellar peduncles with similar pattern of gadolinium enhancement . arrows depict areas used for immunohistochemical stainings for cd45 , cd3 , cd4 , cd8 , and cd20 in brainstem ( b f ) and cerebellum ( l p ) with punctate gadolinium enhancements and parietal ( g k ) and insular cortex ( q u ) with normal mri appearance . we found prominent cd4 + t - cell infiltration in the brainstem ( d ) and cerebellum ( n ) but also in insular cortex ( s ) and parietal cortex ( i ) . the magnitude of infiltration shows a gradient with less infiltration with greater distance from the brainstem . similarly , we found less infiltration of cd8 + t cells ( e , j , o , and t ) and only a very limited number of cd20 + b cells ( f , k , p , and u ) with a similar gradient in cell numbers and degree of infiltration into the tissue . hematoxylin & eosin staining reveals inflammation also in cranial nerve roots from the brainstem ( v and w ) . magnification 5 ( b and f ) ( v and w ) , 10 ( g u ) . scale bar : 200 m ( b f ) , 100 m ( g u ) , 200 m ( v , w ) . the clinical course , mri data , and follow - up of the 3 patients have been recently described elsewhere . patient 2 is a 23-year - old man who developed subacute nystagmus and gait ataxia ( table ) . he was treated with methylprednisolone and azathioprine , but had 6 relapses in 14 years with gradual worsening of cerebellar dysarthria and gait ataxia . no systemic autoantibodies were found . at present , the patient is ambulatory ( modified rankin scale [ mrs ] 2 ) . patient 3 is a 58-year - old woman who developed subacute gait ataxia , dysarthria , and paresthesia of the face , hands , and feet ( table ) . brain biopsy indicated parenchymal and perivascular inflammatory infiltrates predominantly composed of cd3 + t cells . no systemic autoantibodies were found . at present , the patient is severely dysarthric , wheelchair - bound , and has mild involuntary movements ( mrs 4 ) . patient 4 is a 42-year - old man who developed subacute dysarthria , horizontal nystagmus , diplopia , jerky eye movements , ataxia , tetraspasticity , and paraparesis ( table ) . brain biopsy indicated parenchymal and perivascular inflammatory infiltrates predominantly composed of cd3 + t cells . mri showed multiple punctate gadolinium - enhancing lesions in the cerebellum , brainstem , and midbrain . he was treated with methotrexate and azathioprine , and had only a single relapse during the follow - up period of 19 months ( mrs 2 ) . patient 5 is a 48-year - old woman who developed subacute numbness in the left half of her body , double vision , gait instability , dysarthria , facial pain , numbness of the oral cavity , and fatigue over a period of 2 weeks in september 2013 ( table ) . during the weeks before the onset of these symptoms the patient was admitted to another hospital in october 2013 , where a cranial 1.5 t mri demonstrated multiple small t2 hyperintense lesions with punctate and curvilinear contrast enhancement in the pons and cerebellum , and a bigger right cerebellar lesion . as this case has not been published before , the brain mri findings of this patient are shown in figure e-1 at neurology.org/nn . a spinal cord mri demonstrated a small longitudinal contrast - enhancing lesion in the cervical cord . histologic analysis revealed cerebellitis with prominent cd4 + t - cellular infiltrates , compatible with clippers syndrome . antinuclear antibodies , antibodies to extractable nuclear antigens , and antineutrophil cytoplasmic antibodies as well as antibodies to aquaporin-4 were negative . she was initially treated with high - dose iv methylprednisolone ( 1 g / day for 3 days followed by 1 g / day for 5 days ) and subsequently with oral prednisolone ( starting dose 25 mg / day with a subsequent taper to 5 mg / day ) and azathioprine ( 3 50 mg ) , which was changed to methotrexate in may 2014 , when a cranial mri demonstrated constant to minimally progressive clippers lesions . at the time of the 7.0 t mri examination , she was treated with prednisolone ( 5 mg daily ) and methotrexate ( 15 mg weekly ) , and patient 6 is a 73-year - old woman who was diagnosed with clippers in october 2010 and whose clinical and paraclinical details were previously reported in detail . the patient had mildly elevated antinuclear antibodies ( 1:320 ) and double - stranded dna antibodies ( immunoglobulin g ) . antibodies against aquaporin-4 and extractable nuclear antigens were negative . at the time of the 7.0 t mri examination , she was treated with methylprednisolone ( 4 mg daily ) and methotrexate ( 15 mg weekly ) and was in clinical remission . three - micrometer sections from formalin - fixed , paraffin - embedded tissue blocks were stained with hematoxylin & eosin ( h&e ) and for cd45 ( clone : 2b11&pd7/26 ventana - lca , ventana medical systems , tucson , az ) , cd3 ( clone : 2gv6 , ventana medical systems ) , cd4 ( sp35 , ventana medical systems ) , cd8 ( clone : c8/144b , dako , glostrup , denmark ) , cd20 ( clone : cyl26 , ventana medical systems ) , and neurofilament heavy - chain ( nf smi31 , affinity , exeter , uk ) . selected sections from brainstem and insular adjacent formalin - fixed tissue section from brainstem tissue ( autopsy ) as well as sections from 3 additional brainstem biopsies were similarly stained for light - chain restriction ( kappa - smi and lambda - smi , smlg reaction , dako ) , the zinc - finger transcription factor , b - cell lymphoma 6 protein ( bcl-6 , clone : ln22 novocastra , leica biosystems , newcastle , uk ) , the large b - cell lymphoma marker , multiple myeloma oncogene 1 ( mum-1 , irf4 , dako ) , and the precursor b lymphoma marker , cd10 ( clone : 56c6-calla , novocastra , leica biosystems ) . all immunohistochemical stainings were performed using the benchmark ultra ihc staining system ( ventana medical systems ) . for detection , digital images were obtained at a magnification of 5 or 10 ( brainstem ) or 10 ( cerebellum , parietal cortex , insular cortex / basal ganglia , nerve root , and biopsies 13 ) using a leica microscope ( leica 4000b led , leica microsystems , wetzlar , germany ) equipped with a leica digital camera ( leica dfc420 , leica microsystems ) . ultra - high - field mri were acquired using a 7.0 t whole body mri scanner ( magnetom , siemens , erlangen , germany ) applying a 24-channel receive head coil ( nova medical , wilmington , ma ) equipped with a birdcage volume coil used for transmission . the imaging protocol included 2d t2 * -weighted ( t2*w ) fast low angle shot ( echo time [ te ] = 25.0 ms , repetition time [ tr ] = 1,820 ms , spatial resolution = 0.2 0.2 2 mm , supratentorial coverage ) , pregadolinium and postgadolinium 3d t1-weighted magnetization - prepared rapid gradient echo ( t1w mprage ) ( te = 2.98 ms , tr = 2,300 ms , inversion time = 900 ms , spatial resolution = 1.0 1.0 1.0 mm , whole brain coverage ) , and 3d flow - compensated gradient echo based susceptibility - weighted imaging ( swi ) ( te = 14 ms , tr = 25 ms , flip angle = 12 , spatial resolution = 0.5 0.5 1.0 mm , supratentorial and infratentorial coverage ) . for infratentorial imaging , postgadolinium volumetric interpolated brain examination ( vibe ) sequence was used ( te = 3.3 ms , tr = 19.7 ms , spatial resolution = 0.5 0.5 1.0 mm ) . to identify vascular structures within contrast - enhancing clippers lesions , we calculated the sum of ( 1 ) coregistered t1w mprage with supratentorial t2*w images , and ( 2 ) coregistered vibe with infratentorial swi images using the image calculator function of the mipav software package ( version 7.0.1 , bethesda , md ) . first , we investigated inflammation in different brain regions corresponding to gadolinium - enhancing and normal - appearing brain areas on 3.0 t mri ( autopsy tissue of patient 1 ) : brainstem and cerebellum were compared to parietal and insular cortex ( figure 1 ) . perivascular accumulation of cd45 + lymphocytes ( figure 1 , b , g , l , and q ) with predominance of cd3 + t cells ( figure 1 , c , h , m and r ) and cd4 + t cells were detected in brainstem and cerebellum as expected ( figure 1 , d and n ) . cd4 + t cells also infiltrated the parenchyma , which was most pronounced in the brainstem ( figure 1d ) . accumulation and infiltration by cd8 + t cells were less pronounced ( figure 1 , e and q ) , and only a few cd20 + b cells were observed in close proximity to the vessel wall ( figure 1 , f and p ) . however , insular and parietal cortex with normal appearance on 3.0 t mri revealed a similar , albeit less prominent perivascular accumulation of cd4 + t cells ( figure 1 , i and s ) , fewer cd8 + t cells ( figure 1 , j and t ) , and a very limited number of cd20 + b cells ( figure 1 , k and u ) . cd4 + t cells also infiltrated the parenchyma the insular cortex ( figure 1s ) . perivascular inflammation was also found in the roots of cranial nerves ( figure 1 , v and w ) . these data suggested that brain areas with normal appearance on 3.0 t mri may be affected by inflammation in clippers syndrome . next , brainstem samples ( patients 14 ) were analyzed for the possibility of lymphoma or prelymphoma . we found no evidence for monoclonal cell expansion using staining for light chain restriction ( figure 2 , a b , f l , and p q ) and lymphoma markers cd10 , bcl-6 , and mum-1 ( figure 2 , c e , h j , m o , and r follow - up of the 3 biopsy cases ( patients 24 ; mean of 75 months ) did not indicate clinical or paraclinical evidence of lymphoma either . immunohistochemical stainings for light chain restriction ( lambda - smi and kappa - smi : a , b , f , g , k , l , p , and q ) , cd10 ( c , h , m , and r ) , bcl-6 ( d , i , n , and s ) , and mum-1 ( e , j , o , and t ) in samples from an autopsy ( a e ) and biopsy specimens of 3 patients ( f j , k we found no evidence for light chain restriction or significant staining for lymphoma markers cd10 , bcl-6 , or mum-1 suggesting a prelymphoma or lymphoma state . scale bar : 200 m ( a e ) , 100 m ( f t ) . since pathologic examination indicated perivascular inflammation in brain areas with normal appearance on 3.0 t mri , albeit less pronounced than in brainstem and cerebellum , we next examined 2 patients with clippers by 7.0 t mri during clinical remission . contrast - enhancing lesions ( n = 20 ) were only depicted in patient 5 . of those , 2 lesions were found in a supratentorial localization affecting ( n = 1 ) or bordering ( n = 1 ) the thalamus ( figure 3d ) . supratentorial t2 * -weighted ( a , b ) and postgadolinium t1-weighted images ( d ) of patient 5 obtained at 7.0 t are displayed . postgadolinium t1-weighted imaging depicts a small contrast - enhancing lesion ( black arrow , d ) that is only marginally delineated on corresponding t2 * -weighted images ( zoom , b ) despite using a very high spatial resolution of 0.08 mm . fusion ( c ) of coregistered postgadolinium t1-weighted ( d ) and t2 * -weighted ( b ) identifies a small brain vessel within the center of the chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids ( clippers ) lesion ( white arrow , c ) . * -weighted fast low angle shot at 7.0 t , echo time ( te ) = 25.0 ms , repetition time ( tr ) = 1,820 ms , spatial resolution = 0.2 0.2 2 mm ; t1-gad = postgadolinium 3d t1-weighted magnetization - prepared rapid gradient echo at 7.0 t , te = 2.98 ms , tr = 2,300 ms , inversion time = 900 ms , spatial resolution = 1.0 1.0 1.0 mm . to visualize the perivascular distribution of lesions , we fused contrast - enhanced t1w and supratentorial t2*w images with a volume resolution of 0.08 mm to display small t2*w hypointense venous structures within gadolinium - enhancing hyperintense clippers lesions ( figure 3 ) . indeed , a distinct venous vessel could be seen within the center of the thalamic clippers lesion in vivo ( figure 3c ) . due to technical limitations , a different approach with a lower spatial resolution of 0.25 mm was used for infratentorial areas . even with this approach , a very small venous vessel could be depicted within 8 of 10 contrast - enhancing clippers lesions on fused postgadolinium vibe and swi ( data not shown ) . owing to substantially increased signal - to - noise ratio , 7.0 t t1-weighted magnetization - prepared rapid gradient echo delineates numerous t1-weighted hypointense lesions within the pons and cerebellum ( arrowheads , e in contrast , corresponding areas appear normal ( c , d ) or only marginally hypointense on t1-weighted images at 3.0 t ( circle , a , b ) . t1-weighted hypointensity within non - contrast - enhancing chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids lesions may indicate postinflammatory structural brain damage including axonal loss on top of demyelination . immunohistochemical stainings of autopsy case for neurofilament ( nf ) revealed massive axonal injury in the inflammatory lesion of the brainstem ( h ; cd3 red , nf brown ) compared to insular cortex with limited inflammation ( j ) . the presence of phosphorylated nf in the perinuclear cytoplasm of neurons suggests retrograde neuronal degeneration due to axonal injury in the lesions ( h ) . similarly , severe loss of perivascular myelin was found in areas of severe inflammation ( i ) , compared to areas with limited inflammation ( k ) , with luxol fast blue and hematoxylin & eosin . 7.0 t = 3d t1-weighted magnetization - prepared rapid gradient echo at 7.0 t , echo time = 2.98 ms , repetition time = 2,300 ms , inversion time = 900 ms , spatial resolution = 1.0 1.0 1.0 mm ; 3.0 t = standard t1-weighted axial images ( b d ) and t1-weighted magnetization - prepared rapid gradient echo at 3.0 t ( a ) . scale bar = 100 m . in addition , 7.0 t t1w mprage depicted numerous t1w hypointense lesions within the pons and cerebellum ( figure 4 , e g ) in patient 5 , which were not visible on conventional 3.0 t mri ( figure 4 , a d ) , indicating severe tissue destruction within postinflammatory clippers lesions ( figure 4 ) . multiple pontine postinflammatory t1w hypointense lesions were also found in patient 6 by using t1w imaging at 7.0 t ( data not shown ) . corresponding to the 7.0 t mri findings , neurofilament and myelin staining revealed axonal injury and myelin loss in brainstem areas with severe inflammation of the autopsy case ( figure 4 , h and i ) . preservation of axons and myelin was seen in areas with limited inflammation ( figure 4 , j and k ) . first , we investigated inflammation in different brain regions corresponding to gadolinium - enhancing and normal - appearing brain areas on 3.0 t mri ( autopsy tissue of patient 1 ) : brainstem and cerebellum were compared to parietal and insular cortex ( figure 1 ) . perivascular accumulation of cd45 + lymphocytes ( figure 1 , b , g , l , and q ) with predominance of cd3 + t cells ( figure 1 , c , h , m and r ) and cd4 + t cells were detected in brainstem and cerebellum as expected ( figure 1 , d and n ) . cd4 + t cells also infiltrated the parenchyma , which was most pronounced in the brainstem ( figure 1d ) . accumulation and infiltration by cd8 + t cells were less pronounced ( figure 1 , e and q ) , and only a few cd20 + b cells were observed in close proximity to the vessel wall ( figure 1 , f and p ) . however , insular and parietal cortex with normal appearance on 3.0 t mri revealed a similar , albeit less prominent perivascular accumulation of cd4 + t cells ( figure 1 , i and s ) , fewer cd8 + t cells ( figure 1 , j and t ) , and a very limited number of cd20 + b cells ( figure 1 , k and u ) . cd4 + t cells also infiltrated the parenchyma the insular cortex ( figure 1s ) . perivascular inflammation was also found in the roots of cranial nerves ( figure 1 , v and w ) . these data suggested that brain areas with normal appearance on 3.0 t mri may be affected by inflammation in clippers syndrome . next , brainstem samples ( patients 14 ) were analyzed for the possibility of lymphoma or prelymphoma . we found no evidence for monoclonal cell expansion using staining for light chain restriction ( figure 2 , a b , f l , and p q ) and lymphoma markers cd10 , bcl-6 , and mum-1 ( figure 2 , c e , h j , m follow - up of the 3 biopsy cases ( patients 24 ; mean of 75 months ) did not indicate clinical or paraclinical evidence of lymphoma either . immunohistochemical stainings for light chain restriction ( lambda - smi and kappa - smi : a , b , f , g , k , l , p , and q ) , cd10 ( c , h , m , and r ) , bcl-6 ( d , i , n , and s ) , and mum-1 ( e , j , o , and t ) in samples from an autopsy ( a e ) and biopsy specimens of 3 patients ( f j , k we found no evidence for light chain restriction or significant staining for lymphoma markers cd10 , bcl-6 , or mum-1 suggesting a prelymphoma or lymphoma state . scale bar : 200 m ( a e ) , 100 m ( f t ) . since pathologic examination indicated perivascular inflammation in brain areas with normal appearance on 3.0 t mri , albeit less pronounced than in brainstem and cerebellum , we next examined 2 patients with clippers by 7.0 t mri during clinical remission . contrast - enhancing lesions ( n = 20 ) were only depicted in patient 5 . of those , 2 lesions were found in a supratentorial localization affecting ( n = 1 ) or bordering ( n = 1 ) the thalamus ( figure 3d ) . supratentorial t2 * -weighted ( a , b ) and postgadolinium t1-weighted images ( d ) of patient 5 obtained at 7.0 t are displayed . postgadolinium t1-weighted imaging depicts a small contrast - enhancing lesion ( black arrow , d ) that is only marginally delineated on corresponding t2 * -weighted images ( zoom , b ) despite using a very high spatial resolution of 0.08 mm . fusion ( c ) of coregistered postgadolinium t1-weighted ( d ) and t2 * -weighted ( b ) identifies a small brain vessel within the center of the chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids ( clippers ) lesion ( white arrow , c ) . t2*w = 2d t2 * -weighted fast low angle shot at 7.0 t , echo time ( te ) = 25.0 ms , repetition time ( tr ) = 1,820 ms , spatial resolution = 0.2 0.2 2 mm ; t1-gad = postgadolinium 3d t1-weighted magnetization - prepared rapid gradient echo at 7.0 t , te = 2.98 ms , tr = 2,300 ms , inversion time = 900 ms , spatial resolution = 1.0 1.0 1.0 mm . to visualize the perivascular distribution of lesions , we fused contrast - enhanced t1w and supratentorial t2*w images with a volume resolution of 0.08 mm to display small t2*w hypointense venous structures within gadolinium - enhancing hyperintense clippers lesions ( figure 3 ) . indeed , a distinct venous vessel could be seen within the center of the thalamic clippers lesion in vivo ( figure 3c ) . due to technical limitations , a different approach with a lower spatial resolution of 0.25 mm even with this approach , a very small venous vessel could be depicted within 8 of 10 contrast - enhancing clippers lesions on fused postgadolinium vibe and swi ( data not shown ) . t1-weighted images of patient 5 at 3.0 t and 7.0 t are shown . owing to substantially increased signal - to - noise ratio , 7.0 t t1-weighted magnetization - prepared rapid gradient echo delineates numerous t1-weighted hypointense lesions within the pons and cerebellum ( arrowheads , e in contrast , corresponding areas appear normal ( c , d ) or only marginally hypointense on t1-weighted images at 3.0 t ( circle , a , b ) . t1-weighted hypointensity within non - contrast - enhancing chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids lesions may indicate postinflammatory structural brain damage including axonal loss on top of demyelination . immunohistochemical stainings of autopsy case for neurofilament ( nf ) revealed massive axonal injury in the inflammatory lesion of the brainstem ( h ; cd3 red , nf brown ) compared to insular cortex with limited inflammation ( j ) . the presence of phosphorylated nf in the perinuclear cytoplasm of neurons suggests retrograde neuronal degeneration due to axonal injury in the lesions ( h ) . similarly , severe loss of perivascular myelin was found in areas of severe inflammation ( i ) , compared to areas with limited inflammation ( k ) , with luxol fast blue and hematoxylin & eosin . 7.0 t = 3d t1-weighted magnetization - prepared rapid gradient echo at 7.0 t , echo time = 2.98 ms , repetition time = 2,300 ms , inversion time = 900 ms , spatial resolution = 1.0 1.0 1.0 mm ; 3.0 t = standard t1-weighted axial images ( b d ) and t1-weighted magnetization - prepared rapid gradient echo at 3.0 t ( a ) . in addition , 7.0 t t1w mprage depicted numerous t1w hypointense lesions within the pons and cerebellum ( figure 4 , e g ) in patient 5 , which were not visible on conventional 3.0 t mri ( figure 4 , a d ) , indicating severe tissue destruction within postinflammatory clippers lesions ( figure 4 ) . multiple pontine postinflammatory t1w hypointense lesions were also found in patient 6 by using t1w imaging at 7.0 t ( data not shown ) . corresponding to the 7.0 t mri findings , neurofilament and myelin staining revealed axonal injury and myelin loss in brainstem areas with severe inflammation of the autopsy case ( figure 4 , h and i ) . preservation of axons and myelin was seen in areas with limited inflammation ( figure 4 , j and k ) . in this article , we describe neuropathologic as well as high - field / ultra - high - field mri features of 6 patients with clippers syndrome . the diagnosis of clippers syndrome was supported by ( 1 ) postcontrast t1-weighted mri with typical punctate , nodular , or curvilinear and occasionally more confluent gadolinium - enhancing lesions , ( 2 ) perivascular inflammation in the brainstem dominated by t cells , ( 3 ) clinical course with several steroid - responsive relapses , and ( 4 ) absence of other diseases . in addition to punctate and curvilinear lesions , case 5 had a larger confluent lesion in the cerebellum . similar lesions were previously described in patients with clippers and a biopsy of the cerebellar lesion of case 5 demonstrated histopathologic features of clippers . we found widespread perivascular inflammation in brain areas with normal appearance on 3.0 t mri in an autopsy case of clippers syndrome . the inflammatory pattern of these regions was similar to the original and later neuropathologic descriptions of the disease characterized by pronounced cd4 + t - cell infiltration of brain tissue and less predominant angiocentric cd8 + t - cell and cd20 + b - cell inflammation . although t2w and gadolinium - enhancing lesions were found only in the pons , midbrain , and cerebellar regions , inflammation could be seen as distant as the parietal cortex and even involved the cranial nerve roots . the degree of inflammation , however , showed a gradient of less inflammation with greater distance from the primarily affected areas ( brainstem > cerebellum > insular cortex > parietal cortex ) , suggesting that the inflammation seen on conventional 1.5t3.0 t mri only depicts the most severely affected brain regions . consistent with these neuropathologic findings , cognitive impairment such as dysexecutive syndromes , language disturbance , frontal release signs and cortical atrophy were described in a case series of clippers syndrome , suggesting that cortical involvement may be more prominent than initially thought . since our pathologic examinations revealed widespread inflammatory changes in brain tissue appearing normal on conventional 3.0 t mri , we performed ultra - high - field 7.0 t mri in 2 additional clippers cases to investigate such possibilities in vivo . indeed , these efforts revealed contrast - enhancing lesions in supratentorial locations , supporting widespread inflammation in brain areas , in contrast to the only other autopsy report published recently . the number of such contrast - enhancing lesions was , however , limited , which is likely due to the immunosuppressive treatment : mri changes in clippers respond rapidly to immunosuppression . remarkably , when combining t1w and supratentorial t2*w images with a resolution as good as 0.08 mm , we could show a vascular structure within most contrast - enhancing lesions . the 7.0 t mri thus enabled visualization of perivascular inflammation , the neuropathologic hallmark of clippers , in vivo . the hypointense appearance of these intralesional vascular structures on t2*w and swi images indicates the presence of a paramagnetic material . thus , these intralesional vascular structures are most likely small veins filled with paramagnetic deoxyhemoglobin . a central intralesional vein was previously reported to be a characteristic finding in ms lesions . on the contrary , a central vein is only rarely depictable in brain lesions of other origin such as neuromyelitis optica , cerebral small vessel disease , and susac syndrome , which is considered a microangiopathy of the brain , cochlea , and retina . evidence for the presence of mri - detectable intralesional veins in other inflammatory vasculopathies such as wegner granulomatosis , behet syndrome , primary cns vasculitis , and vasculitis in systemic lupus erythematosus is , however , limited . of note , highly resolving imaging techniques were needed to visualize the intralesional venous vessel in clippers lesions , which thus appear much smaller in diameter than central veins commonly visualized in ms lesions on 7.0 t t2*w images . however , as no systematic analysis of vessel diameters in clippers vs ms could be performed owing to the low sample size , future comparative studies will have to investigate whether this feature is consistently detectable in other clippers cases and distinct from ms . in addition , we utilized the higher sensitivity of 7.0 t t1w mprage vs t1w imaging at 1.5 t and 3.0 t to detect brain lesions and to investigate brain areas that appeared normal at lower field strengths . although 1.5t3.0 t mri and 7.0 t mri were done at different time points , therefore direct comparison was not possible , patients had no signs of clinical activity at the time of the mri examinations . ultra - high - field mri revealed numerous t1w hypointense lesions within the pons and cerebellum in one of the patients , indicating postinflammatory brain tissue destruction , which were not visible on conventional 3.0 t mri . indeed , axonal injury with evidence of axonal spheroids and torpedoes was found in our autopsy case , similar to previous results . however , axonal injury and myelin loss was observed only in areas with severe inflammation , which may explain the restricted localization of t1w hypointense lesions on 7.0 t mri . since emerging cases of clippers with subsequent diagnosis of lymphoma have been recently described , we investigated this possibility in the autopsy case and available biopsy samples from 3 other danish cases . we found no evidence for prelymphoma or lymphoma when using immunohistochemical staining for light chain restriction or lymphoma markers cd10 , bcl-6 , and mum-1 . since the analyses were done on the initial diagnostic biopsies , this might not exclude per se that the patients progressed into a lymphoma . however , 3 of the patients have been already followed for a mean of more than 6 years without any evidence of lymphoma . altogether , our present results strengthen the concept that clippers is a distinct disease entity that can be discerned from alternative diagnoses such as lymphoma or vasculitis . we found perivascular inflammation predominated by cd4 + t cells even in areas with normal appearance on 3.0 t mri and extending to the cranial nerve roots , suggesting widespread inflammation in clippers syndrome , though most prominent in the primarily affected brainstem . we found no evidence of lymphoma in our 4 investigated patients . when applying ultra - high - field mri at 7.0 t , supratentorial lesions could be confirmed , and most of the contrast - enhancing lesions contained a vessel structure indicating the pathologic hallmark of clippers syndrome in vivo . the 7.0 t mri also indicated prominent t1w hypointensities suggesting tissue injury , which could not be seen on 3.0 t mri but corresponded to axonal injury and loss of myelin in the autopsy specimen . one limitation of the study might be the application of immunosuppressive treatment at the time of biopsy or mri examination ( table 1 ) . morten blaabjerg : design and conceptualization , clinical data , analysis and interpretation of the data , drafting and revising the manuscript . klemens ruprecht : mri design and conceptualization , clinical data , analysis and interpretation of the data , drafting and revising the manuscript . bjrg morell kerrn - jespersen : clinical data , interpretation of the data , revising the manuscript . bjarne winther kristensen : pathological analysis and interpretation of the data , revising the manuscript . friedemann paul : mri design and conceptualization , analysis and interpretation of the data , drafting and revising the manuscript . zsolt illes : design and conceptualization , analysis and interpretation of the data , drafting and revising the manuscript . lundbeckfonden and scleroseforeningen ( denmark ) to z.i . ; deutsche forschungsgemeinschaft ( exc 257 ) and the german ministry for education and research ( bmbf competence network multiple sclerosis ) to f.p . ; german ministry for education and research ( bmbf competence network multiple sclerosis ) to k.r . k. ruprecht served on the scientific advisory board for sanofi - aventis / genzyme , novartis , and roche ; received travel funding and/or speaker honoraria from bayer healthcare , biogen idec , merck serono , sanofi - aventis / genzyme , teva pharmaceuticals , novartis , and guthy jackson charitable foundation ; is an academic editor for plos one ; receives publishing royalties from elsevier ; and received research support from novartis and the german ministry of education and research . d. kondziella received speaker honoraria and/or travel funding from bristol meyer squibb and ucb and has consulted for pfizer sweden . t. niendorf received travel funding and/or speaker funding from siemens healthcare , is founder and ceo of mri tools gmbh , and received research support from siemens healthcare and helmholtz alliance . lassmann received travel funding and speaker honoraria from biogen idec , novartis , and teva ; is on the editorial board for several journals in the fields of neurology and neuroscience ; has consulted for biogen idec and amgen ; and received research support from austrian science fund , european union . f. paul is on the steering committees for novartis and medimmune ; received speaker honoraria and travel funding from bayer , novartis , biogen idec , teva , sanofi - aventis / genzyme , and merck serono ; is an academic editor for plos one ; is an associate editor for neurology : neuroimmunology & neuroinflammation ; has consulted for sanofigenzyme , biogenidec , and medimmune ; and received research support from bayer , novartis , biogen idec , teva , sanofi - aventis / genzyme , merck serono , german research council , werth stiftung of the city of cologne , german ministry of education and research , arthur arnstein stiftung berlin , eu fp7 framework program , guthy jackson charitable foundation , and national multiple sclerosis society of the usa . z. illes served on the scientific advisory board for biogen idec , novartis , sanofi - aventis / genzyme , and teva pharmaceuticals ; received travel funding and/or speaker honoraria from biogen idec , novartis , sanofi - aventis / genzyme , teva pharmaceuticals , bayer healthcare , and merck serono ; is on the editorial board for clinical and experimental neuroimmunology ; holds a patent for fumaric acide derivates for medical use ; and received research support from biogen idec , region of southern denmark , odense university hospital , scleroseforeningen , lundbeckfonden , direktor enjar jonasson , kaldet jonsen , og hustru 's mindelegat .

objective : to examine if there is widespread inflammation in the brain of patients with chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids ( clippers ) syndrome by using histology and ultra - high - field mri at 7.0t.methods:we performed a detailed neuropathologic examination in 4 cases , including 1 autopsy case , and studied 2 additional patients by mri at 7.0 t to examine ( 1 ) extension of inflammation to areas appearing normal on 3.0 t mri , ( 2 ) potential advantages of 7.0 t mri compared to 3.0 t mri in reflecting widespread inflammation , perivascular pathology , and axonal damage , and ( 3 ) the possibility of lymphoma.results:in the autopsy case , perivascular inflammation dominated by cd4 + t cells was not only detected in the brainstem and cerebellum but also in brain areas with normal appearance on 3.0 t mri , including supratentorial regions and cranial nerve roots . there was no evidence of lymphoma in any of the 4 patients . the 7.0 t mri in clinical remission also revealed supratentorial lesions and perivascular pathology in vivo with contrast - enhancing lesions centered around a small venous vessel . ultra - high - field mri at 7.0 t disclosed prominent t1 hypointensities in the brainstem , which were not seen on 3.0 t mri . this corresponded to neuropathologic detection of axonal injury in the autopsy case.conclusion:our findings suggest more widespread perivascular inflammation and postinflammatory axonal injury in patients with clippers .

METHODS Standard approvals, registrations, and patients. Description of autopsy case (patient 1). Description of the 3 cases with brainstem biopsy (patients 24). Patients examined by 7T MRI (patients 5 and 6). Neuropathologic examination. Ultra-high-field MRI data acquisition and postprocessing. RESULTS Perivascular inflammation in CLIPPERS extends to areas with normal appearance on 3.0T MRI: Analysis of the autopsy case. No evidence of lymphoma and prelymphoma state: Analysis of 4 cases. Ultra-high-field MRI reveals perivascular lesions outside the brainstem/cerebellum and tissue damage. DISCUSSION Supplementary Material AUTHOR CONTRIBUTIONS STUDY FUNDING DISCLOSURE

mri showed patchy t2 signal hyperintensities in the brainstem and cerebellar peduncles with punctate gadolinium enhancements ( figure 1 ) . there was no evidence of malignant cells and viral examinations of the csf for herpesviruses were negative . there was no evidence of lymphoma or vessel destruction . we found prominent cd4 + t - cell infiltration in the brainstem ( d ) and cerebellum ( n ) but also in insular cortex ( s ) and parietal cortex ( i ) . hematoxylin & eosin staining reveals inflammation also in cranial nerve roots from the brainstem ( v and w ) . mri showed multiple punctate gadolinium - enhancing lesions in the cerebellum , brainstem , and midbrain . during the weeks before the onset of these symptoms the patient was admitted to another hospital in october 2013 , where a cranial 1.5 t mri demonstrated multiple small t2 hyperintense lesions with punctate and curvilinear contrast enhancement in the pons and cerebellum , and a bigger right cerebellar lesion . a spinal cord mri demonstrated a small longitudinal contrast - enhancing lesion in the cervical cord . she was initially treated with high - dose iv methylprednisolone ( 1 g / day for 3 days followed by 1 g / day for 5 days ) and subsequently with oral prednisolone ( starting dose 25 mg / day with a subsequent taper to 5 mg / day ) and azathioprine ( 3 50 mg ) , which was changed to methotrexate in may 2014 , when a cranial mri demonstrated constant to minimally progressive clippers lesions . at the time of the 7.0 t mri examination , she was treated with prednisolone ( 5 mg daily ) and methotrexate ( 15 mg weekly ) , and had residual facial paresthesiae but no clinical signs of new disease activity . at the time of the 7.0 t mri examination , she was treated with methylprednisolone ( 4 mg daily ) and methotrexate ( 15 mg weekly ) and was in clinical remission . ultra - high - field mri were acquired using a 7.0 t whole body mri scanner ( magnetom , siemens , erlangen , germany ) applying a 24-channel receive head coil ( nova medical , wilmington , ma ) equipped with a birdcage volume coil used for transmission . to identify vascular structures within contrast - enhancing clippers lesions , we calculated the sum of ( 1 ) coregistered t1w mprage with supratentorial t2*w images , and ( 2 ) coregistered vibe with infratentorial swi images using the image calculator function of the mipav software package ( version 7.0.1 , bethesda , md ) . mri showed patchy t2 signal hyperintensities in the brainstem and cerebellar peduncles with punctate gadolinium enhancements ( figure 1 ) . there was no evidence of malignant cells and viral examinations of the csf for herpesviruses were negative . there was no evidence of lymphoma or vessel destruction . we found prominent cd4 + t - cell infiltration in the brainstem ( d ) and cerebellum ( n ) but also in insular cortex ( s ) and parietal cortex ( i ) . hematoxylin & eosin staining reveals inflammation also in cranial nerve roots from the brainstem ( v and w ) . mri showed multiple punctate gadolinium - enhancing lesions in the cerebellum , brainstem , and midbrain . during the weeks before the onset of these symptoms the patient was admitted to another hospital in october 2013 , where a cranial 1.5 t mri demonstrated multiple small t2 hyperintense lesions with punctate and curvilinear contrast enhancement in the pons and cerebellum , and a bigger right cerebellar lesion . a spinal cord mri demonstrated a small longitudinal contrast - enhancing lesion in the cervical cord . she was initially treated with high - dose iv methylprednisolone ( 1 g / day for 3 days followed by 1 g / day for 5 days ) and subsequently with oral prednisolone ( starting dose 25 mg / day with a subsequent taper to 5 mg / day ) and azathioprine ( 3 50 mg ) , which was changed to methotrexate in may 2014 , when a cranial mri demonstrated constant to minimally progressive clippers lesions . at the time of the 7.0 t mri examination , she was treated with prednisolone ( 5 mg daily ) and methotrexate ( 15 mg weekly ) , and patient 6 is a 73-year - old woman who was diagnosed with clippers in october 2010 and whose clinical and paraclinical details were previously reported in detail . at the time of the 7.0 t mri examination , she was treated with methylprednisolone ( 4 mg daily ) and methotrexate ( 15 mg weekly ) and was in clinical remission . ultra - high - field mri were acquired using a 7.0 t whole body mri scanner ( magnetom , siemens , erlangen , germany ) applying a 24-channel receive head coil ( nova medical , wilmington , ma ) equipped with a birdcage volume coil used for transmission . to identify vascular structures within contrast - enhancing clippers lesions , we calculated the sum of ( 1 ) coregistered t1w mprage with supratentorial t2*w images , and ( 2 ) coregistered vibe with infratentorial swi images using the image calculator function of the mipav software package ( version 7.0.1 , bethesda , md ) . first , we investigated inflammation in different brain regions corresponding to gadolinium - enhancing and normal - appearing brain areas on 3.0 t mri ( autopsy tissue of patient 1 ) : brainstem and cerebellum were compared to parietal and insular cortex ( figure 1 ) . perivascular accumulation of cd45 + lymphocytes ( figure 1 , b , g , l , and q ) with predominance of cd3 + t cells ( figure 1 , c , h , m and r ) and cd4 + t cells were detected in brainstem and cerebellum as expected ( figure 1 , d and n ) . cd4 + t cells also infiltrated the parenchyma , which was most pronounced in the brainstem ( figure 1d ) . however , insular and parietal cortex with normal appearance on 3.0 t mri revealed a similar , albeit less prominent perivascular accumulation of cd4 + t cells ( figure 1 , i and s ) , fewer cd8 + t cells ( figure 1 , j and t ) , and a very limited number of cd20 + b cells ( figure 1 , k and u ) . these data suggested that brain areas with normal appearance on 3.0 t mri may be affected by inflammation in clippers syndrome . we found no evidence for monoclonal cell expansion using staining for light chain restriction ( figure 2 , a b , f l , and p q ) and lymphoma markers cd10 , bcl-6 , and mum-1 ( figure 2 , c e , h j , m o , and r follow - up of the 3 biopsy cases ( patients 24 ; mean of 75 months ) did not indicate clinical or paraclinical evidence of lymphoma either . since pathologic examination indicated perivascular inflammation in brain areas with normal appearance on 3.0 t mri , albeit less pronounced than in brainstem and cerebellum , we next examined 2 patients with clippers by 7.0 t mri during clinical remission . fusion ( c ) of coregistered postgadolinium t1-weighted ( d ) and t2 * -weighted ( b ) identifies a small brain vessel within the center of the chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids ( clippers ) lesion ( white arrow , c ) . even with this approach , a very small venous vessel could be depicted within 8 of 10 contrast - enhancing clippers lesions on fused postgadolinium vibe and swi ( data not shown ) . owing to substantially increased signal - to - noise ratio , 7.0 t t1-weighted magnetization - prepared rapid gradient echo delineates numerous t1-weighted hypointense lesions within the pons and cerebellum ( arrowheads , e in contrast , corresponding areas appear normal ( c , d ) or only marginally hypointense on t1-weighted images at 3.0 t ( circle , a , b ) . t1-weighted hypointensity within non - contrast - enhancing chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids lesions may indicate postinflammatory structural brain damage including axonal loss on top of demyelination . immunohistochemical stainings of autopsy case for neurofilament ( nf ) revealed massive axonal injury in the inflammatory lesion of the brainstem ( h ; cd3 red , nf brown ) compared to insular cortex with limited inflammation ( j ) . 7.0 t = 3d t1-weighted magnetization - prepared rapid gradient echo at 7.0 t , echo time = 2.98 ms , repetition time = 2,300 ms , inversion time = 900 ms , spatial resolution = 1.0 1.0 1.0 mm ; 3.0 t = standard t1-weighted axial images ( b d ) and t1-weighted magnetization - prepared rapid gradient echo at 3.0 t ( a ) . in addition , 7.0 t t1w mprage depicted numerous t1w hypointense lesions within the pons and cerebellum ( figure 4 , e g ) in patient 5 , which were not visible on conventional 3.0 t mri ( figure 4 , a d ) , indicating severe tissue destruction within postinflammatory clippers lesions ( figure 4 ) . multiple pontine postinflammatory t1w hypointense lesions were also found in patient 6 by using t1w imaging at 7.0 t ( data not shown ) . corresponding to the 7.0 t mri findings , neurofilament and myelin staining revealed axonal injury and myelin loss in brainstem areas with severe inflammation of the autopsy case ( figure 4 , h and i ) . first , we investigated inflammation in different brain regions corresponding to gadolinium - enhancing and normal - appearing brain areas on 3.0 t mri ( autopsy tissue of patient 1 ) : brainstem and cerebellum were compared to parietal and insular cortex ( figure 1 ) . perivascular accumulation of cd45 + lymphocytes ( figure 1 , b , g , l , and q ) with predominance of cd3 + t cells ( figure 1 , c , h , m and r ) and cd4 + t cells were detected in brainstem and cerebellum as expected ( figure 1 , d and n ) . cd4 + t cells also infiltrated the parenchyma , which was most pronounced in the brainstem ( figure 1d ) . however , insular and parietal cortex with normal appearance on 3.0 t mri revealed a similar , albeit less prominent perivascular accumulation of cd4 + t cells ( figure 1 , i and s ) , fewer cd8 + t cells ( figure 1 , j and t ) , and a very limited number of cd20 + b cells ( figure 1 , k and u ) . these data suggested that brain areas with normal appearance on 3.0 t mri may be affected by inflammation in clippers syndrome . we found no evidence for monoclonal cell expansion using staining for light chain restriction ( figure 2 , a b , f l , and p q ) and lymphoma markers cd10 , bcl-6 , and mum-1 ( figure 2 , c e , h j , m follow - up of the 3 biopsy cases ( patients 24 ; mean of 75 months ) did not indicate clinical or paraclinical evidence of lymphoma either . since pathologic examination indicated perivascular inflammation in brain areas with normal appearance on 3.0 t mri , albeit less pronounced than in brainstem and cerebellum , we next examined 2 patients with clippers by 7.0 t mri during clinical remission . fusion ( c ) of coregistered postgadolinium t1-weighted ( d ) and t2 * -weighted ( b ) identifies a small brain vessel within the center of the chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids ( clippers ) lesion ( white arrow , c ) . due to technical limitations , a different approach with a lower spatial resolution of 0.25 mm even with this approach , a very small venous vessel could be depicted within 8 of 10 contrast - enhancing clippers lesions on fused postgadolinium vibe and swi ( data not shown ) . t1-weighted hypointensity within non - contrast - enhancing chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids lesions may indicate postinflammatory structural brain damage including axonal loss on top of demyelination . immunohistochemical stainings of autopsy case for neurofilament ( nf ) revealed massive axonal injury in the inflammatory lesion of the brainstem ( h ; cd3 red , nf brown ) compared to insular cortex with limited inflammation ( j ) . 7.0 t = 3d t1-weighted magnetization - prepared rapid gradient echo at 7.0 t , echo time = 2.98 ms , repetition time = 2,300 ms , inversion time = 900 ms , spatial resolution = 1.0 1.0 1.0 mm ; 3.0 t = standard t1-weighted axial images ( b d ) and t1-weighted magnetization - prepared rapid gradient echo at 3.0 t ( a ) . in addition , 7.0 t t1w mprage depicted numerous t1w hypointense lesions within the pons and cerebellum ( figure 4 , e g ) in patient 5 , which were not visible on conventional 3.0 t mri ( figure 4 , a d ) , indicating severe tissue destruction within postinflammatory clippers lesions ( figure 4 ) . multiple pontine postinflammatory t1w hypointense lesions were also found in patient 6 by using t1w imaging at 7.0 t ( data not shown ) . corresponding to the 7.0 t mri findings , neurofilament and myelin staining revealed axonal injury and myelin loss in brainstem areas with severe inflammation of the autopsy case ( figure 4 , h and i ) . in this article , we describe neuropathologic as well as high - field / ultra - high - field mri features of 6 patients with clippers syndrome . the diagnosis of clippers syndrome was supported by ( 1 ) postcontrast t1-weighted mri with typical punctate , nodular , or curvilinear and occasionally more confluent gadolinium - enhancing lesions , ( 2 ) perivascular inflammation in the brainstem dominated by t cells , ( 3 ) clinical course with several steroid - responsive relapses , and ( 4 ) absence of other diseases . similar lesions were previously described in patients with clippers and a biopsy of the cerebellar lesion of case 5 demonstrated histopathologic features of clippers . we found widespread perivascular inflammation in brain areas with normal appearance on 3.0 t mri in an autopsy case of clippers syndrome . although t2w and gadolinium - enhancing lesions were found only in the pons , midbrain , and cerebellar regions , inflammation could be seen as distant as the parietal cortex and even involved the cranial nerve roots . the degree of inflammation , however , showed a gradient of less inflammation with greater distance from the primarily affected areas ( brainstem > cerebellum > insular cortex > parietal cortex ) , suggesting that the inflammation seen on conventional 1.5t3.0 t mri only depicts the most severely affected brain regions . since our pathologic examinations revealed widespread inflammatory changes in brain tissue appearing normal on conventional 3.0 t mri , we performed ultra - high - field 7.0 t mri in 2 additional clippers cases to investigate such possibilities in vivo . indeed , these efforts revealed contrast - enhancing lesions in supratentorial locations , supporting widespread inflammation in brain areas , in contrast to the only other autopsy report published recently . the number of such contrast - enhancing lesions was , however , limited , which is likely due to the immunosuppressive treatment : mri changes in clippers respond rapidly to immunosuppression . the 7.0 t mri thus enabled visualization of perivascular inflammation , the neuropathologic hallmark of clippers , in vivo . on the contrary , a central vein is only rarely depictable in brain lesions of other origin such as neuromyelitis optica , cerebral small vessel disease , and susac syndrome , which is considered a microangiopathy of the brain , cochlea , and retina . of note , highly resolving imaging techniques were needed to visualize the intralesional venous vessel in clippers lesions , which thus appear much smaller in diameter than central veins commonly visualized in ms lesions on 7.0 t t2*w images . in addition , we utilized the higher sensitivity of 7.0 t t1w mprage vs t1w imaging at 1.5 t and 3.0 t to detect brain lesions and to investigate brain areas that appeared normal at lower field strengths . although 1.5t3.0 t mri and 7.0 t mri were done at different time points , therefore direct comparison was not possible , patients had no signs of clinical activity at the time of the mri examinations . ultra - high - field mri revealed numerous t1w hypointense lesions within the pons and cerebellum in one of the patients , indicating postinflammatory brain tissue destruction , which were not visible on conventional 3.0 t mri . indeed , axonal injury with evidence of axonal spheroids and torpedoes was found in our autopsy case , similar to previous results . however , axonal injury and myelin loss was observed only in areas with severe inflammation , which may explain the restricted localization of t1w hypointense lesions on 7.0 t mri . since emerging cases of clippers with subsequent diagnosis of lymphoma have been recently described , we investigated this possibility in the autopsy case and available biopsy samples from 3 other danish cases . we found perivascular inflammation predominated by cd4 + t cells even in areas with normal appearance on 3.0 t mri and extending to the cranial nerve roots , suggesting widespread inflammation in clippers syndrome , though most prominent in the primarily affected brainstem . we found no evidence of lymphoma in our 4 investigated patients . when applying ultra - high - field mri at 7.0 t , supratentorial lesions could be confirmed , and most of the contrast - enhancing lesions contained a vessel structure indicating the pathologic hallmark of clippers syndrome in vivo . the 7.0 t mri also indicated prominent t1w hypointensities suggesting tissue injury , which could not be seen on 3.0 t mri but corresponded to axonal injury and loss of myelin in the autopsy specimen .

intracerebral hemorrhage ( ich ) accounts from 10 to 15% of all strokes , and this cerebral vascular disease is related to a low degree of neurorehabilitation . much of the knowledge about brain plasticity mechanisms and physical rehabilitation after stroke comes from brain ischemic strokes studies . it is not possible to assume that findings in ischemic rehabilitation will apply to ich since there are many differences in pathophysiology , location , and extension of the injury . upper limb motor impairment is an important functional limitation associated with diminished health - related quality of life that can persist in the long term , even with rehabilitation treatment . the study of experimental stroke in animal models has provided better understanding of pathological and recovery mechanisms . experimental rehabilitative therapies may influence synaptogenesis , neurogenesis , and neuron and glial responses in addition to functional recovery [ 6 , 7 ] . although positive effects of skilled training as a rehabilitative modality have been reported , neurobiological mechanisms that support motor and functional recovery are not completely elucidated [ 8 , 9 ] . motor skill learning has the ability to induce synaptogenesis in both cortical and subcortical brain regions [ 10 , 11 ] , the neuroplasticity in motor cortex is related to the skill task training , and this morphophysiological changes may also mediate functional recovery after cerebral vascular disease . cellular mechanisms of plasticity and motor recovery after stroke involve coordinated neuronal changes including regulation of growth factors , increase in protein synthesis , and cortical map reorganization [ 5 , 12 ] . there is evidence that the amount of microtubule - associated protein-2 ( map-2 ) might be associated with sensorimotor rehabilitation in both penumbral zone and contralesional hemisphere after cerebral vascular disease [ 13 , 14 ] . map-2 is one of the most important cytoskeleton proteins which is selectively located in the somatodendritic compartment of neurons and predominantly expressed in dendrites . map-2 expression is high in the early stage after brain lesion , probably due to compensatory regeneration , and low in later stage after ischemia . the increase of the map-2 expression also occurs in a neural organization induced by exercise after cerebral ischemia or cerebral physiological conditions . map-2 also regulates neuronal polarity and dendritic extension , and it promotes structure modulation and morphological stabilization in neuronal cells [ 15 , 20 ] . the acquisition of new motor skills involves dendritic reorganization in pyramidal neurons in motor cortex which is correlated to the increase of protein synthesis and enhanced expression of map-2 . it was demonstrated that treadmill running training improves motor function after ich , and this improvement may be related to changes in dendritic morphology in the striatum . still , previous studies found that synapse formation in the motor cortex begins immediately when learning a new task . the increase of map-2 expression is related to dendritic growth , and the increased dendritic branching is related to changes in sensorimotor behavior . although studies have shown functional changes in the upper limb and cerebral plasticity , the relationship between different motor skills practice and map-2 expression is not completely understood . besides , outcomes of previous studies can not be generalized due to the different methodological approaches . in this study , we investigated whether different rehabilitation modalities ( skilled or unskilled training ) would result in a differential forelimb motor behavior and in morphological adaptations evaluated by the map-2 immunoreactivity in forelimb representation in motor cortex . we hypothesized that functional motor recovery and the increase of map-2 expression are greater after skilled as compared to unskilled training . adult male wistar rats ( n = 48 ) were obtained from a local colony at approximately 3 months of age ( 300350 g ) and housed in groups of 4 - 5 in plexiglas cages under standard laboratory conditions ( 12 h light / dark cycle with lights off 7:30 pm and controlled temperature in 22 2c ) . water and standard laboratory chow were provided ad libitum except during behavioral adaptation , testing , and training periods . the experimental design and all procedures were approved by the committee of research ethics of federal university of rio grande do sul ( identifier number 2008015 ) and were in accordance with the guidelines for care and use of laboratory animals adopted by the national institute of health ( usa ) and with the federation of brazilian societies for experimental biology . beginning on the day before , and continuing for the duration of the staircase test period , animals were mildly food deprived ( in order to increase interest for new food ) . after each training session , rats were provided with a measured amount of standard laboratory chow ( 1520 g ) to maintain approximately 8595% of their free - feeding weight . three weeks before surgery ( 5 days per week ) , rats were trained to reach sucrose pellets ( 4.6 mm/65 mg 10% ) in staircase boxes ( 2 trials per day , 15 min each trial ) . this test provides a sensitive measurement of independent forelimb skilled reaching [ 25 , 26 ] . rats that did not obtain an average of 14 pellets with at least one limb over the final 2 days of training ( baseline measure ) were excluded from the study . pellets from third to the seventh degree were colored differently to enhance the quantitative discrimination of the skilled reaching . testing sessions consisted in 4 trials ( two trials per day with at least 4 hours interval , 15 min each ) conduced before ( baseline ) and after surgery ( postsurgery , week 2 and week 4 ) . animals were anesthetized with 4% halothane in 30% oxygen and 70% nitrous oxide and maintained in a stereotaxic frame with 2% halothane for ich surgery . a midline incision was made in the scalp , and a burr hole was drilled in the skull 3.6 mm lateral to bregma . surgery side was contralateral to the preferred paw , which was determined according to staircase evaluation ( baseline ) . then , a 26-gauge needle ( hamilton , reno , nv , usa ) was inserted 6.0 mm deep into the hole , and 0.2 u of bacterial collagenase type iv ( sigma - aldrich , usa ) diluted in 1.0 l saline buffer was infused into the striatum over 5 min . the needle was kept in position for an additional 5 min and then slowly removed to prevent backflow . in sham surgeries , collagenase was replaced with sterile saline . body temperature was maintained between 36.5c and 37.5c throughout the surgery using a self - regulating heating blanket ( letica , spain ) . a local anesthetic ( lidocaine , 3 m , brazil ) was applied to the wound at the end of surgery . animals received one session for habituation in skilled and unskilled tasks prior to surgery . before the surgery , rats were grouped according to their reaching success ( baseline in the staircase evaluation ) and randomly designated to one of six groups : sham no task ( s , n = 08 ) , sham skilled task ( s - sk , n = 08 ) , sham unskilled task ( s - us , n = 08 ) , ich no task ( ich , n = 08 ) , ich skilled task ( ich - sk , n = 08 ) , and ich unskilled task ( ich - us , n = 08 ) . seven days after sham / ich surgery , s - sk and ich - sk rats received daily skilled reaching session ( 5 days per week ) . these animals were removed from their housing cages and placed into standard rodent cages containing a plexiglass reaching apparatus . the shelf below to the unaffected forelimb was left empty , whereas the shelf below to the impaired forelimb was filled with the same sucrose pellets used for staircase test ( in a sufficient amount to prevent tongue and unaffected forelimb retrieval ) . animals had access to sugar pellets ( 15 g ) for 40 min per day , and the total of pellets retrieved was measured at the end of each session ( measurement by total weight ) . during the session , rats had free access to water , but no other type of food was available . at the end of each training day , the s , s - us , ich , and ich - us animals received the same average of pellets provided for s - sk and ich - sk rats . for unskilled training , s - us and ich - us groups received daily walking session ( 5 days per week ) on adapted motorized rodent treadmill ( inbramed tk 01 , porto alegre , brazil ) . each session consisted of walking at speed 1.8 m / min during all time ( 40 min per day ) . this session duration was chosen to maximize the comparison among all groups , and the slow speed was selected to incentive animals to walk ( not to run ) and to limit the possible effects of aerobic conditioning . the grade of the treadmill remained at 0% , and no aversive stimulus was used . after 4 weeks , animals were deeply anesthetized with chloride hydrate ( 30% , 10 ml / kg , i.p . ) and injected with 1000 ui heparin ( cristlia , brazil ) . then , animals were perfused through the left ventricle , using a peristaltic pump ( control company , so paulo , brazil ) with 200 ml of saline solution 0.9% followed by 150 ml of fixative solution composed of 4% paraformaldehyde ( pfa ) ( reagen , rio de janeiro , brazil ) in 0.1 m phosphate buffer ( pbs ) ph 7.4 at room temperature . brains were postfixed in 4% pfa at room temperature for 4 h , kept in 30% sucrose in pbs 0.1 m at 4c for 3 days , and then frozen in isopentane and liquid nitrogen . coronal sections ( 40 m ) were obtained using a cryostat ( leica , germany ) . slices obtained from the motor cortex were selected according to paxinos and watson and stored in a solution of freezing ( 40% pbs 0.1 m ph 7.4 ; 30% ethylene glycol ; and 30% glycol ) until the immunohistochemistry staining procedure . immunohistochemistry staining was performed for map-2 ( m9942 , sigma , usa , monoclonal produced in mouse ) . briefly , free - floating sections were washed in pbs , fixed in 4% pfa , pretreated with cooled 100% methanol in 3% h2o2 , and then carefully washed and blocked with 5% normal goat serum ( ngs ) ( g9023 , sigma , usa ) in pbs containing 0.3% triton x-100 ( pbstx , t9284 , sigma aldrich , usa ) for 30 min . brain slices were then incubated with primary antibody diluted in pbs - tx ( 1 : 1000 ) overnight at 4c . sections were washed in pbs and incubated in goat anti - mouse biotinylated secondary antibody ( 1 : 500 ) diluted in 0.3% pbs - tx and 5% ngs for 1 hour at room temperature . after slices had been washed in pbs , they were treated along 30 min with kit vectastain abc elite ( vector labs ) , washed in pbs , and exposed for 5 min in 3.3 diaminobenzidine ( dab , d8001 , sigma , usa ) and h2o2 . sections were raised , mounted on gelatinized slides , dehydrated with ethanol , cleared in xylene , and covered with dpx mount ( sigma ) and coverslips . control sections were simultaneously processed without the primary antibody addition in order to serve as a background control . brain slices were analyzed ( 400x magnification ) in a nikon eclipse e-600 microscope ( japan ) coupled to a nikon dxm 1200c ccd camera and to nis elements ar 2.30 software . the estimation of hemispheric and striatal areas as well as the cortical thickness ( ipsilesional and contralesional to injury ) was conducted in one slice per animal ( n = 5 for each experimental group ) in the same brain slices used in the immunohistochemistry staining procedure . images were scanned in 1200 dpi , and scion image j 4.0 program ( scion corporation , frederick , md , usa ) [ 31 , 32 ] was used for the morphometric analysis . estimation of cortical thickness was calculated using the average of three measurements obtained from the upper edge of the corpus callosum until the upper edge of the hemispheres . for map analyses , two motor cortex images were used between m1 and m2 ( at the level + 2.16 to 0.36 mm from bregma ) preferably in layer v , and cortical thicknesses were captured from both hemispheres ( 6 animals per group ) . picture elements ( pixels ) employed to measure relative optical densities were obtained from region of interest ( roi ) squares with 82.145,43 m overlaid on the gray scale image , with background correction . all lighting conditions and magnifications behavioral data were analyzed by one - way repeated measures analysis of variance ( anova ) . morphometric measurements and map-2 relative optical densities were analyzed by one - way anova followed by tukey post hoc tests when appropriated . all ich rats showed motor behavior compatible with surgery success , which included spontaneous rotation towards to the contralateral surgery side when held by the tail . a total number of animals were used for behavioral analysis ( n = 48 ) , and 36 animals were randomly chosen for morphological assessment ( 6 animals per group ) . [ f(3,126 ) = 114.77 , p < 0.01 ] , effect of group [ f(1,42 ) = 8.22 , p < 0.01 ] , and time group interaction [ f(15,126 ) = 20.78 , p < 0.01 ] . baseline evaluation performed before surgery showed no difference between experimental groups ( p > 0.05 ) . all ich groups retrieved significantly fewer pellets than s animals in postsurgery and 2nd and 4th week evaluations ( p < 0.01 ) . there was no difference between the number of pellets collected by s - sk and s - uk animals at any point ( p > 0.05 ) . tukey post hoc tests showed difference between ich and ich - sk and between ich - sk and ich - us groups ( p < 0.01 ) in the 4th week . at this point , there was no difference between ich and ich - us groups ( p > 0.05 ) as shown in table 1 . for pellets retrieved from deeper stairs ( 5th to 7th ) , repeated measures anova showed time [ f(3,126 ) = 94.27 , p < 0.01 ] , group [ f(5,42 ) = 77.73 , p < 0.01 ] , and time group interaction effects [ f(15,126 ) = 17.38 , p < 0.01 ] . differences were evidenced among ich and all sham groups ( p < 0.01 ) in all evaluation times . the last point measurement ( 4th week ) revealed difference between ich and ich - sk and between ich - sk and ich - us groups ( p < 0.01 ) as depicted in figure 1(a ) and table 2 . one - way anova of morphometric data showed significant main effects on total hemisphere area [ f(5,29 ) = 24.27 , p < 0.01 ] , lesional area [ f(5,29 ) = 19.35 , p < 0.01 ] , and cortical thickness [ f(5,29 ) = 35.18 , p < 0.01 ] only in the injured hemisphere . differences among all sham and ich groups were revealed by tukey post hoc tests ( p < 0.01 ) . as displayed in table 3 , no task - related effects on morphometric data were found ( p > 0.05 ) . there was a significant effect of group in both hemispheres , ipsilesional ( injured side ) [ f(5,35 ) = 14.25 ( p < 0.01 ) ] , and contralesional side [ f(5,35 ) = 9.70 ( p < 0.01 ) ] . differences among s and s - sk ( p < 0.001 ) , ich ( p = 0.02 ) , s - us , ich - us , and ich - sk ( p < 0.001 ) and between s - us and ich - us ( p = 0.03 ) , ich , and ich - sk ( p < 0.01 ) when the contralesional hemisphere was analyzed , differences were found among s and s - sk , ich , ich - sk ( p < 0.01 ) , and ich - us ( p = 0.02 ) and between s - us and ich - sk ( p < 0.01 ) , ich - sk , and ich - us ( p = 0.04 ) . the purpose of the current study was to investigate whether different rehabilitation modalities ( skilled or unskilled training ) would result in a differential upper limb motor behavior and in morphological adaptations as evaluated by the means of map-2 immunoreactivity in the forelimb representation in motor cortex . the significance of investigating this approach refers to the major limitations of recovery after stroke and functional disability of upper limb [ 4 , 33 ] . the persistence of this deficit impacts directly in an index of independence and quality of life . present results show that skill task training performed with the impaired forelimb was able to increase map-2 immunoreactivity in motor cortex either in sham or in ich groups in both cortices . behavioral evaluation revealed that ich - sk performed better than ich and ich - un animals in the staircase test . physical motion is between therapeutic interventions that can improve brain recovery after stroke [ 3 , 12 ] . the type of treatment , intensity and duration of the protocol , and the period in which it is applied are factors related to beneficial effects on functional recovery [ 3537 ] . this analysis revealed that after ich , only animals submitted to skill task training showed significant improvement in motor performance at the end of the 4th week of treatment , as evidenced by the total number of retrieved pellets . color - coded pellets can be used in staircase test aiming to increase the sensitivity of the instrument since it allows taking into consideration the levels of reaching difficulty . all ich animals exhibit difficulties in reaching pellets from the last steps ( 5th to 7th ) immediately after ischemia and at the end of the 2nd week of treatment . ich - sk group presented superior reaching rate when compared to ich and ich - us groups at the end of the 4th week . this may indicate that skill task training is able to induce superior upper limb recovery after intracerebral hemorrhage when compared to unskilled task training . in agreement with our results , another study demonstrated that skilled reaching training of impaired forelimb can induce brain plasticity which was observed in association with enhanced sensorimotor recovery after stroke in sensorimotor cortex and striatum [ 13 , 38 , 39 ] . previous investigations also have found that activity - based therapies to promote forelimb use after stroke are able to develop cortical adaptations as protein synthesis , synaptogenesis , and increase in cortical representation of hand and fingers [ 12 , 37 ] . these modifications correlated with functional recovery are usually proportional to the complexity of the task and do not occur in the same intensity when movement or gestures are executed in a simple and repetitive pattern [ 10 , 36 , 40 ] . our behavioral results showed that physical motion can induce motor recovery , but that skill task training as a rehabilitative strategy has the capacity to improve motor performance . therefore , food obtained as a reward may be considered a motivational factor that contributes for morphophysiological changes associated with behavioral responses [ 13 , 14 ] . even though behavioral assessment evidenced motor recovery , morphometric analysis showed differences only between injured and uninjured animals . histological and biochemical studies have demonstrated a mild correlation between motor performance and these measurements in collagenase - induced intrastriatal hemorrhage and focal ischemic models [ 8 , 29 ] . our finding reinforces the idea that morphometrical parameters can not be applied as a prognostic factor of motor recovery in experimental studies . in view of the lack of correlation between morphometrical / macroscopic analysis and behavioral results , probably the supporting for behavioral changes observed in our results map-2 immunoreactivity in the motor cortex was increased by skilled task training in both cerebral hemispheres either in physiological or pathological ( ich ) conditions . motor learning is a set of internal processes associated with practice or experience that involves a coordinate system in which a planned task is encoded and combined with appropriate recruitment of motor units to execute a movement goal [ 41 , 42 ] . the synaptogenesis induced by motor practice or synaptic reinforcing transmission may consolidate the motor gesture learned in both intact and stroke - injured brains [ 11 , 36 ] . however , our behavioral and morphological results suggest that the ich model is adequate for future studies regarding brain injury and motor recovery by greater understanding between the morphological changes of the nervous cells and motor neurorehabilitation techniques . map-2 is a structural protein that acts with other intracellular components to maintain neuroarchitecture , preferably in dendrites . furthermore , map-2 plays in the growth , differentiation , and plasticity of neurons , with key roles in neuronal responses to growth factors , neurotransmitters , synaptic activity , and neurotoxins [ 30 , 45 ] . the increase of map-2-positive dendrites is connected with neural activity regeneration after injury cerebrovascular [ 17 , 46 ] , and dendritic morphological changes may be involved in motor learning . since modification and rearrangement of map-2 are obligatory steps in many processes that modify neuronal function , we decided to investigate the effects of skilled and unskilled training after ich on functional recovery and map-2 expression . the motor recovery after ischemia techniques for inducing restoration of excitability in motor cortex can exert positive effects on rehabilitation therapy by means of changes in dendritic structure and expression of map-2 [ 23 , 48 ] . dendritic growth process seems to be related to the amount of motor cortex inputs and partially depends on map-2 expression . relative optical densities analysis revealed that the amount of map-2 was significantly higher in ich animals submitted to skill task training ( ich - sk ) when compared to ich and ich - un in the ipsilesional and contralesional cortices , respectively . even though ich and ich - us groups have shown increased map-2 immunoreactivity in both ipsi- and contralesional motor cortices , no behavioral improving outcome was observed . since map-2 is involved with neuronal plasticity under physiological and pathological conditions , those levels of immunoreactivity after ich were not surprising . on the other hand , motor cortex lesion causes damage in cortical networks and their projections for movement and results in contralateral limb disuse . compensatory use of the unaffected limb can be followed by the increasing of the cortical activity in the contralesional hemisphere , which can explain levels of map-2 immunoreactivity in contralesional cortex in ich nonrehabilitation group . this limitation can be related to the strategy chosen as rehabilitative program since each approach can stimulate different patterns of cns plasticity [ 39 , 50 ] . our results reinforce previous findings and reaffirm that skill task training is a good option for rehabilitation after stroke as demonstrated by the behavioral motor recovery and morphological changes . our main results demonstrated that skill task can drive brain plasticity in motor cortex by the increasing of map-2 expression in both hemispheres and with positive functional outcomes after collagenase - induced intrastriatal unilateral hemorrhage in a superior manner when compared to unskilled training . these findings may offer insights for improving functional recovery in stroke patients since that rehabilitation depends on type , intensity , and duration of approaches used as physical treatment .

motor skill learning may induce behavioral and neurophysiological adaptations after intracerebral hemorrhage ( ich ) . learning a new motor skill is associated with dendritic reorganization and requires protein synthesis and expression of map-2 . the purpose of this study was to evaluate motor performance and expression of map-2 in the motor cortex of rats submitted to intracerebral hemorrhage model ( ich ) and skill task training ( sk ) or unskilled training ( us ) during 4 weeks . the staircase test was used for behavioral evaluation , and relative optical densities and morphometrical analysis were used to estimate map-2 immunoreactivity and parameters of brain tissue in both motor cortices . results show that skill task training performed with the impaired forelimb was able to increase map-2 immunoreactivity in the motor cortex either in sham or in ich groups in both cortices : ipsilesional [ f(5,35 ) = 14.25 ( p < 0.01 ) ] and contralesional hemispheres [ f(5,35 ) = 9.70 ( p < 0.01 ) ] . ich alone also increased map-2 immunoreactivity despite the absence of functional gains . behavioral evaluation revealed that ich - sk group performed better than ich and ich - us animals in the staircase test . data suggest that motor skill training induces plastic modifications in both motor cortices , either in physiological or pathological conditions and that skill motor training produces higher brain plasticity and positive functional outcomes than unskilled training after experimental intracerebral hemorrhage .

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

intracerebral hemorrhage ( ich ) accounts from 10 to 15% of all strokes , and this cerebral vascular disease is related to a low degree of neurorehabilitation . much of the knowledge about brain plasticity mechanisms and physical rehabilitation after stroke comes from brain ischemic strokes studies . it is not possible to assume that findings in ischemic rehabilitation will apply to ich since there are many differences in pathophysiology , location , and extension of the injury . upper limb motor impairment is an important functional limitation associated with diminished health - related quality of life that can persist in the long term , even with rehabilitation treatment . experimental rehabilitative therapies may influence synaptogenesis , neurogenesis , and neuron and glial responses in addition to functional recovery [ 6 , 7 ] . motor skill learning has the ability to induce synaptogenesis in both cortical and subcortical brain regions [ 10 , 11 ] , the neuroplasticity in motor cortex is related to the skill task training , and this morphophysiological changes may also mediate functional recovery after cerebral vascular disease . cellular mechanisms of plasticity and motor recovery after stroke involve coordinated neuronal changes including regulation of growth factors , increase in protein synthesis , and cortical map reorganization [ 5 , 12 ] . there is evidence that the amount of microtubule - associated protein-2 ( map-2 ) might be associated with sensorimotor rehabilitation in both penumbral zone and contralesional hemisphere after cerebral vascular disease [ 13 , 14 ] . map-2 is one of the most important cytoskeleton proteins which is selectively located in the somatodendritic compartment of neurons and predominantly expressed in dendrites . map-2 expression is high in the early stage after brain lesion , probably due to compensatory regeneration , and low in later stage after ischemia . map-2 also regulates neuronal polarity and dendritic extension , and it promotes structure modulation and morphological stabilization in neuronal cells [ 15 , 20 ] . the acquisition of new motor skills involves dendritic reorganization in pyramidal neurons in motor cortex which is correlated to the increase of protein synthesis and enhanced expression of map-2 . it was demonstrated that treadmill running training improves motor function after ich , and this improvement may be related to changes in dendritic morphology in the striatum . still , previous studies found that synapse formation in the motor cortex begins immediately when learning a new task . the increase of map-2 expression is related to dendritic growth , and the increased dendritic branching is related to changes in sensorimotor behavior . although studies have shown functional changes in the upper limb and cerebral plasticity , the relationship between different motor skills practice and map-2 expression is not completely understood . in this study , we investigated whether different rehabilitation modalities ( skilled or unskilled training ) would result in a differential forelimb motor behavior and in morphological adaptations evaluated by the map-2 immunoreactivity in forelimb representation in motor cortex . we hypothesized that functional motor recovery and the increase of map-2 expression are greater after skilled as compared to unskilled training . adult male wistar rats ( n = 48 ) were obtained from a local colony at approximately 3 months of age ( 300350 g ) and housed in groups of 4 - 5 in plexiglas cages under standard laboratory conditions ( 12 h light / dark cycle with lights off 7:30 pm and controlled temperature in 22 2c ) . water and standard laboratory chow were provided ad libitum except during behavioral adaptation , testing , and training periods . the experimental design and all procedures were approved by the committee of research ethics of federal university of rio grande do sul ( identifier number 2008015 ) and were in accordance with the guidelines for care and use of laboratory animals adopted by the national institute of health ( usa ) and with the federation of brazilian societies for experimental biology . beginning on the day before , and continuing for the duration of the staircase test period , animals were mildly food deprived ( in order to increase interest for new food ) . rats that did not obtain an average of 14 pellets with at least one limb over the final 2 days of training ( baseline measure ) were excluded from the study . testing sessions consisted in 4 trials ( two trials per day with at least 4 hours interval , 15 min each ) conduced before ( baseline ) and after surgery ( postsurgery , week 2 and week 4 ) . a midline incision was made in the scalp , and a burr hole was drilled in the skull 3.6 mm lateral to bregma . then , a 26-gauge needle ( hamilton , reno , nv , usa ) was inserted 6.0 mm deep into the hole , and 0.2 u of bacterial collagenase type iv ( sigma - aldrich , usa ) diluted in 1.0 l saline buffer was infused into the striatum over 5 min . in sham surgeries , collagenase was replaced with sterile saline . before the surgery , rats were grouped according to their reaching success ( baseline in the staircase evaluation ) and randomly designated to one of six groups : sham no task ( s , n = 08 ) , sham skilled task ( s - sk , n = 08 ) , sham unskilled task ( s - us , n = 08 ) , ich no task ( ich , n = 08 ) , ich skilled task ( ich - sk , n = 08 ) , and ich unskilled task ( ich - us , n = 08 ) . seven days after sham / ich surgery , s - sk and ich - sk rats received daily skilled reaching session ( 5 days per week ) . the shelf below to the unaffected forelimb was left empty , whereas the shelf below to the impaired forelimb was filled with the same sucrose pellets used for staircase test ( in a sufficient amount to prevent tongue and unaffected forelimb retrieval ) . animals had access to sugar pellets ( 15 g ) for 40 min per day , and the total of pellets retrieved was measured at the end of each session ( measurement by total weight ) . at the end of each training day , the s , s - us , ich , and ich - us animals received the same average of pellets provided for s - sk and ich - sk rats . for unskilled training , s - us and ich - us groups received daily walking session ( 5 days per week ) on adapted motorized rodent treadmill ( inbramed tk 01 , porto alegre , brazil ) . this session duration was chosen to maximize the comparison among all groups , and the slow speed was selected to incentive animals to walk ( not to run ) and to limit the possible effects of aerobic conditioning . the grade of the treadmill remained at 0% , and no aversive stimulus was used . after 4 weeks , animals were deeply anesthetized with chloride hydrate ( 30% , 10 ml / kg , i.p . ) brains were postfixed in 4% pfa at room temperature for 4 h , kept in 30% sucrose in pbs 0.1 m at 4c for 3 days , and then frozen in isopentane and liquid nitrogen . slices obtained from the motor cortex were selected according to paxinos and watson and stored in a solution of freezing ( 40% pbs 0.1 m ph 7.4 ; 30% ethylene glycol ; and 30% glycol ) until the immunohistochemistry staining procedure . briefly , free - floating sections were washed in pbs , fixed in 4% pfa , pretreated with cooled 100% methanol in 3% h2o2 , and then carefully washed and blocked with 5% normal goat serum ( ngs ) ( g9023 , sigma , usa ) in pbs containing 0.3% triton x-100 ( pbstx , t9284 , sigma aldrich , usa ) for 30 min . brain slices were then incubated with primary antibody diluted in pbs - tx ( 1 : 1000 ) overnight at 4c . after slices had been washed in pbs , they were treated along 30 min with kit vectastain abc elite ( vector labs ) , washed in pbs , and exposed for 5 min in 3.3 diaminobenzidine ( dab , d8001 , sigma , usa ) and h2o2 . sections were raised , mounted on gelatinized slides , dehydrated with ethanol , cleared in xylene , and covered with dpx mount ( sigma ) and coverslips . the estimation of hemispheric and striatal areas as well as the cortical thickness ( ipsilesional and contralesional to injury ) was conducted in one slice per animal ( n = 5 for each experimental group ) in the same brain slices used in the immunohistochemistry staining procedure . images were scanned in 1200 dpi , and scion image j 4.0 program ( scion corporation , frederick , md , usa ) [ 31 , 32 ] was used for the morphometric analysis . for map analyses , two motor cortex images were used between m1 and m2 ( at the level + 2.16 to 0.36 mm from bregma ) preferably in layer v , and cortical thicknesses were captured from both hemispheres ( 6 animals per group ) . picture elements ( pixels ) employed to measure relative optical densities were obtained from region of interest ( roi ) squares with 82.145,43 m overlaid on the gray scale image , with background correction . all lighting conditions and magnifications behavioral data were analyzed by one - way repeated measures analysis of variance ( anova ) . morphometric measurements and map-2 relative optical densities were analyzed by one - way anova followed by tukey post hoc tests when appropriated . a total number of animals were used for behavioral analysis ( n = 48 ) , and 36 animals were randomly chosen for morphological assessment ( 6 animals per group ) . [ f(3,126 ) = 114.77 , p < 0.01 ] , effect of group [ f(1,42 ) = 8.22 , p < 0.01 ] , and time group interaction [ f(15,126 ) = 20.78 , p < 0.01 ] . baseline evaluation performed before surgery showed no difference between experimental groups ( p > 0.05 ) . all ich groups retrieved significantly fewer pellets than s animals in postsurgery and 2nd and 4th week evaluations ( p < 0.01 ) . there was no difference between the number of pellets collected by s - sk and s - uk animals at any point ( p > 0.05 ) . tukey post hoc tests showed difference between ich and ich - sk and between ich - sk and ich - us groups ( p < 0.01 ) in the 4th week . at this point , there was no difference between ich and ich - us groups ( p > 0.05 ) as shown in table 1 . for pellets retrieved from deeper stairs ( 5th to 7th ) , repeated measures anova showed time [ f(3,126 ) = 94.27 , p < 0.01 ] , group [ f(5,42 ) = 77.73 , p < 0.01 ] , and time group interaction effects [ f(15,126 ) = 17.38 , p < 0.01 ] . differences were evidenced among ich and all sham groups ( p < 0.01 ) in all evaluation times . the last point measurement ( 4th week ) revealed difference between ich and ich - sk and between ich - sk and ich - us groups ( p < 0.01 ) as depicted in figure 1(a ) and table 2 . one - way anova of morphometric data showed significant main effects on total hemisphere area [ f(5,29 ) = 24.27 , p < 0.01 ] , lesional area [ f(5,29 ) = 19.35 , p < 0.01 ] , and cortical thickness [ f(5,29 ) = 35.18 , p < 0.01 ] only in the injured hemisphere . differences among all sham and ich groups were revealed by tukey post hoc tests ( p < 0.01 ) . as displayed in table 3 , no task - related effects on morphometric data were found ( p > 0.05 ) . there was a significant effect of group in both hemispheres , ipsilesional ( injured side ) [ f(5,35 ) = 14.25 ( p < 0.01 ) ] , and contralesional side [ f(5,35 ) = 9.70 ( p < 0.01 ) ] . differences among s and s - sk ( p < 0.001 ) , ich ( p = 0.02 ) , s - us , ich - us , and ich - sk ( p < 0.001 ) and between s - us and ich - us ( p = 0.03 ) , ich , and ich - sk ( p < 0.01 ) when the contralesional hemisphere was analyzed , differences were found among s and s - sk , ich , ich - sk ( p < 0.01 ) , and ich - us ( p = 0.02 ) and between s - us and ich - sk ( p < 0.01 ) , ich - sk , and ich - us ( p = 0.04 ) . the purpose of the current study was to investigate whether different rehabilitation modalities ( skilled or unskilled training ) would result in a differential upper limb motor behavior and in morphological adaptations as evaluated by the means of map-2 immunoreactivity in the forelimb representation in motor cortex . the persistence of this deficit impacts directly in an index of independence and quality of life . present results show that skill task training performed with the impaired forelimb was able to increase map-2 immunoreactivity in motor cortex either in sham or in ich groups in both cortices . behavioral evaluation revealed that ich - sk performed better than ich and ich - un animals in the staircase test . the type of treatment , intensity and duration of the protocol , and the period in which it is applied are factors related to beneficial effects on functional recovery [ 3537 ] . this analysis revealed that after ich , only animals submitted to skill task training showed significant improvement in motor performance at the end of the 4th week of treatment , as evidenced by the total number of retrieved pellets . color - coded pellets can be used in staircase test aiming to increase the sensitivity of the instrument since it allows taking into consideration the levels of reaching difficulty . ich - sk group presented superior reaching rate when compared to ich and ich - us groups at the end of the 4th week . this may indicate that skill task training is able to induce superior upper limb recovery after intracerebral hemorrhage when compared to unskilled task training . in agreement with our results , another study demonstrated that skilled reaching training of impaired forelimb can induce brain plasticity which was observed in association with enhanced sensorimotor recovery after stroke in sensorimotor cortex and striatum [ 13 , 38 , 39 ] . previous investigations also have found that activity - based therapies to promote forelimb use after stroke are able to develop cortical adaptations as protein synthesis , synaptogenesis , and increase in cortical representation of hand and fingers [ 12 , 37 ] . these modifications correlated with functional recovery are usually proportional to the complexity of the task and do not occur in the same intensity when movement or gestures are executed in a simple and repetitive pattern [ 10 , 36 , 40 ] . our behavioral results showed that physical motion can induce motor recovery , but that skill task training as a rehabilitative strategy has the capacity to improve motor performance . therefore , food obtained as a reward may be considered a motivational factor that contributes for morphophysiological changes associated with behavioral responses [ 13 , 14 ] . histological and biochemical studies have demonstrated a mild correlation between motor performance and these measurements in collagenase - induced intrastriatal hemorrhage and focal ischemic models [ 8 , 29 ] . in view of the lack of correlation between morphometrical / macroscopic analysis and behavioral results , probably the supporting for behavioral changes observed in our results map-2 immunoreactivity in the motor cortex was increased by skilled task training in both cerebral hemispheres either in physiological or pathological ( ich ) conditions . motor learning is a set of internal processes associated with practice or experience that involves a coordinate system in which a planned task is encoded and combined with appropriate recruitment of motor units to execute a movement goal [ 41 , 42 ] . the synaptogenesis induced by motor practice or synaptic reinforcing transmission may consolidate the motor gesture learned in both intact and stroke - injured brains [ 11 , 36 ] . however , our behavioral and morphological results suggest that the ich model is adequate for future studies regarding brain injury and motor recovery by greater understanding between the morphological changes of the nervous cells and motor neurorehabilitation techniques . furthermore , map-2 plays in the growth , differentiation , and plasticity of neurons , with key roles in neuronal responses to growth factors , neurotransmitters , synaptic activity , and neurotoxins [ 30 , 45 ] . the increase of map-2-positive dendrites is connected with neural activity regeneration after injury cerebrovascular [ 17 , 46 ] , and dendritic morphological changes may be involved in motor learning . since modification and rearrangement of map-2 are obligatory steps in many processes that modify neuronal function , we decided to investigate the effects of skilled and unskilled training after ich on functional recovery and map-2 expression . the motor recovery after ischemia techniques for inducing restoration of excitability in motor cortex can exert positive effects on rehabilitation therapy by means of changes in dendritic structure and expression of map-2 [ 23 , 48 ] . dendritic growth process seems to be related to the amount of motor cortex inputs and partially depends on map-2 expression . relative optical densities analysis revealed that the amount of map-2 was significantly higher in ich animals submitted to skill task training ( ich - sk ) when compared to ich and ich - un in the ipsilesional and contralesional cortices , respectively . even though ich and ich - us groups have shown increased map-2 immunoreactivity in both ipsi- and contralesional motor cortices , no behavioral improving outcome was observed . since map-2 is involved with neuronal plasticity under physiological and pathological conditions , those levels of immunoreactivity after ich were not surprising . on the other hand , motor cortex lesion causes damage in cortical networks and their projections for movement and results in contralateral limb disuse . compensatory use of the unaffected limb can be followed by the increasing of the cortical activity in the contralesional hemisphere , which can explain levels of map-2 immunoreactivity in contralesional cortex in ich nonrehabilitation group . our results reinforce previous findings and reaffirm that skill task training is a good option for rehabilitation after stroke as demonstrated by the behavioral motor recovery and morphological changes . our main results demonstrated that skill task can drive brain plasticity in motor cortex by the increasing of map-2 expression in both hemispheres and with positive functional outcomes after collagenase - induced intrastriatal unilateral hemorrhage in a superior manner when compared to unskilled training . these findings may offer insights for improving functional recovery in stroke patients since that rehabilitation depends on type , intensity , and duration of approaches used as physical treatment .

understanding the physical processes that control the dynamics of energetic electrons in the inner magnetosphere is important for the protection of satellite and groundbased technologies . electron fluxes that intensify during storm time could damage satellites in two important ways : ( 1 ) lower energy ( kev ) electrons cause surface charging by nonuniformly depositing their charge in spacecraft surfaces and driving potential differences which can damage electronics [ thomsen et al . , 2013 ] and ( 2 ) higherenergy ( above 1 mev ) electrons can penetrate the surface and accumulate within internal dielectrics causing deepdielectric charging , the subsequent breakdown of which can lead to component failures [ e.g. , wrenn et al . , accurate modeling and , ultimately , predicting electron fluxes that could cause hazardous space weather events , however , is challenging since the nearearth system is strongly coupled . relativistic electron fluxes can change drastically during active conditions , and their response to solar wind driving is not simple . although initial analysis of mev electron fluxes at geosynchronous orbit [ paulikas and blake , 1979 ] indicated that they are well correlated with solar wind velocity , later studies [ reeves et al . , 2011 ] found that only the lower flux limit depends linearly on the solar wind speed while highestelectron fluxes can occur for any solar wind speed . the statistical distribution of kev electron fluxes was found to be remarkably similar to the distribution of mev fluxes and showed distinctly nonlinear correlations to solar wind driving . the van allen probes mission ( previously known as the radiation belt storm probes , rbsp ) was thus launched in august 2012 to investigate the dynamics of nearearth fluxes of relativistic electrons and to help the development of models for specification and prediction of their space weather effects [ kessel et al . , there are several processes that lead to the enhancement of electron fluxes in the nearearth space environment . a traditional heating mechanism for the radiation belt ( mev ) electrons is the inward transport of electrons across the geomagnetic field by radial diffusion leading to significant energization [ schulz and lanzerotti , 1974 ] , but the timescales are typically too slow to account for the observed variations . this process could be enhanced by ultralow frequency ( ulf ) waves [ e.g. , hudson et al . , 2000 ; ulf waves can efficiently accelerate electrons through a driftresonant interaction , and a strong correlation between electron fluxes and ulf activity has been observed , with large increases in wave power preceding electron flux enhancement at geosynchronous orbit [ rostoker et al . similarly , the ring current ( kev ) electron fluxes increase after injection from the plasma sheet during substormassociated dipolarization events [ e.g. , baker et al . , 1996 ; birn et al . , 1997 ; fu et al . , 2011b ] and earthward transport and energization [ e.g. , jordanova and miyoshi , 2005 ; liu et al . , the injected 10 kev electrons provide the energy source for the generation of whistler mode waves [ e.g. , jordanova , 2012 , and references therein ] which transfer the energy back to the particles and accelerate them to higher energies . the tens of kev ring current electrons are thus often referred to as the source population of the radiation belts [ e.g. , li and temerin , 2001 ; jaynes et al . , population for the radiation belts as they may be accelerated to mev energies by linear [ e.g. , horne and thorne , 1998 ; summers et al . , interactions with very low frequency ( vlf ) whistlers and by interactions with fast magnetosonic waves [ e.g. , horne et al . , 2007 ] . on the other hand , the decrease of the magnetic field strength during the main phase of a storm due to the ring current buildup causes an outward motion of energetic electrons through conservation of the third adiabatic invariant and a decrease of relativistic electron fluxes , the dst effect , first demonstrated for flux variations near geosynchronous orbit by kim and chan . additional processes contributing to the loss of energetic electrons are outward radial diffusion combined with magnetopause shadowing [ e.g. , shprits et al . , 2006 ; morley et al . , 2010 ] and precipitation into earth 's atmosphere via waveparticle interactions [ e.g. , walt and macdonald , 1962 ] . scattering into the atmospheric loss cone may be significant and includes electron precipitation by plasmaspheric hiss [ e.g. , lyons et al . , 1972 ; meredith et al . , 2006 ] , by scattering by vlf chorus [ e.g. , o'brien et al . , 2004 ; bortnik and thorne , 2007 ] , and by electromagnetic ion cyclotron ( emic ) waves [ e.g. , thorne and kennel , 1971 ; jordanova et al . , the electron losses balance the energization processes [ e.g. , fu et al . , 2011a ] and thus constrain the rebuilding of the ring current and the radiation belts . recent studies using highquality particle and wave data from the van allen probes mission provided improved understanding of many of the physical processes discussed above that drive relativistic electron dynamics in nearearth space . for example , the importance of local acceleration by plasma waves was demonstrated by reeves et al . and thorne et al . , while the importance of gradual acceleration by radial diffusion was shown by baker et al . . significant physical insight on the simultaneously acting and highly variable transport , acceleration , and loss mechanisms was also gained from model simulations and their comparison with observations . it was thus shown that the enhancement of mev electrons is higher when a selfconsistent electric field model is used [ fok et al . , 2014 ] and that parameterized pitch angle loss rates could account for the trapped electron fluxes observed at geosynchronous orbit as well as for the precipitating fluxes observed in the ionosphere [ chen et al . , 2015 ] . doubledip large geomagnetic storm , which is also the subject of this study , occurred shortly after the rbsp launch in october 2012 [ reeves et al . , previous numerical modeling simulations in two dimensions ( 2d ) [ thorne et al . , we investigate the dynamics of lower energy ( kev ) electrons , the generation of whistler mode waves , and the effects from subsequent waveparticle interactions in the inner magnetosphere using a fourdimensional ( 4d ) physicsbased model , the ring currentatmosphere interactions model with selfconsistent magnetic field ( ramscb ) [ jordanova et al . the ring current electron fluxes represent the source ( approximately tens of kev ) and seed ( approximately hundreds of kev ) populations for relativistic electrons and are crucial elements for radiation belt evolution . we present quantitative comparisons between our model predictions and van allen probes and noaa polarorbiting operational environmental satellites ( poes ) observations available during the investigated storm period . interplanetary observations and geomagnetic indices during 710 october 2012 taken from the omniweb 5 min database are shown in figure 1 . sharp increases in solar wind density , speed , and dynamic pressure are observed around 05 ut 8 october ( hour 29 ) , indicative of the arrival of an interplanetary shock ( figures 1a1c ) . the subsequent southward excursion of the interplanetary magnetic field ( imf ) b z component to about 15 nt ( figure 1d ) triggered the first dip of a large geomagnetic storm with minimum symh 100 nt at 10 ut 8 october ( hour 34 ) ( figure 1f ) . after an intermediate recovery to symh 50 nt at 18 ut ( hour 42 ) during a shortlived northward imf turning , the storm intensified again reaching a second dip of symh 115 nt at 02 ut 9 october ( hour 50 ) . the dynamic pressure was stronger during the first dip due to the larger solar wind density and compressed the magnetopause to < 8 r e ; during the second dip the solar wind dynamic pressure decreased and the magnetopause moved out to > 10 r e [ thorne et al . , 2013 ] . the substorm activity intensified during both dips as shown by the a l index decreasing below 1000 nt ( figure 1e ) . the planetary k p index reached maxima of 6 and 7 at hour 30 and hour 48 , while the cross polar cap potential ( cpcp ) drop obtained with the imfdependent weimer ( w05 ) ionospheric electric potential model peaked at approximately hour 30 and hour 42 , respectively ( figure 1 g ) . the solar wind speed fluctuated around 400 km / s until approximately hour 68 when it began a slow rise while the storm recovered to symh 0 . ( a ) proton density , ( b ) solar wind speed , ( c ) dynamic pressure , and ( d ) b z ( gsm ) component of the magnetic field . ( e ) the a l index , ( f ) measured ( solid ) and pressurecorrected ( dashdotted ) symh , ( g ) the k p index ( solid ) and the w05 cross polar cap potential drop ( dashdotted ) . the vertical dashed lines mark the time period of ramscb simulation . measurements during this period from the magnetic electron ion spectrometer ( mageis ) [ blake et al . , 2013 ] and electric and magnetic field instrument suite and integrated science ( emfisis ) investigation [ kletzing et al . , 2013 ] on board the van allen probe a , along an elliptical orbit on the morning side with apogee at 6 r e and mlt7 , are displayed in figure 2 . the panels show , from top to bottom , the spinaveraged differential electron flux as a function of energy from 30 to 350 kev , the electron flux magnitude at four representative energies , the whistler wave power spectral density , and the l shell [ mcilwain , 1966 ] as a function of time ( horizontal axis ) . as seen from the mageis flux data , during 7 october the satellite traversed several times at low l < 4 the electron ring current characterized by quiet time fluxes extending from 30 to 350 kev ; the 40 kev flux was of the order of 10 cm s sr kev , and the flux decreased at larger energies . this trapped population was clearly separated from the < 100 kev population on open drift paths at larger l shells . with the development of substorm activity , a dispersionless electron injection was observed at < 100 kev energies at 01 ut on 8 october that reached l 5 at mlt 5 . the next nightside satellite pass occurred during the storm main phase from 07 to 12 ut 8 october , and mageis measured enhanced electron fluxes typical of a strong ring current with fluxes at higher energies ( > 100 kev ) confined to lower l shells . the electron fluxes decreased at all energies from 14 ut to 18 ut as the storm recovered . several electron injections occurred after 20 ut 8 october and throughout 9 october during the second symh dip when the ring current intensified even more but the electron flux enhancement remained at e 150 kev at apogee . emfisis observations of interest to this study showed weakly enhanced lower band chorus ( f < 0.5f ce ) concurrent with the enhancement of 3050 kev electrons during the initial substorm activity on 0105 ut 8 october . the power spectral density increased significantly in this frequency band during the first and second symh minima , while it was reduced during the intermediate storm recovery . on the other hand , lowfrequency ( f < 0.1f ce ) hiss was observed mostly at small radial distances on the dayside when the satellite passed briefly through the plasmasphere . van allen probe a observations during 79 october 2012 including ( top to bottom ) mageis differential electron flux spectrogram ; spinaveraged electron flux at 37 kev ( red ) , 111 kev ( green ) , 211 kev ( light blue ) , and 317 kev ( dark blue ) ; emfisis magnetic field power spectral density spectrogram with lines denoting fractions of the electron gyrofrequency at 0.1 f ce ( blue ) , 0.5 f ce ( red ) and 1.0 f ce ( black ) ; and the mcilwain l parameter along the satellite orbit . figure 3 shows observations from one of the noaa poes satellites which have circular sunsynchronous orbits ( altitude 800850 km with an inclination of 98 ) and a period of 102 min . the space environment monitor 2 ( sem2 ) [ evans and greer , 2000 ] on board each threeaxis stabilized poes spacecraft includes a set of solidstate medium energy proton and electron detectors ( meped ) . for electrons , there are two solidstate detector telescopes , one pointing toward zenith ( i.e. , the 0 telescope ) and the other to view at about 90 to the first ( i.e. , the 90 telescope ) , to measure electrons in three energy ranges ( e1 = 0.032.5 mev , e2 = 0.12.5 mev , and e3 = 0.32.5 mev ) . in the outer belt region , the 0 telescope measures precipitating electrons inside the loss cone , while the 90 telescope measures electrons that are locally trapped [ rodger et al . , the electrons ' radial distributions measured by noaa 15 , which samples in the predawn and afternoon sectors , over a 3 day period , are presented in figure 3 . here the electron fluxes are binned into 3 h intervals and sorted by precipitating versus trapped populations for three energy ranges ( e1 , e2 , and e3 ) and two local time sectors . in comparison with the prestorm quiet time flux level , both the precipitating and trapped fluxes intensified at 3<l<6 predawn in the lowest energy range ( e1 ) during the first symh dip . on the other hand , the fluxes increased at all energies and all local times during the second symh dip and the intensification of the trapped fluxes at higher energies lasted throughout the recovery phase . the noaa poes precipitating and trapped electron fluxes ( in units of 1/cm / s / sr ) during 79 october 2012 binned into three energy ranges ( > 30 kev , > 100 kev , and > 300 kev ) , ( left column ) for mlt = 06 and ( right ) for mlt = 1218 local time sectors . by comparing to meped proton data following the method used by chen et al . , we confirmed that protons have no significant contaminations on electron data during the period . the symh index is given for reference , and the vertical lines mark the start of ramscb simulation . electron and ion flux measurements from the mageis instruments on the van allen probes a and b and from the magnetospheric plasma analyzer ( mpa ) and synchronous orbit particle analyzer ( sopa ) instruments on lanlgeo satellites during 3 days of the october 2012 storm are plotted in figure 4 . for these comparisons , the lanlgeo fluxes at 6.6 r e are interpolated in time and mlt to the epoch and mlt of van allen probes ' apogee . it is evident that the fluxes from the different instruments agree reasonably well , especially at lower ( < 200 kev ) energies of interest to this study , when interpolated to the nearly same epoch and location in the magnetosphere . the agreement is better during the prestorm period on 7 october than during the more disturbed time on 8 and 9 october when temporal flux variations are clearly seen . the lanlgeo fluxes are used as boundary conditions for the standalone ramscb simulations . a comparison between ( left column ) electron and ( right column ) ion fluxes from the mageis instruments ( dotted ) on the van allen probes a and b and from the mpa and sopa instruments ( solid ) on lanlgeo satellites during 79 october 2012 . the lanlgeo fluxes are interpolated in time and mlt to the epoch and mlt of van allen probes ' apogee . we simulate the ring current evolution during the doubledip storm of 79 october 2012 using our ramscb model which couples the ring currentatmosphere interactions model ( ram ) [ jordanova et al . , 1997 , 2010b ] with the eulerpotentialbased plasma equilibrium code this model has been used successfully in many ring current studies over the past decade ; therefore , only a brief description of ramscb focusing on its recent development is given below . the 4d ram solves the kinetic equation for h , o , he , and electron phase space distributions q l(r o,,e, o , t ) in the magnetic equatorial plane as a function of time t , radial distance from earth ( r o from 2 to 6.5 r e ) , geomagnetic east longitude , kinetic energy e from 100 ev to 400 kev , and equatorial pitch angle o from 0 to 90 : ( 1)qlt+1ro2roro2drodtql+ddtql+1pepdedtql+1hoohododtql=qltloss here the brackets denote bounceaveraging , o = cos(o ) , p is the relativistic momentum of the particle , and is the relativistic factor . the integral h=1/(2ro)ds/(1b(s)/bm ) is calculated along the magnetic field lines and between the mirror points where the field intensity is b m. the equatorial anisotropic plasma pressure produced by the ring current particles is mapped along the field lines and used by the 3d eulerpotentialbased equilibrium code to calculate the resulting forcebalanced magnetic field [ e.g. , zaharia et al . , 2006 ] . this selfconsistent magnetic field is passed back to ram in order to propagate the phase space distributions . in the standalone ramscb the electric field is provided from the latest version of the solar winddriven w05 ionospheric model ( figure 1 g ) by mapping it along the scb field lines to the equatorial plane and adding corotation ; the process of radial diffusion is not considered . the nightside boundary conditions are determined from plasma sheet flux measurements from the mpa and sopa instruments on the lanl geosynchronous spacecraft ( figure 4 ) . in this study we assume isotropic plasma boundary conditions , although anisotropic electron pitch angle distributions like cigartype or pancaketype have been observed in the plasma sheet behind dipolarization fronts [ e.g. , fu et al . , 2011b , 2012 ] ; their effect will be considered in future work . to set the initial conditions , we use quiet time measurements during october 2012 from the mageis and hope [ funsten et al . , 2013 ] instruments which are part of the energetic particle , composition , and thermal plasma ( ect ) suite [ spence et al . , 2013 ] on the van allen probes , starting the simulation at 10 ut 7 october well before the storm commencement ( figure 1 ) . additional loss processes included in this study are charge exchange of ring current ions with geocoronal hydrogen , and ion and electron precipitation due to collisions with the dense atmosphere assuming a loss cone boundary of 200 km altitude . after injection from the plasma sheet , the electron population becomes unstable as the particles drift through the inner magnetosphere and undergo further acceleration and loss processes . to investigate the free energy available for the excitation of whistler mode waves during the 79 october 2012 storm period , we performed initial simulations of electron dynamics considering a simplified loss term ( q e/ wp ) where ( wp ) is the waveparticle interactions lifetime . in this case electron losses inside the plasmasphere were included using lifetimes calculated after albert , while losses due to whistler mode wave scattering outside the plasmasphere were incorporated using empirical lifetimes for diffusion that is not strong everywhere ( for further details , see jordanova et al . [ 2008 , 2010a , and references therein ] ) . the linear growth rate of whistler mode chorus was calculated in the equatorial plane using the electron distributions and selfconsistent magnetic field simulated with ramscb and cold plasma density simulated with the coupled plasmasphere model [ rasmussen et al . these ramscb simulation results are compared with the wave proxy derived from poes data at three representative times ( prestorm , main , and recovery phase ) in figure 5 ( left , middle , and right columns , respectively ) . the linear growth rate of parallel propagating chorus in the lower band frequency range ( figure 5a ) intensifies significantly during the storm main phase . this is due to the increased effective electron anisotropy ( figure 5b , as defined in jordanova et al . [ 2010a , equation ( 2 ) ] ) that develops at outer l shells when particles drift in realistic magnetic field and undergo large acceleration and drift shell splitting [ jordanova et al . , 2012 ] . in agreement with previous studies , the growth rate maximizes in the morning sector outside the plasmasphere , as indicated by the cold plasma density ( figure 5c ) ; the corresponding parallel energy of resonant electrons is from 2 to 5 kev . during the prestorm and recovery phases the electron fluxes are smaller and figure 5d shows 2d snapshots of equatorial chorus wave amplitudes inferred from the precipitating 30 kev electron fluxes measured at low altitude by multiple noaa poes satellites [ chen et al . , specifically , the measured precipitating electron fluxes are related with an adjustable parameter to the equatorial wave measurements made by emfisis onboard van allen probes to infer the global distribution of chorus waves . a previous study by tu et al . has shown that the obtained amplitudes are in good agreement with in situ emfisis observations during this storm ( figure 2 ) ; their spatial and temporal evolutions also agree reasonably well with the global patterns of chorus wave growth simulated with ramscb ( figure 5a ) . ( a ) normalized growth rate of whistler mode chorus with frequency 0.45 f ce calculated with ramscb , ( b ) effective anisotropy of ring current electrons , and ( c ) cold plasma density . ( d ) proxy for chorus wave amplitude ( defined as the square root of wave intensity integrated over [ 0.10.8 ] f ce ) ; data gaps are due to the limited spatial coverage of poes satellites . all parameters are plotted in the equatorial plane at hours 24 , 32 , and 42 after 00 ut 7 october 2012 indicated with stars in the symh plot . to implement in ramscb for the first time quasilinear diffusion of relativistic electrons by whistler mode waves , we include diffusion in pitch angle and energy as the most important terms ( mixed diffusion will be considered in future extensions of this work ) on the righthand side of equation ( 1 ) following jordanova et al . [ 1997 , 1998 ] : ( 2)qltwp=1hoohodooqlo+1pepdeeqle here the bounceaveraged quasilinear pitch angle and energy diffusion coefficients are : ( 3)doo=(1o2)2ho2rosmsmbobdoop2ds=(1o2)p2doo ( 4)dee=p22h2mo2rosmsmdppds = p2mo22dpp where m o is the rest mass of the resonating particle and s m denotes the mirror point . in this study the bounceaveraged pitch angle doo and momentum d pp diffusion coefficients are calculated following the method in summers assuming fieldaligned waves . global wave intensity distributions in magnetic latitude ( mlat ) , magnetic local time ( mlt ) , and l are required for the calculation . for hiss waves inside the plasmasphere we use the empirical wave distributions statistically derived from the combined release and radiation effects satellite ( crres ) wave data , which are binned in a e * ( the mean value of a e over the previous 1 h ) . these wave databases were built at lanl and validated against similar models from meredith et al . [ 2003 , 2004 ] . the same empirical plasma density models and wave spectral properties were used in tu et al . [ however , for whistler mode chorus outside the plasmasphere , which is critical to simulate the strong enhancement of relativistic electrons during the october 2012 storm , we use the global chorus wave proxy of chen et al . to produce the eventspecific chorus wave amplitudes ( figure 5d ) as a function of mlt , l , and time on a 3 h basis . since the chorus wave distribution in mlat can not be resolved by the proxy , the mlat dependence from a statistical model is used ( also based on crres wave data from tu et al . ) . figure 6 shows the equatorial 2d distribution of the bounceaveraged diffusion coefficients calculated for 100 kev electrons at three representative times . as expected from previous studies , the pitch angle diffusion coefficients are larger than the momentum ones and maximize in the dawn sector during the storm main phase where the chorus wave proxy is most enhanced , corresponding to a minimum electron lifetime of about an hour . eventspecific ( top row ) pitch angle < doo / p2 > and ( bottom row ) momentum < d pp / p > diffusion coefficients calculated for 100 kev electrons and o=50 at hours 24 , 32 , and 42 after 00 ut 7 october 2012 . the plasmapause location is plotted with a dashdotted line for reference . ring current electron simulations with ramscb during the october 2012 doubledip storm are displayed in figure 7 where the electron flux was plotted along the van allen probe a trajectory in the same format as in figure 2 to ease the comparison with mageis observations . to this end the spacecraft location was mapped along the 3d scb magnetic field lines to the equatorial plane and the ram flux was obtained by interpolation from neighboring grid points . figure 7 shows the electron flux averaged over pitch angle obtained with ramscb ( version 1 ) that uses empirical lifetimes for electron loss ( middle row ) , as well as results from the newly developed in this study ramscb ( version 2 ) that includes quasilinear plasma wave scattering with eventspecific diffusion coefficients ( bottom row ) ; results from a ramscb test simulation that considers only transport and precipitation losses due to atmospheric collisions at low altitude are shown in the top row for comparison . the modeled fluxes increase during the storm main phase at low energies due to convective transport of newly injected electrons from the plasma sheet in agreement with mageis observations ( figure 2 ) , but the model underestimates significantly the fluxes at high ( > 100 kev ) energies in this case . with storm development the flux at lower energies overestimates mageis observations when plasma wave scattering losses are not included ( figure 7 , top row ) . on the other hand , the empirical lifetimes used in ramscb version 1 ( figure 7 , middle row ) are too short and the electron injection is not large enough to overcome the losses , especially during quiet time and at high energies . simulations with ramscb version 2 ( figure 7 , bottom row ) indicate larger fluxes and better agreement with mageis data at all energies during quiet time ( note the different scales in the plots ) . as the storm develops the fluxes increase not only at low but also at high energies due to the local acceleration by chorus waves , and while the lowenergy flux agrees reasonably well with observations , the flux at e>100 kev becomes about an order of magnitude larger than mageis observations near the first symh dip . contrary to observations , the simulated fluxes do not decrease much in the morning sector during the intermediate recovery phase and increase even further during the second symh dip significantly overestimating mageis fluxes . simulations of electron fluxes averaged over pitch angle and plotted along van allen probe a orbit , ( top row ) from a ramscb test run considering only transport and loss by atmospheric collisions , ( middle row ) from ramscb ( version 1 ) using empirical lifetimes for electron loss , and ( bottom row ) from ramscb ( version 2 ) using quasilinear diffusion . the vertical white bands indicate successive perigee passes when the satellite is outside the computational domain . the line plots show the electron fluxes at four representative energies : 40 kev ( red ) , 100 kev ( green ) , 210 kev ( light blue ) , and 335 kev ( dark blue ) . in figure 8 we compare results from several ramscb simulations at l = 4 and mlt = 7 with mageis data as a function of time . electron fluxes from the mageis spectrometer when the van allen probe a passes through the morning sector at l 4 are interpolated onto the ram energy grid and plotted in figure 8a . ramscb results considering timedependent drift and electron loss due to atmospheric collisions at low altitude are shown in figure 8b , adding pitch angle diffusion by plasma waves in figure 8c , and adding energy diffusion in figure 8d . in the observations the flux increases at all energies with about 2 orders of magnitude at 06 ut 8 october during the storm main phase . this is followed by an order of magnitude decrease at 18 ut during the intermediate recovery phase and a subsequent increase during the second symh dip at 04 ut 9 october to approximately the same magnitude as during the first dip . the first sharp increase is reproduced well by the simulated ramscb flux at 30 kev ( figure 8b ) . the dynamics of the lowenergy electrons is dominated by magnetospheric convection , and the flux rises significantly as the w05 cross polar cap potential drop , indicative of convection strength , and rises at 06 ut ( figure 1 g ) . with storm development higherenergy electrons penetrate closer to earth and the 50 kev flux rises at 10 ut ; another rise occurs at 18 ut . the 100 kev flux rises at 24 ut , while the increase by convective transport is negligible at higher energies . note that the smallscale fluctuations clearly seen in the 50 kev flux are representative of drift echoes , i.e. , bunches of injected electrons drifting longitudinally [ lanzerotti et al . , 1967 ] , and correspond to the electron drift period of 3.5 h at this energy . when pitch angle scattering is considered ( figure 8c ) , the enhancement at these energies is substantial when energy diffusion by plasma waves is included ( figure 8d ) , and the simulated energy spectra shapes agree better with observations . in this case , however , the fluxes at e>50 kev increase by about 2 orders of magnitude and reach larger values than mageis observations during the storm main phase , remain at this elevated level during the intermediate recovery phase , and rise again during the second symh dip . similarly , tu et al . found that the local heating by chorus during the first symh dip overwhelmed the mev electron loss from outward radial diffusion and the electron distribution did not show a decrease as in the observations but rather an early increase . this indicates that either the local acceleration by chorus waves is overestimated at this time in the eventspecific diffusion model or additional loss mechanisms have to be considered . ( a ) electron flux measured with mageis when the van allen probe a crosses r o=4 0.1r e during 710 october 2012 interpolated onto ram energy and pitch angle grids . simulated ramscb electron flux at l = 4 and mlt = 7 ( b ) considering transport and loss due to atmospheric collisions , ( c ) adding pitch angle scattering , and ( d ) adding energy scattering , at five representative energies and o=50. to provide better understanding of the role various processes have in ring current dynamics , in figure 9 we plot the electron phase space distribution at l = 4 and mlt = 7 during prestorm quiet time at 00 ut 8 october ( hour 24 ) and during the storm main phase at 10 ut 8 october ( hour 34 ) . the increasing with geomagnetic storm activity crosstail electric field moves the boundary between open and closed drift paths inward and allows access of electrons with successively higher energies , from 20 kev at hour 24 ( figure 9a ) to 80 kev at hour 34 ( figure 9d ) . strong anisotropies thus develop in energy due to the fact that particles with higher energies can not penetrate the open / closed drift boundary . the process of pitch angle diffusion scatters electrons from larger to smaller pitch angles and reduces the distributions at e > 5 kev near the loss cone ( figures 9b and 9e ) ; it does not remove the anisotropy in energy , but it shifts it toward lower energy . electrons are scattered by energy diffusion from lower to higher energy to reduce this anisotropy ( figures 9c and 9f ) ; this process is more effective at hour 34 due to the stronger wave activity and larger diffusion coefficients . this acceleration effect is more significant than expected because of the large gradients in the distribution function , even though the energy diffusion coefficients are much smaller than the pitch angle diffusion coefficients . simulated ramscb electron phase space distribution ( s / km ) as a function of energy and pitch angle at l = 4 and mlt = 7 ( a , d ) considering transport and loss due to atmospheric collisions , ( b , e ) adding pitch angle scattering , and ( c , f ) adding energy scattering . figures 9a9c are at hour 24 , while figures 9d9f are at hour 34 after 00 ut 7 october 2012 . finally , global patterns of electron fluxes simulated with ramscb are plotted in figures 10 and 11 in the same l time format as the noaa poes observations in figure 3 . from top to bottom are shown fluxes at 30 kev , 100 kev , and 335 kev , at mlt = 3 ( figure 10 ) and mlt = 15 ( figure 11 ) . fluxes of trapped ( o=50 ) and precipitating ( mirroring at 200 km altitude ) electrons calculated at every hour are displayed ; these correspond roughly to the temporal and spatial resolution and pitch angles sampled by poes detectors . when energy diffusion is not included ( left column ) , the 30 kev electrons penetrate to l 3 on the morning side during the storm main phase after convective transport from the plasma sheet and eastward drift , forming an asymmetric ring current in good agreement with poes observations . the electron fluxes at higher energies are very small and considerably underestimate the observations . the fluxes increase due to local acceleration by chorus waves when energy diffusion is included ( middle column ) , the enhancement being larger at higher energies , and the electron distribution becomes symmetric with mlt at e>100 kev as seen in poes observations . during the intermediate recovery phase the trapped highenergy flux decreases at large ( l>5 ) shells but remains enhanced between l=4 and l=5 ; it increases by an order of magnitude during the second symh dip and overestimates poes data . the precipitating flux ( right column ) follows the temporal and spatial evolution of the trapped flux but is more than an order of magnitude smaller ( note the different scales ) and reproduces better poes observations . simulated ramscb flux ( 1/cm / s / sr / kev ) for 30 kev , 100 kev , and 335 kev ( from top to bottom ) at mlt = 3 as a function of radial distance from earth and time after 00 ut 7 october 2012 for trapped electrons ( o=50 ) ( left column ) considering transport , loss due to atmospheric collisions , and pitch angle scattering and ( middle column ) adding energy scattering . ( right column ) simulated fluxes including all processes for precipitating ( mirroring at 200 km altitude ) electrons . simulated ramscb electron flux ( 1/cm / s / sr / kev ) in the same format as figure 10 but at mlt = 15 . mechanisms for particle injection in the nearearth space environment and their subsequent trapping or loss have been the subject of detailed investigation recently , thanks to the highresolution data available from the van allen probes . their theoretical evaluation and numerical implementation in global models with highfidelity predictive capabilities , however , remains challenging . we studied the combined effects from timedependent transport and scattering by whistler mode chorus and hiss on the source ( approximately tens of kev ) and seed ( approximately hundreds of kev ) populations of the radiation belts during the october 2012 doubledip storm . we used our kinetic ring currentatmosphere interactions model coupled with a eulerpotentialbased threedimensional plasma equilibrium code ( ramscb ) , including for the first time pitch angle and energy diffusion using l and mltdependent eventspecific wave models . the chorus wave amplitudes were thus inferred from correlations of poes / meped precipitating electron fluxes and rbsp / emfisis wave observations ; the spatial and temporal evolutions of peak wave amplitudes was in reasonable agreement with chorus growth rate maxima calculated with ramscb . comparisons of ramscb simulations with complementary data sets from the van allen probes in the morning mlt sector and noaa poes satellites in the afternoon and predawn sectors indicated the following : during quiet time ramscb electron simulations using l and mltdependent eventspecific pitch angle and energy diffusion coefficients reproduced much better the in situ satellite observations of ring current electron flux than ramscb simulations using empirical lifetimes , since these lifetimes were too short and overestimated the electron losses.the simulated electron flux at lower ( e < 100 kev ) energies increased during the first and second symh dips and decreased during the intermediate recovery phase due to earthward transport in timedependent convective electric field and nondipolar magnetic field . this electron dynamics was in good agreement with observations of both trapped and precipitating electron fluxes and showed the initial formation of an asymmetric ring current that evolved into a more symmetric one with storm development.at higher ( e100 kev ) energies ramscb simulations that considered only convective transport and loss substantially underestimated the observations . when local acceleration by chorus was included , the electron fluxes increased by about 2 orders of magnitude during the first symh dip , remained enhanced during the intermediate recovery phase at l = 4 to l = 5 , and increased further during the second symh dip thus considerably overestimating the satellite observations . during quiet time ramscb electron simulations using l and mltdependent eventspecific pitch angle and energy diffusion coefficients reproduced much better the in situ satellite observations of ring current electron flux than ramscb simulations using empirical lifetimes , since these lifetimes were too short and overestimated the electron losses . the simulated electron flux at lower ( e < 100 kev ) energies increased during the first and second symh dips and decreased during the intermediate recovery phase due to earthward transport in timedependent convective electric field and nondipolar magnetic field . this electron dynamics was in good agreement with observations of both trapped and precipitating electron fluxes and showed the initial formation of an asymmetric ring current that evolved into a more symmetric one with storm development . at higher ( e100 kev ) energies ramscb simulations that considered only convective transport and loss substantially underestimated the observations . when local acceleration by chorus was included , the electron fluxes increased by about 2 orders of magnitude during the first symh dip , remained enhanced during the intermediate recovery phase at l = 4 to l = 5 , and increased further during the second symh dip thus considerably overestimating the satellite observations . ramscb model results that reproduced significant acceleration by plasma waves at energies as low as 50 kev were initially unexpected since the energy diffusion coefficients are much smaller than the pitch angle diffusion coefficients . this energization effect is enhanced by the presence of a large energy anisotropy that develops in the electron phase space distribution after particles are injected from the plasma sheet . these results indicate that either additional loss mechanisms ( like enhanced losses through the dayside magnetopause or coulomb collisions ) or a better representation of plasma wave scattering taking into account nonlinear effects is needed . a selfconsistently calculated electric field , with higher temporal and spatial resolution , will influence both the injection of particles from the tail and their dayside outflow and could result in reduced gradients in the distribution function . note that the diffusion coefficients themselves depend on many parameters ( i.e. , wave amplitude , frequency and wave normal distributions , and cold plasma density ) which need to be specified with improved temporal and spatial resolution . in addition , other types of plasma waves like magnetosonic and emic waves , as well as oblique chorus waves , could produce more scattering and thus influence ring current dynamics . this problem could also be alleviated by including mixed diffusion in ramscb , and this will be considered in future work .

abstractmechanisms for electron injection , trapping , and loss in the nearearth space environment are investigated during the october 2012 doubledip storm using our ring currentatmosphere interactions model with selfconsistent magnetic field ( ramscb ) . pitch angle and energy scattering are included for the first time in ramscb using l and magnetic local time ( mlt)dependent eventspecific chorus wave models inferred from noaa polarorbiting operational environmental satellites ( poes ) and van allen probes electric and magnetic field instrument suite and integrated science observations . the dynamics of the source ( approximately tens of kev ) and seed ( approximately hundreds of kev ) populations of the radiation belts simulated with ramscb is compared with van allen probes magnetic electron ion spectrometer observations in the morning sector and with measurements from noaa 15 satellite in the predawn and afternoon mlt sectors . we find that although the lowenergy ( e < 100 kev ) electron fluxes are in good agreement with observations , increasing significantly by magnetospheric convection during both symh dips while decreasing during the intermediate recovery phase , the injection of highenergy electrons is underestimated by this mechanism throughout the storm . local acceleration by chorus waves intensifies the electron fluxes at e50 kev considerably , and ramscb simulations overestimate the observed trapped fluxes by more than an order of magnitude ; the precipitating fluxes simulated with ramscb are weaker , and their temporal and spatial evolutions agree well with poes / medium energy proton and electron detectors data .

Introduction Observations During the October 2012 DoubleDip Storm RAMSCB Model Developments Comparison of RAMSCB Results With Observations Summary and Conclusions

the van allen probes mission ( previously known as the radiation belt storm probes , rbsp ) was thus launched in august 2012 to investigate the dynamics of nearearth fluxes of relativistic electrons and to help the development of models for specification and prediction of their space weather effects [ kessel et al . , there are several processes that lead to the enhancement of electron fluxes in the nearearth space environment . the tens of kev ring current electrons are thus often referred to as the source population of the radiation belts [ e.g. on the other hand , the decrease of the magnetic field strength during the main phase of a storm due to the ring current buildup causes an outward motion of energetic electrons through conservation of the third adiabatic invariant and a decrease of relativistic electron fluxes , the dst effect , first demonstrated for flux variations near geosynchronous orbit by kim and chan . , 2014 ] and that parameterized pitch angle loss rates could account for the trapped electron fluxes observed at geosynchronous orbit as well as for the precipitating fluxes observed in the ionosphere [ chen et al . , we investigate the dynamics of lower energy ( kev ) electrons , the generation of whistler mode waves , and the effects from subsequent waveparticle interactions in the inner magnetosphere using a fourdimensional ( 4d ) physicsbased model , the ring currentatmosphere interactions model with selfconsistent magnetic field ( ramscb ) [ jordanova et al . the ring current electron fluxes represent the source ( approximately tens of kev ) and seed ( approximately hundreds of kev ) populations for relativistic electrons and are crucial elements for radiation belt evolution . we present quantitative comparisons between our model predictions and van allen probes and noaa polarorbiting operational environmental satellites ( poes ) observations available during the investigated storm period . , 2013 ] and electric and magnetic field instrument suite and integrated science ( emfisis ) investigation [ kletzing et al . the panels show , from top to bottom , the spinaveraged differential electron flux as a function of energy from 30 to 350 kev , the electron flux magnitude at four representative energies , the whistler wave power spectral density , and the l shell [ mcilwain , 1966 ] as a function of time ( horizontal axis ) . as seen from the mageis flux data , during 7 october the satellite traversed several times at low l < 4 the electron ring current characterized by quiet time fluxes extending from 30 to 350 kev ; the 40 kev flux was of the order of 10 cm s sr kev , and the flux decreased at larger energies . the next nightside satellite pass occurred during the storm main phase from 07 to 12 ut 8 october , and mageis measured enhanced electron fluxes typical of a strong ring current with fluxes at higher energies ( > 100 kev ) confined to lower l shells . van allen probe a observations during 79 october 2012 including ( top to bottom ) mageis differential electron flux spectrogram ; spinaveraged electron flux at 37 kev ( red ) , 111 kev ( green ) , 211 kev ( light blue ) , and 317 kev ( dark blue ) ; emfisis magnetic field power spectral density spectrogram with lines denoting fractions of the electron gyrofrequency at 0.1 f ce ( blue ) , 0.5 f ce ( red ) and 1.0 f ce ( black ) ; and the mcilwain l parameter along the satellite orbit . the space environment monitor 2 ( sem2 ) [ evans and greer , 2000 ] on board each threeaxis stabilized poes spacecraft includes a set of solidstate medium energy proton and electron detectors ( meped ) . , the electrons ' radial distributions measured by noaa 15 , which samples in the predawn and afternoon sectors , over a 3 day period , are presented in figure 3 . here the electron fluxes are binned into 3 h intervals and sorted by precipitating versus trapped populations for three energy ranges ( e1 , e2 , and e3 ) and two local time sectors . in comparison with the prestorm quiet time flux level , both the precipitating and trapped fluxes intensified at 3<l<6 predawn in the lowest energy range ( e1 ) during the first symh dip . on the other hand , the fluxes increased at all energies and all local times during the second symh dip and the intensification of the trapped fluxes at higher energies lasted throughout the recovery phase . the noaa poes precipitating and trapped electron fluxes ( in units of 1/cm / s / sr ) during 79 october 2012 binned into three energy ranges ( > 30 kev , > 100 kev , and > 300 kev ) , ( left column ) for mlt = 06 and ( right ) for mlt = 1218 local time sectors . electron and ion flux measurements from the mageis instruments on the van allen probes a and b and from the magnetospheric plasma analyzer ( mpa ) and synchronous orbit particle analyzer ( sopa ) instruments on lanlgeo satellites during 3 days of the october 2012 storm are plotted in figure 4 . we simulate the ring current evolution during the doubledip storm of 79 october 2012 using our ramscb model which couples the ring currentatmosphere interactions model ( ram ) [ jordanova et al . the 4d ram solves the kinetic equation for h , o , he , and electron phase space distributions q l(r o,,e, o , t ) in the magnetic equatorial plane as a function of time t , radial distance from earth ( r o from 2 to 6.5 r e ) , geomagnetic east longitude , kinetic energy e from 100 ev to 400 kev , and equatorial pitch angle o from 0 to 90 : ( 1)qlt+1ro2roro2drodtql+ddtql+1pepdedtql+1hoohododtql=qltloss here the brackets denote bounceaveraging , o = cos(o ) , p is the relativistic momentum of the particle , and is the relativistic factor . the linear growth rate of whistler mode chorus was calculated in the equatorial plane using the electron distributions and selfconsistent magnetic field simulated with ramscb and cold plasma density simulated with the coupled plasmasphere model [ rasmussen et al . in agreement with previous studies , the growth rate maximizes in the morning sector outside the plasmasphere , as indicated by the cold plasma density ( figure 5c ) ; the corresponding parallel energy of resonant electrons is from 2 to 5 kev . during the prestorm and recovery phases the electron fluxes are smaller and figure 5d shows 2d snapshots of equatorial chorus wave amplitudes inferred from the precipitating 30 kev electron fluxes measured at low altitude by multiple noaa poes satellites [ chen et al . , specifically , the measured precipitating electron fluxes are related with an adjustable parameter to the equatorial wave measurements made by emfisis onboard van allen probes to infer the global distribution of chorus waves . has shown that the obtained amplitudes are in good agreement with in situ emfisis observations during this storm ( figure 2 ) ; their spatial and temporal evolutions also agree reasonably well with the global patterns of chorus wave growth simulated with ramscb ( figure 5a ) . to implement in ramscb for the first time quasilinear diffusion of relativistic electrons by whistler mode waves , we include diffusion in pitch angle and energy as the most important terms ( mixed diffusion will be considered in future extensions of this work ) on the righthand side of equation ( 1 ) following jordanova et al . [ 1997 , 1998 ] : ( 2)qltwp=1hoohodooqlo+1pepdeeqle here the bounceaveraged quasilinear pitch angle and energy diffusion coefficients are : ( 3)doo=(1o2)2ho2rosmsmbobdoop2ds=(1o2)p2doo ( 4)dee=p22h2mo2rosmsmdppds = p2mo22dpp where m o is the rest mass of the resonating particle and s m denotes the mirror point . global wave intensity distributions in magnetic latitude ( mlat ) , magnetic local time ( mlt ) , and l are required for the calculation . as expected from previous studies , the pitch angle diffusion coefficients are larger than the momentum ones and maximize in the dawn sector during the storm main phase where the chorus wave proxy is most enhanced , corresponding to a minimum electron lifetime of about an hour . eventspecific ( top row ) pitch angle < doo / p2 > and ( bottom row ) momentum < d pp / p > diffusion coefficients calculated for 100 kev electrons and o=50 at hours 24 , 32 , and 42 after 00 ut 7 october 2012 . ring current electron simulations with ramscb during the october 2012 doubledip storm are displayed in figure 7 where the electron flux was plotted along the van allen probe a trajectory in the same format as in figure 2 to ease the comparison with mageis observations . figure 7 shows the electron flux averaged over pitch angle obtained with ramscb ( version 1 ) that uses empirical lifetimes for electron loss ( middle row ) , as well as results from the newly developed in this study ramscb ( version 2 ) that includes quasilinear plasma wave scattering with eventspecific diffusion coefficients ( bottom row ) ; results from a ramscb test simulation that considers only transport and precipitation losses due to atmospheric collisions at low altitude are shown in the top row for comparison . the modeled fluxes increase during the storm main phase at low energies due to convective transport of newly injected electrons from the plasma sheet in agreement with mageis observations ( figure 2 ) , but the model underestimates significantly the fluxes at high ( > 100 kev ) energies in this case . on the other hand , the empirical lifetimes used in ramscb version 1 ( figure 7 , middle row ) are too short and the electron injection is not large enough to overcome the losses , especially during quiet time and at high energies . simulations with ramscb version 2 ( figure 7 , bottom row ) indicate larger fluxes and better agreement with mageis data at all energies during quiet time ( note the different scales in the plots ) . as the storm develops the fluxes increase not only at low but also at high energies due to the local acceleration by chorus waves , and while the lowenergy flux agrees reasonably well with observations , the flux at e>100 kev becomes about an order of magnitude larger than mageis observations near the first symh dip . contrary to observations , the simulated fluxes do not decrease much in the morning sector during the intermediate recovery phase and increase even further during the second symh dip significantly overestimating mageis fluxes . simulations of electron fluxes averaged over pitch angle and plotted along van allen probe a orbit , ( top row ) from a ramscb test run considering only transport and loss by atmospheric collisions , ( middle row ) from ramscb ( version 1 ) using empirical lifetimes for electron loss , and ( bottom row ) from ramscb ( version 2 ) using quasilinear diffusion . the line plots show the electron fluxes at four representative energies : 40 kev ( red ) , 100 kev ( green ) , 210 kev ( light blue ) , and 335 kev ( dark blue ) . electron fluxes from the mageis spectrometer when the van allen probe a passes through the morning sector at l 4 are interpolated onto the ram energy grid and plotted in figure 8a . in the observations the flux increases at all energies with about 2 orders of magnitude at 06 ut 8 october during the storm main phase . this is followed by an order of magnitude decrease at 18 ut during the intermediate recovery phase and a subsequent increase during the second symh dip at 04 ut 9 october to approximately the same magnitude as during the first dip . the dynamics of the lowenergy electrons is dominated by magnetospheric convection , and the flux rises significantly as the w05 cross polar cap potential drop , indicative of convection strength , and rises at 06 ut ( figure 1 g ) . when pitch angle scattering is considered ( figure 8c ) , the enhancement at these energies is substantial when energy diffusion by plasma waves is included ( figure 8d ) , and the simulated energy spectra shapes agree better with observations . in this case , however , the fluxes at e>50 kev increase by about 2 orders of magnitude and reach larger values than mageis observations during the storm main phase , remain at this elevated level during the intermediate recovery phase , and rise again during the second symh dip . found that the local heating by chorus during the first symh dip overwhelmed the mev electron loss from outward radial diffusion and the electron distribution did not show a decrease as in the observations but rather an early increase . this indicates that either the local acceleration by chorus waves is overestimated at this time in the eventspecific diffusion model or additional loss mechanisms have to be considered . ( a ) electron flux measured with mageis when the van allen probe a crosses r o=4 0.1r e during 710 october 2012 interpolated onto ram energy and pitch angle grids . simulated ramscb electron flux at l = 4 and mlt = 7 ( b ) considering transport and loss due to atmospheric collisions , ( c ) adding pitch angle scattering , and ( d ) adding energy scattering , at five representative energies and o=50. to provide better understanding of the role various processes have in ring current dynamics , in figure 9 we plot the electron phase space distribution at l = 4 and mlt = 7 during prestorm quiet time at 00 ut 8 october ( hour 24 ) and during the storm main phase at 10 ut 8 october ( hour 34 ) . finally , global patterns of electron fluxes simulated with ramscb are plotted in figures 10 and 11 in the same l time format as the noaa poes observations in figure 3 . from top to bottom are shown fluxes at 30 kev , 100 kev , and 335 kev , at mlt = 3 ( figure 10 ) and mlt = 15 ( figure 11 ) . when energy diffusion is not included ( left column ) , the 30 kev electrons penetrate to l 3 on the morning side during the storm main phase after convective transport from the plasma sheet and eastward drift , forming an asymmetric ring current in good agreement with poes observations . the fluxes increase due to local acceleration by chorus waves when energy diffusion is included ( middle column ) , the enhancement being larger at higher energies , and the electron distribution becomes symmetric with mlt at e>100 kev as seen in poes observations . during the intermediate recovery phase the trapped highenergy flux decreases at large ( l>5 ) shells but remains enhanced between l=4 and l=5 ; it increases by an order of magnitude during the second symh dip and overestimates poes data . the precipitating flux ( right column ) follows the temporal and spatial evolution of the trapped flux but is more than an order of magnitude smaller ( note the different scales ) and reproduces better poes observations . simulated ramscb flux ( 1/cm / s / sr / kev ) for 30 kev , 100 kev , and 335 kev ( from top to bottom ) at mlt = 3 as a function of radial distance from earth and time after 00 ut 7 october 2012 for trapped electrons ( o=50 ) ( left column ) considering transport , loss due to atmospheric collisions , and pitch angle scattering and ( middle column ) adding energy scattering . mechanisms for particle injection in the nearearth space environment and their subsequent trapping or loss have been the subject of detailed investigation recently , thanks to the highresolution data available from the van allen probes . we studied the combined effects from timedependent transport and scattering by whistler mode chorus and hiss on the source ( approximately tens of kev ) and seed ( approximately hundreds of kev ) populations of the radiation belts during the october 2012 doubledip storm . we used our kinetic ring currentatmosphere interactions model coupled with a eulerpotentialbased threedimensional plasma equilibrium code ( ramscb ) , including for the first time pitch angle and energy diffusion using l and mltdependent eventspecific wave models . the chorus wave amplitudes were thus inferred from correlations of poes / meped precipitating electron fluxes and rbsp / emfisis wave observations ; the spatial and temporal evolutions of peak wave amplitudes was in reasonable agreement with chorus growth rate maxima calculated with ramscb . comparisons of ramscb simulations with complementary data sets from the van allen probes in the morning mlt sector and noaa poes satellites in the afternoon and predawn sectors indicated the following : during quiet time ramscb electron simulations using l and mltdependent eventspecific pitch angle and energy diffusion coefficients reproduced much better the in situ satellite observations of ring current electron flux than ramscb simulations using empirical lifetimes , since these lifetimes were too short and overestimated the electron losses.the simulated electron flux at lower ( e < 100 kev ) energies increased during the first and second symh dips and decreased during the intermediate recovery phase due to earthward transport in timedependent convective electric field and nondipolar magnetic field . this electron dynamics was in good agreement with observations of both trapped and precipitating electron fluxes and showed the initial formation of an asymmetric ring current that evolved into a more symmetric one with storm development.at higher ( e100 kev ) energies ramscb simulations that considered only convective transport and loss substantially underestimated the observations . when local acceleration by chorus was included , the electron fluxes increased by about 2 orders of magnitude during the first symh dip , remained enhanced during the intermediate recovery phase at l = 4 to l = 5 , and increased further during the second symh dip thus considerably overestimating the satellite observations . during quiet time ramscb electron simulations using l and mltdependent eventspecific pitch angle and energy diffusion coefficients reproduced much better the in situ satellite observations of ring current electron flux than ramscb simulations using empirical lifetimes , since these lifetimes were too short and overestimated the electron losses . the simulated electron flux at lower ( e < 100 kev ) energies increased during the first and second symh dips and decreased during the intermediate recovery phase due to earthward transport in timedependent convective electric field and nondipolar magnetic field . this electron dynamics was in good agreement with observations of both trapped and precipitating electron fluxes and showed the initial formation of an asymmetric ring current that evolved into a more symmetric one with storm development . when local acceleration by chorus was included , the electron fluxes increased by about 2 orders of magnitude during the first symh dip , remained enhanced during the intermediate recovery phase at l = 4 to l = 5 , and increased further during the second symh dip thus considerably overestimating the satellite observations . a selfconsistently calculated electric field , with higher temporal and spatial resolution , will influence both the injection of particles from the tail and their dayside outflow and could result in reduced gradients in the distribution function .

there is increasing need to design and develop suitable drug carrier systems to control , localize and improve drug delivery . however , designing a drug delivery system is challenging in terms of targeting the drug to specific sites and also to improve its therapeutic value with minimal side effects . many different drug carriers can be used depending on the route of administration , among other factors . oral drug administration is the most commonly employed route owing to its safety as well as convenience and ease of administration to the patient . however , the development of oral forms of many drugs remains a challenge either on account of their stability or their absorption from the gastrointestinal tract ( git ) thus leading to sub - therapeutic bioavailability . to overcome the poor absorption capacity of such drugs an array of lipid systems such as emulsions , micellar solutions , liposomes , lipid nanoparticles , structured lipid carriers , self - emulsifying lipid formulations , solid dispersions , dry emulsions , solid - liquid compacts , and drug - lipid conjugates is available to drug formulators . among the various lipid systems , solid lipid microparticles ( slms ) have been developed to address some issues such as stability and low payload capacity of some lipid systems . slms have a lower risk of the reaction of the substance to be delivered to the vehicle than in emulsion system . in addition , the release rate of substance from the slms can be manipulated by altering either one or both the inner solid vesicle or the outer phospholipid layer . the combination of the solid inner core with phospholipid exterior ensures high dispersibility in an aqueous medium , and a release rate for the entrapped substance that is controlled by phospholipid coating and carrier , among other advantages . in addition , they have been widely investigated as potential drug delivery systems for hydrophilic drugs which encounter penetration and absorption problems . gentamicin , a broad - spectrum hydrophilic bactericidal antibiotic of the aminoglycoside group , acts by inhibition of protein synthesis after binding to specific 30s - subunit ribosomal proteins . it is very poorly absorbed from the git and is unstable in acidic ph of the stomach . more so , its cationic nature affects its penetration into the mucosal walls of the git . gentamicin is active against a wide range of human bacterial infections , mostly gram - negative bacteria including pseudomonas , proteus , serratia , escherichia coli and the gram - positive bacteria such as staphylococcus aureus . like other aminoglycosides , gentamicin is toxic to the sensory cells of the ear and also causes nephrotoxicity by inhibiting protein synthesis in renal cells . this mechanism specifically causes necrosis of cells in the proximal tubule , resulting in acute tubular necrosis that can lead to acute renal failure . by tactical engineering of lipid - based drug delivery systems ( lbdds ) such as solidified reverse micellar solution ( srms)-based slms , the toxicity researchers have used this novel technology ( srmds ) to increase the overall efficacy while minimizing the toxicity of gentamicin . tallow fat is an edible animal fat derived from tallow fat ( bos indicus ) . slms based on bos indicus has been evaluated as a basis for delivery of piroxicam . thus , the aims of this study were to formulate srms ( lipid matrix ) consisting of p90 g and bos indicus , and srms - based slms containing gentamicin using melt - emulsification technique and evaluate the in vitro dissolution and bioactivity of gentamicin from such a delivery system . the following materials were used : gentamicin pure sample ( juhel pharmaceutical limited , awka , nigeria ) , tallow fat ( a biodegradable homolipid was obtained from bos indicus and purified in our laboratory ) , phospholipon 90 g ( phospholipid gmbh , kln , nattermann , germany ) , poloxamer 188 ( sigma aldrich , spain ) , polyethylene glycol 4000 ( peg ) ( acros organics , usa ) , monobasic potassium phosphate , sodium hydroxide and concentrated hydrochloric acid ( bdh , england ) and distilled water ( lion water , unn , nigeria ) . all other reagents and solvents were analytical grade and were used as supplied . the homolipid was extracted from the adipose tissue of bos indicus by wet rendering following standard procedures . briefly , tallow fat was extracted from the adipose tissue of bos indicus which was collected from freshly slaughtered cow , manually freed of extraneous materials , crushed and boiled in distilled water for 45 min , filtered through a muslin cloth and allowed to solidify at room temperature . the solid fat was manually removed and bleached / deodorized by passing it through a mixture of activated charcoal and bentonite ( 2:1 ) at 100c at a ratio of 10 g of the fat and 1 g of the column material . lipid matrix consisting of a mixture of 35% w / w tallow fat ( homolipid ) and 15% w / w phospholipon 90 g ( p90 g ) was prepared by fusion method . briefly , the tallow fat and p90 g were weighed using electronic balance ( mettler h8 , switzerland ) , placed into a crucible , melted together at 75c on a thermo - regulated water bath shaker ( heto , denmark ) and stirred thoroughly . thereafter , the mixture was allowed to cool and solidify at room temperature to obtain the lipid matrix ( srms ) . for the preparation of the slms , the melt - emulsification technique was adopted . in each case , the srms was melted at 75c , and the aqueous phase containing peg 4000 and poloxamer 188 at the same temperature was added to the srms with gentle stirring with a magnetic stirrer ( sr 1 um 52188 , remi equip . , india ) , and the mixture was further dispersed with a mixer ( t 25 digital ultra - turrax ; ika , staufen , germany ) at 8000 rpm for 5 min . the slms suspension obtained after cooling at room temperature was then lyophilized using a freeze - dryer ( amsco gt3 , germany ) . the above procedure was repeated using peg 4000 and gentamicin ( 1.0 , 2.0 and 3.0% w / w ) and lipid matrix ( 4.0 , 3.0 and 2.0% w / w ) , to obtained gentamicin - loaded slms ( batches a1 -a3 , b1 -b3 and c1 -c3 ) . formulation compositions of the slms the particle size and morphology of the slms were determined by computerized image analysis . briefly , approximately 5.0 mg of the slms from each batch was dispersed in distilled water and smeared on a slide ( marinfield , germany ) using a glass rod . it was then covered with a cover slip and viewed with a photomicroscope ( hund , weltzlar , germany ) attached with a digital camera at a magnification of 1000 . with the aid of the software in the photomicroscope , approximately 0.5% w / v dispersion of the slms in distilled water was prepared , allowed to equilibrate for 48 h at room temperature , shaken , and filtered . the filtrate was adequately analyzed for gentamicin content spectrophotometrically ( unico 2102 pc uv / vis4 spectrophotometer , usa ) at 203 nm . the amount of drug encapsulated in the slms was calculated with reference to a standard beer 's plot for gentamicin to obtain the ee % using the formula : lc expresses the ratio between the entrapped active pharmaceutical ingredient ( api ) and total weight of the lipids . it is determined as follows : where wl is the weight of lipid added in the formulation and wa is the amount of api entrapped by the lipid . the ph of dispersions of the slms from each batch was determined using a ph meter ( suntex ts-2 , taiwan ) after 1-week , 1-month , and 3 months of storage . phosphate buffered saline ( pbs , ph 7.4 ) and the usp xxii rotating paddle apparatus ( erweka , germany ) were employed for this release study . the dissolution medium consisted of 500 ml of freshly prepared pbs maintained at 37 1c by means of a thermostatically controlled water bath . the polycarbonate dialysis membrane used was pretreated by soaking it in pbs for 24 h prior to the commencement of each release experiment . in each case , 300 mg of the formulated slms was placed in the dialysis membrane containing 5 ml of the pbs , securely tied with a thermo - resistant thread and then immersed in pbs under agitation provided by the paddle at 100 rpm . at 60 min intervals , 10 ml portions of pbs were withdrawn and replaced with equal volume of pbs to maintain a sink condition , filtered , and analyzed spectrophotometrically at 341 nm . the amount of drug released at each time interval was determined with reference to the standard beer 's plot for gentamicin in pbs . this test was replicated for all the batches , gentamicin pure sample , and commercial gentamicin injection . the antimicrobial activity of the slms was tested against clinical isolates of s. aureus and e. coli by agar diffusion technique using samples withdrawn during the in vitro drug release studies . it was mixed thoroughly , poured into sterile petri dishes and rotated for even distribution of the organism . the agar plates were allowed to set and a sterile cork borer was used to bore three cups in the seeded agar medium . using a sterile syringe , a definite volume withdrawn from the receptor compartment of the diffusion apparatus at predetermined time intervals the plates were allowed to stand at room temperature before incubating at 37 1c for 24 h. the diameter of each inhibition zone was measured and the average determined . anova and student 's t - test were performed on the data sets generated using spss ( version 17 , spss inc . the following materials were used : gentamicin pure sample ( juhel pharmaceutical limited , awka , nigeria ) , tallow fat ( a biodegradable homolipid was obtained from bos indicus and purified in our laboratory ) , phospholipon 90 g ( phospholipid gmbh , kln , nattermann , germany ) , poloxamer 188 ( sigma aldrich , spain ) , polyethylene glycol 4000 ( peg ) ( acros organics , usa ) , monobasic potassium phosphate , sodium hydroxide and concentrated hydrochloric acid ( bdh , england ) and distilled water ( lion water , unn , nigeria ) . all other reagents and solvents were analytical grade and were used as supplied . the homolipid was extracted from the adipose tissue of bos indicus by wet rendering following standard procedures . briefly , tallow fat was extracted from the adipose tissue of bos indicus which was collected from freshly slaughtered cow , manually freed of extraneous materials , crushed and boiled in distilled water for 45 min , filtered through a muslin cloth and allowed to solidify at room temperature . the solid fat was manually removed and bleached / deodorized by passing it through a mixture of activated charcoal and bentonite ( 2:1 ) at 100c at a ratio of 10 g of the fat and 1 g of the column material . lipid matrix consisting of a mixture of 35% w / w tallow fat ( homolipid ) and 15% w / w phospholipon 90 g ( p90 g ) was prepared by fusion method . briefly , the tallow fat and p90 g were weighed using electronic balance ( mettler h8 , switzerland ) , placed into a crucible , melted together at 75c on a thermo - regulated water bath shaker ( heto , denmark ) and stirred thoroughly . thereafter , the mixture was allowed to cool and solidify at room temperature to obtain the lipid matrix ( srms ) . for the preparation of the slms , the melt - emulsification technique was adopted . in each case , the srms was melted at 75c , and the aqueous phase containing peg 4000 and poloxamer 188 at the same temperature was added to the srms with gentle stirring with a magnetic stirrer ( sr 1 um 52188 , remi equip . , india ) , and the mixture was further dispersed with a mixer ( t 25 digital ultra - turrax ; ika , staufen , germany ) at 8000 rpm for 5 min . the slms suspension obtained after cooling at room temperature was then lyophilized using a freeze - dryer ( amsco gt3 , germany ) . the above procedure was repeated using peg 4000 and gentamicin ( 1.0 , 2.0 and 3.0% w / w ) and lipid matrix ( 4.0 , 3.0 and 2.0% w / w ) , to obtained gentamicin - loaded slms ( batches a1 -a3 , b1 -b3 and c1 -c3 ) . the particle size and morphology of the slms were determined by computerized image analysis . briefly , approximately 5.0 mg of the slms from each batch was dispersed in distilled water and smeared on a slide ( marinfield , germany ) using a glass rod . it was then covered with a cover slip and viewed with a photomicroscope ( hund , weltzlar , germany ) attached with a digital camera at a magnification of 1000 . with the aid of the software in the photomicroscope , approximately 0.5% w / v dispersion of the slms in distilled water was prepared , allowed to equilibrate for 48 h at room temperature , shaken , and filtered . the filtrate was adequately analyzed for gentamicin content spectrophotometrically ( unico 2102 pc uv / vis4 spectrophotometer , usa ) at 203 nm . the amount of drug encapsulated in the slms was calculated with reference to a standard beer 's plot for gentamicin to obtain the ee % using the formula : lc expresses the ratio between the entrapped active pharmaceutical ingredient ( api ) and total weight of the lipids . it is determined as follows : where wl is the weight of lipid added in the formulation and wa is the amount of api entrapped by the lipid . the ph of dispersions of the slms from each batch was determined using a ph meter ( suntex ts-2 , taiwan ) after 1-week , 1-month , and 3 months of storage . phosphate buffered saline ( pbs , ph 7.4 ) and the usp xxii rotating paddle apparatus ( erweka , germany ) were employed for this release study . the dissolution medium consisted of 500 ml of freshly prepared pbs maintained at 37 1c by means of a thermostatically controlled water bath . the polycarbonate dialysis membrane used was pretreated by soaking it in pbs for 24 h prior to the commencement of each release experiment . in each case , 300 mg of the formulated slms was placed in the dialysis membrane containing 5 ml of the pbs , securely tied with a thermo - resistant thread and then immersed in pbs under agitation provided by the paddle at 100 rpm . at 60 min intervals , 10 ml portions of pbs were withdrawn and replaced with equal volume of pbs to maintain a sink condition , filtered , and analyzed spectrophotometrically at 341 nm . the amount of drug released at each time interval was determined with reference to the standard beer 's plot for gentamicin in pbs . this test was replicated for all the batches , gentamicin pure sample , and commercial gentamicin injection . the antimicrobial activity of the slms was tested against clinical isolates of s. aureus and e. coli by agar diffusion technique using samples withdrawn during the in vitro drug release studies . it was mixed thoroughly , poured into sterile petri dishes and rotated for even distribution of the organism . the agar plates were allowed to set and a sterile cork borer was used to bore three cups in the seeded agar medium . using a sterile syringe , a definite volume withdrawn from the receptor compartment of the diffusion apparatus at predetermined time intervals the plates were allowed to stand at room temperature before incubating at 37 1c for 24 h. the diameter of each inhibition zone was measured and the average determined . anova and student 's t - test were performed on the data sets generated using spss ( version 17 , spss inc . some physicochemical properties of the slms are presented in table 2 while the photomicrographs of the formulations are depicted in figure 1 . the results indicate that the mean particle sizes of the gentamicin - loaded slms and unloaded slms were in the range of 21.35 1.57 - 50.62 2.37 m , and 18.62 1.24 - 20.59 1.36 m , respectively . more so , the results revealed that after 3 months of storage , drug - loaded slms , and unloaded slms had a mean ph range of 2.23 0.19 - 4.18 0.79 and 2.27 0.17 to 2.29 0.09 , respectively . the photomicrographs [ figure 1 ] showed that the slms were discrete , spherical , and greenish brown . the ee % [ table 2 ] increased with increase in gentamicin concentration in the formulations . thus , sub - batches c1 -c3 gave highest ee % while sub - batches a1 -a3 gave the least . table 2 also shows that maximum lc of 52.50 , 57.90 , and 62.70 g of gentamicin per 100 g of lipid were obtained for sub - batches c1 -c3 respectively containing 3% w / w gentamicin . key : batches a1 , b1 , and c1 are gentamicin - loaded solid lipid microparticles while batch d1 is unloaded ( zero - drug ) solid lipid microparticles results of the physicochemical tests on the slms showed that high drug loading resulted in large particle sizes , consistent with earlier reports . the stability tests , which were carried out to determine the ph stability of the slms when stored at different time intervals , revealed an insignificant change ( p > 0.05 ) in the ph of the slms over a period of 3 months , implying that there was little or no degradation of the drug and/or the excipients used in the formulations within this period . it was discernible from the ee % results that the lipid contents improved the ee % of gentamicin in the slms . the implication of the lc results is improved solubility of gentamicin in the lipid matrix . further incorporation of p90 g in the slms led to the formation of structured lipid matrix , which invariably enhanced gentamicin encapsulation in the core of the slms , consistent with previous reports . in addition , peg 4000 being a hydrophilic surfactant improved the solubilization of the drug within the core lipids . similarly , poloxamer 188 is a nonionic tri - block copolymer composed of a central hydrophobic chain of polyoxypropylene flanked by two hydrophilic chains of polyoxyethylene . this amphiphilic nature and surfactant properties of the polymer , in addition to the lipid components of bos indicus increased the solubility of the drug within the lipid core . the determination of drug loading ( or drug incorporation ) is an important tool to evaluate a potential drug carrier system . it is obviously desirable to formulate slms with high drug content to decrease the amount of slms to be administered . the prerequisite to obtain a sufficient lc is a sufficiently high solubility of the drug in the lipid melt . the highest drug encapsulation of 86.60 3.17% was obtained from the slms which means that the pegylated lipid matrix of p90 g and tallow fat had enormous spaces which accommodated the gentamicin ( enhanced dissolution ) . figures 2 - 4 depict the in vitro release profiles of gentamicin from the slms in pbs . drug release from the slms followed the order : c1 -c3 > b1 -b3 > a1 -a3 . the in vitro release profiles indicate controlled release of gentamicin from the slms . in batch a formulations , sub - batch a3 gave a maximum release of 92% while sub - batch a1 gave the least ( maximum release of 70% ) . similarly , in batch b slms , sub - batch b3 released the highest amount ( i.e. , 96% ) of gentamicin while sub - batch b1 released the least amount ( 77% of gentamicin ) . furthermore , in batch c formulations , sub - batch c3 recorded complete drug release while sub - batch c1 released 83% of the encapsulated drug . commercial gentamicin injection ( g1 ) and gentamicin pure sample ( g2 ) gave 65% and 62% drug release , respectively . the high and rapid release of gentamicin from the slms may be related to high rate of hydration and swelling of the slms in the medium , which might be attributed to the lipophilicity imparted on the drug by the excipients used in preparing the slms . the controlled release observed in the study could be a consequence of the decreasing residual amount of drug in the slms and the build - up of drug concentration in the dissolution medium in the course of time . in vitro release profile of gentamicin from batch a solid lipid microparticles in phosphate bufferred saline , key : a1-a3 contain 1.0%w / w of gentamicin while g1 and g2 are commercial gentamicin injection and plain gentamicin , respectively in vitro release profile of gentamicin from batch b solid lipid microparticles in phosphate bufferred saline , ph 7.4 ( n = 3 ) . key : b1-b3 contain 2.0%w / w of gentamicin while g1 and g2 are commercial gentamicin injection and plain gentamicin , respectively in vitro release profile of gentamicin from batch c solid lipid microparticles in phosphate bufferred saline , ph 7.4 ( n = 3 ) . key : c1-c3 contain 3.0%w / w of gentamicin while g1 and g2 are commercial gentamicin injection and plain gentamicin , respectively the anti - microbial results recorded as inhibition zone diameter ( izd ) [ tables 3 and 4 ] indicate that gentamicin - loaded slms produced very significant izd ( p < 0.05 ) against gram - positive organism ( s. aureus ) and gram - negative organism ( e. coli ) . the formulations recorded increasing izds against the organisms with time . moreover , gentamicin - loaded slms gave greater izds than the plain gentamicin as well as commercial gentamicin injection against the organisms . overall , sub - batch c3 containing the highest peg 4000 and gentamicin gave the greatest izd against s. aureus ( 27.49 2.38 m ) and e. coli ( 29.40 3.07 m ) after 420 min , whereas plain gentamicin and commercial gentamicin injection respectively gave izds of 13.47 1.87 m and 11.98 1.09 m against s. aureus and 12.87 1.74 m and 10.77 0.90 m against e. coli after 430 min . the bioevaluation was performed to establish that gentamicin did not lose activity during formulation as well as to show an increasing izd over time during the in vitro dissolution study . generally , the slms produced very significant ( p < 0.05 ) izds against the organisms . it was observed that the greater the amount of gentamicin loaded into the slms , the greater the izd produced , in agreement with earlier reports . by implication , the slms exhibited capacity limited bioactivity . similarly , the antibacterial activity of the formulations was concentration and time - dependent , manifested by an increasing izd against the organisms with time . high izds recorded against the organisms early in the study was an indication that these formulations would have exhibited the fastest release of the entrapped drug , hence the fast antibacterial activities ; whereas time - dependent increases in izds within 420 min implies that the slms had potentials for sustained drug release . the improved lipid solubility conferred on the drug by the amphiphilic and surfactant properties of the solubilizers ( active and passive ) ensures sustained delivery of the drug through the gradual and sustained erosion of the lipid core . furthermore , the slms generally gave greater izds than plain gentamicin and commercial gentamicin injection against the organisms . it is equally discernible from the results that the formulations were more active against s. aureus than e. coli . this formulation would be a useful alternative to gentamicin injection for enhanced delivery of gentamicin in the treatment of infections caused by gentamicin - susceptible micro - organisms , thus encouraging further development of this formulation . susceptibility of staphylococcus aureus to gentamicin in the slms susceptibility of escherichia coli to gentamicin in the slms this research has shown that pegylated slms based on a homolipid from tallow fat ( bos indicus ) and phospholipid ( p90 g ) is a potential carrier system for dissolution and bioactivity enhancement of gentamicin . compared with commercial gentamicin injection , the bioactivity and in vitro drug release studies undertaken with the formulations provided a basis to establish that srms - based slms could better be used to control the release of gentamicin . further studies would seek to evaluate these formulations by employing pharmacokinetic models in experimental animals .

background : the aim of this study was to formulate solidified reverse micellar solution ( srms)-based solid lipid microparticles ( slms ) using homolipids from tallow fat ( bos indicus ) and evaluate its potential for enhanced delivery of gentamicin.materials and methods : slms were formulated by melt - emulsification using srms ( 15% w / w phospholipon 90 g in 35% w / w bos indicus ) , polyethylene glycol 4000 ( peg ) and gentamicin ( 1.0 , 2.0 , 3.0% w / w ) , and characterized with respect to size , morphology , encapsulation efficiency % and ph - dependent stability . the in vitro release of gentamicin from the slms was performed in phosphate buffer ( ph 7.4 ) while bioevaluation was carried out using clinical isolates of staphylococcus aureus and escherichia coli.results:results showed that the lipid matrix accommodated gentamicin in a concentration - dependent manner , and that stable and spherical slms with size range of 18.62 1.24 - 20.59 1.36 m and 21.35 1.57 - 50.62 2.37 m respectively for unloaded and drug - loaded formulations were obtained . the in vitro drug release studies revealed that srms - based slms could better be used to control the release of gentamicin than gentamicin injection . results of sensitivity test revealed that the slms time - dependently and capacity - limitedly produced greater inhibition zone diameters ( izds ) than the standards , an indication of improved bioactivity against the test organisms , with greater izds against s. aureus than e. coli . overall , slms containing 2% w / w srms , 3% w / w gentamicin and peg 4000 entrapped the highest amount of drug , achieved complete drug release and gave highest izd against the organisms within 420 min , while plain gentamicin gave the least.conclusion:this research has shown that slms based on bos indicus and p90 g is a potential carrier system for dissolution and bioactivity enhancement of gentamicin .

INTRODUCTION MATERIALS AND METHODS Materials Extraction and purification of tallow fat from Preparation of lipid matrix (solidified reverse micellar solution) and solid lipid microparticles Particle size analysis and morphological characterization of solid lipid microparticles Determination of encapsulation efficiency % and loading capacity Time-resolved pH-dependent stability studies None Antimicrobial studies Statistical analysis RESULTS AND DISCUSSION CONCLUSION Financial support and sponsorship Conflicts of interest

to overcome the poor absorption capacity of such drugs an array of lipid systems such as emulsions , micellar solutions , liposomes , lipid nanoparticles , structured lipid carriers , self - emulsifying lipid formulations , solid dispersions , dry emulsions , solid - liquid compacts , and drug - lipid conjugates is available to drug formulators . among the various lipid systems , solid lipid microparticles ( slms ) have been developed to address some issues such as stability and low payload capacity of some lipid systems . in addition , the release rate of substance from the slms can be manipulated by altering either one or both the inner solid vesicle or the outer phospholipid layer . gentamicin is active against a wide range of human bacterial infections , mostly gram - negative bacteria including pseudomonas , proteus , serratia , escherichia coli and the gram - positive bacteria such as staphylococcus aureus . by tactical engineering of lipid - based drug delivery systems ( lbdds ) such as solidified reverse micellar solution ( srms)-based slms , the toxicity researchers have used this novel technology ( srmds ) to increase the overall efficacy while minimizing the toxicity of gentamicin . tallow fat is an edible animal fat derived from tallow fat ( bos indicus ) . slms based on bos indicus has been evaluated as a basis for delivery of piroxicam . thus , the aims of this study were to formulate srms ( lipid matrix ) consisting of p90 g and bos indicus , and srms - based slms containing gentamicin using melt - emulsification technique and evaluate the in vitro dissolution and bioactivity of gentamicin from such a delivery system . the following materials were used : gentamicin pure sample ( juhel pharmaceutical limited , awka , nigeria ) , tallow fat ( a biodegradable homolipid was obtained from bos indicus and purified in our laboratory ) , phospholipon 90 g ( phospholipid gmbh , kln , nattermann , germany ) , poloxamer 188 ( sigma aldrich , spain ) , polyethylene glycol 4000 ( peg ) ( acros organics , usa ) , monobasic potassium phosphate , sodium hydroxide and concentrated hydrochloric acid ( bdh , england ) and distilled water ( lion water , unn , nigeria ) . the homolipid was extracted from the adipose tissue of bos indicus by wet rendering following standard procedures . briefly , tallow fat was extracted from the adipose tissue of bos indicus which was collected from freshly slaughtered cow , manually freed of extraneous materials , crushed and boiled in distilled water for 45 min , filtered through a muslin cloth and allowed to solidify at room temperature . lipid matrix consisting of a mixture of 35% w / w tallow fat ( homolipid ) and 15% w / w phospholipon 90 g ( p90 g ) was prepared by fusion method . briefly , the tallow fat and p90 g were weighed using electronic balance ( mettler h8 , switzerland ) , placed into a crucible , melted together at 75c on a thermo - regulated water bath shaker ( heto , denmark ) and stirred thoroughly . thereafter , the mixture was allowed to cool and solidify at room temperature to obtain the lipid matrix ( srms ) . for the preparation of the slms , the melt - emulsification technique was adopted . , india ) , and the mixture was further dispersed with a mixer ( t 25 digital ultra - turrax ; ika , staufen , germany ) at 8000 rpm for 5 min . the above procedure was repeated using peg 4000 and gentamicin ( 1.0 , 2.0 and 3.0% w / w ) and lipid matrix ( 4.0 , 3.0 and 2.0% w / w ) , to obtained gentamicin - loaded slms ( batches a1 -a3 , b1 -b3 and c1 -c3 ) . formulation compositions of the slms the particle size and morphology of the slms were determined by computerized image analysis . briefly , approximately 5.0 mg of the slms from each batch was dispersed in distilled water and smeared on a slide ( marinfield , germany ) using a glass rod . with the aid of the software in the photomicroscope , approximately 0.5% w / v dispersion of the slms in distilled water was prepared , allowed to equilibrate for 48 h at room temperature , shaken , and filtered . the amount of drug encapsulated in the slms was calculated with reference to a standard beer 's plot for gentamicin to obtain the ee % using the formula : lc expresses the ratio between the entrapped active pharmaceutical ingredient ( api ) and total weight of the lipids . it is determined as follows : where wl is the weight of lipid added in the formulation and wa is the amount of api entrapped by the lipid . the ph of dispersions of the slms from each batch was determined using a ph meter ( suntex ts-2 , taiwan ) after 1-week , 1-month , and 3 months of storage . phosphate buffered saline ( pbs , ph 7.4 ) and the usp xxii rotating paddle apparatus ( erweka , germany ) were employed for this release study . the amount of drug released at each time interval was determined with reference to the standard beer 's plot for gentamicin in pbs . this test was replicated for all the batches , gentamicin pure sample , and commercial gentamicin injection . the antimicrobial activity of the slms was tested against clinical isolates of s. aureus and e. coli by agar diffusion technique using samples withdrawn during the in vitro drug release studies . using a sterile syringe , a definite volume withdrawn from the receptor compartment of the diffusion apparatus at predetermined time intervals the plates were allowed to stand at room temperature before incubating at 37 1c for 24 h. the diameter of each inhibition zone was measured and the average determined . the following materials were used : gentamicin pure sample ( juhel pharmaceutical limited , awka , nigeria ) , tallow fat ( a biodegradable homolipid was obtained from bos indicus and purified in our laboratory ) , phospholipon 90 g ( phospholipid gmbh , kln , nattermann , germany ) , poloxamer 188 ( sigma aldrich , spain ) , polyethylene glycol 4000 ( peg ) ( acros organics , usa ) , monobasic potassium phosphate , sodium hydroxide and concentrated hydrochloric acid ( bdh , england ) and distilled water ( lion water , unn , nigeria ) . the homolipid was extracted from the adipose tissue of bos indicus by wet rendering following standard procedures . briefly , tallow fat was extracted from the adipose tissue of bos indicus which was collected from freshly slaughtered cow , manually freed of extraneous materials , crushed and boiled in distilled water for 45 min , filtered through a muslin cloth and allowed to solidify at room temperature . lipid matrix consisting of a mixture of 35% w / w tallow fat ( homolipid ) and 15% w / w phospholipon 90 g ( p90 g ) was prepared by fusion method . briefly , the tallow fat and p90 g were weighed using electronic balance ( mettler h8 , switzerland ) , placed into a crucible , melted together at 75c on a thermo - regulated water bath shaker ( heto , denmark ) and stirred thoroughly . thereafter , the mixture was allowed to cool and solidify at room temperature to obtain the lipid matrix ( srms ) . for the preparation of the slms , the melt - emulsification technique was adopted . in each case , the srms was melted at 75c , and the aqueous phase containing peg 4000 and poloxamer 188 at the same temperature was added to the srms with gentle stirring with a magnetic stirrer ( sr 1 um 52188 , remi equip . , india ) , and the mixture was further dispersed with a mixer ( t 25 digital ultra - turrax ; ika , staufen , germany ) at 8000 rpm for 5 min . the above procedure was repeated using peg 4000 and gentamicin ( 1.0 , 2.0 and 3.0% w / w ) and lipid matrix ( 4.0 , 3.0 and 2.0% w / w ) , to obtained gentamicin - loaded slms ( batches a1 -a3 , b1 -b3 and c1 -c3 ) . the particle size and morphology of the slms were determined by computerized image analysis . briefly , approximately 5.0 mg of the slms from each batch was dispersed in distilled water and smeared on a slide ( marinfield , germany ) using a glass rod . with the aid of the software in the photomicroscope , approximately 0.5% w / v dispersion of the slms in distilled water was prepared , allowed to equilibrate for 48 h at room temperature , shaken , and filtered . the amount of drug encapsulated in the slms was calculated with reference to a standard beer 's plot for gentamicin to obtain the ee % using the formula : lc expresses the ratio between the entrapped active pharmaceutical ingredient ( api ) and total weight of the lipids . it is determined as follows : where wl is the weight of lipid added in the formulation and wa is the amount of api entrapped by the lipid . the ph of dispersions of the slms from each batch was determined using a ph meter ( suntex ts-2 , taiwan ) after 1-week , 1-month , and 3 months of storage . phosphate buffered saline ( pbs , ph 7.4 ) and the usp xxii rotating paddle apparatus ( erweka , germany ) were employed for this release study . the amount of drug released at each time interval was determined with reference to the standard beer 's plot for gentamicin in pbs . this test was replicated for all the batches , gentamicin pure sample , and commercial gentamicin injection . the antimicrobial activity of the slms was tested against clinical isolates of s. aureus and e. coli by agar diffusion technique using samples withdrawn during the in vitro drug release studies . using a sterile syringe , a definite volume withdrawn from the receptor compartment of the diffusion apparatus at predetermined time intervals the plates were allowed to stand at room temperature before incubating at 37 1c for 24 h. the diameter of each inhibition zone was measured and the average determined . the results indicate that the mean particle sizes of the gentamicin - loaded slms and unloaded slms were in the range of 21.35 1.57 - 50.62 2.37 m , and 18.62 1.24 - 20.59 1.36 m , respectively . more so , the results revealed that after 3 months of storage , drug - loaded slms , and unloaded slms had a mean ph range of 2.23 0.19 - 4.18 0.79 and 2.27 0.17 to 2.29 0.09 , respectively . the photomicrographs [ figure 1 ] showed that the slms were discrete , spherical , and greenish brown . thus , sub - batches c1 -c3 gave highest ee % while sub - batches a1 -a3 gave the least . table 2 also shows that maximum lc of 52.50 , 57.90 , and 62.70 g of gentamicin per 100 g of lipid were obtained for sub - batches c1 -c3 respectively containing 3% w / w gentamicin . key : batches a1 , b1 , and c1 are gentamicin - loaded solid lipid microparticles while batch d1 is unloaded ( zero - drug ) solid lipid microparticles results of the physicochemical tests on the slms showed that high drug loading resulted in large particle sizes , consistent with earlier reports . the stability tests , which were carried out to determine the ph stability of the slms when stored at different time intervals , revealed an insignificant change ( p > 0.05 ) in the ph of the slms over a period of 3 months , implying that there was little or no degradation of the drug and/or the excipients used in the formulations within this period . it was discernible from the ee % results that the lipid contents improved the ee % of gentamicin in the slms . the implication of the lc results is improved solubility of gentamicin in the lipid matrix . further incorporation of p90 g in the slms led to the formation of structured lipid matrix , which invariably enhanced gentamicin encapsulation in the core of the slms , consistent with previous reports . this amphiphilic nature and surfactant properties of the polymer , in addition to the lipid components of bos indicus increased the solubility of the drug within the lipid core . the determination of drug loading ( or drug incorporation ) is an important tool to evaluate a potential drug carrier system . it is obviously desirable to formulate slms with high drug content to decrease the amount of slms to be administered . the prerequisite to obtain a sufficient lc is a sufficiently high solubility of the drug in the lipid melt . the highest drug encapsulation of 86.60 3.17% was obtained from the slms which means that the pegylated lipid matrix of p90 g and tallow fat had enormous spaces which accommodated the gentamicin ( enhanced dissolution ) . figures 2 - 4 depict the in vitro release profiles of gentamicin from the slms in pbs . drug release from the slms followed the order : c1 -c3 > b1 -b3 > a1 -a3 . the in vitro release profiles indicate controlled release of gentamicin from the slms . in batch a formulations , sub - batch a3 gave a maximum release of 92% while sub - batch a1 gave the least ( maximum release of 70% ) . similarly , in batch b slms , sub - batch b3 released the highest amount ( i.e. furthermore , in batch c formulations , sub - batch c3 recorded complete drug release while sub - batch c1 released 83% of the encapsulated drug . commercial gentamicin injection ( g1 ) and gentamicin pure sample ( g2 ) gave 65% and 62% drug release , respectively . the high and rapid release of gentamicin from the slms may be related to high rate of hydration and swelling of the slms in the medium , which might be attributed to the lipophilicity imparted on the drug by the excipients used in preparing the slms . the controlled release observed in the study could be a consequence of the decreasing residual amount of drug in the slms and the build - up of drug concentration in the dissolution medium in the course of time . in vitro release profile of gentamicin from batch a solid lipid microparticles in phosphate bufferred saline , key : a1-a3 contain 1.0%w / w of gentamicin while g1 and g2 are commercial gentamicin injection and plain gentamicin , respectively in vitro release profile of gentamicin from batch b solid lipid microparticles in phosphate bufferred saline , ph 7.4 ( n = 3 ) . key : b1-b3 contain 2.0%w / w of gentamicin while g1 and g2 are commercial gentamicin injection and plain gentamicin , respectively in vitro release profile of gentamicin from batch c solid lipid microparticles in phosphate bufferred saline , ph 7.4 ( n = 3 ) . key : c1-c3 contain 3.0%w / w of gentamicin while g1 and g2 are commercial gentamicin injection and plain gentamicin , respectively the anti - microbial results recorded as inhibition zone diameter ( izd ) [ tables 3 and 4 ] indicate that gentamicin - loaded slms produced very significant izd ( p < 0.05 ) against gram - positive organism ( s. aureus ) and gram - negative organism ( e. coli ) . the formulations recorded increasing izds against the organisms with time . moreover , gentamicin - loaded slms gave greater izds than the plain gentamicin as well as commercial gentamicin injection against the organisms . overall , sub - batch c3 containing the highest peg 4000 and gentamicin gave the greatest izd against s. aureus ( 27.49 2.38 m ) and e. coli ( 29.40 3.07 m ) after 420 min , whereas plain gentamicin and commercial gentamicin injection respectively gave izds of 13.47 1.87 m and 11.98 1.09 m against s. aureus and 12.87 1.74 m and 10.77 0.90 m against e. coli after 430 min . the bioevaluation was performed to establish that gentamicin did not lose activity during formulation as well as to show an increasing izd over time during the in vitro dissolution study . generally , the slms produced very significant ( p < 0.05 ) izds against the organisms . it was observed that the greater the amount of gentamicin loaded into the slms , the greater the izd produced , in agreement with earlier reports . similarly , the antibacterial activity of the formulations was concentration and time - dependent , manifested by an increasing izd against the organisms with time . high izds recorded against the organisms early in the study was an indication that these formulations would have exhibited the fastest release of the entrapped drug , hence the fast antibacterial activities ; whereas time - dependent increases in izds within 420 min implies that the slms had potentials for sustained drug release . the improved lipid solubility conferred on the drug by the amphiphilic and surfactant properties of the solubilizers ( active and passive ) ensures sustained delivery of the drug through the gradual and sustained erosion of the lipid core . furthermore , the slms generally gave greater izds than plain gentamicin and commercial gentamicin injection against the organisms . it is equally discernible from the results that the formulations were more active against s. aureus than e. coli . this formulation would be a useful alternative to gentamicin injection for enhanced delivery of gentamicin in the treatment of infections caused by gentamicin - susceptible micro - organisms , thus encouraging further development of this formulation . susceptibility of staphylococcus aureus to gentamicin in the slms susceptibility of escherichia coli to gentamicin in the slms this research has shown that pegylated slms based on a homolipid from tallow fat ( bos indicus ) and phospholipid ( p90 g ) is a potential carrier system for dissolution and bioactivity enhancement of gentamicin . compared with commercial gentamicin injection , the bioactivity and in vitro drug release studies undertaken with the formulations provided a basis to establish that srms - based slms could better be used to control the release of gentamicin .

advances in diagnosis and treatment of high blood pressure play an important role in reducing deaths caused by coronary heart disease and stroke in industrialized countries ( 1 , 2 ) . modern approach in the management of hypertension is medication approach , and blood pressure medicines are the most commonly drugs prescribed by doctors . evidence showed that drug treatment of blood pressure can diminish diastolic blood pressure by 5 to 6 mmhg , and then it can likely increase the quality of life , and also decrease the risk of coronary heart disease by 20 to 25 percent and stroke by 35 to 40 per cent ( 3 ) . studies conducted in iran show 48.1% patients with high blood pressure are treated and 21.3% only is controlled ( 4 ) . it is estimated that only 57 percent of patients with high blood pressure have controlled blood pressure across the world ( 5 ) . in the recent years , iran statistics report provides a worse situation in the way that the prevalence of hypertension in tehran suggested that only 36% of patients with hypertension used blood pressure drugs ( 23% of men and 43% of women ) that only 40% had normal blood pressure ( 45% of men and 39% of women ) ( 6 ) . it is supposed that about 50% of patients , who have received blood pressure drugs , do not continue treatment within a year ( 7 ) . the researcher s attention focused on adherence to drug consumption in patients with hypertension due to the low number of patients consuming blood pressure medications as well as lack of control of significant amounts of blood pressure . poor adherence to drug therapy is a major obstacle to achieve afore mentioned health outcomes ; moreover , non - adherence that is one of the main factors of failures to achieve normal blood pressure , refractory blood pressure and sudden loss of control is emphasized ( 8) . evidence emphasized on the possible influence of patient , service providers and providing services in non- adherence . numerous theories and cognitive models have been applied to explain the behavior of adherence to treatment that suggested that patient s beliefs have been regarded as one of the most important predictors of adherence behavior ( 9 , 10 ) . beliefs about medicines can be categorized by general and special beliefs that are associated with different diseases and cultural groups . despite the importance of this belief in adherence to the treatment of blood pressure , the current study showed that the role of beliefs has been rarely considered in iranian society patients that might be caused by the lack of appropriate instrument in relevance with demographic and cultural characteristics of iran society . thus the current survey was aimed to develop and study the beliefs about medicines questionnaire ( bmq ) in patients with hypertension . in a cross - sectional survey , 612 patients with hypertension attended to health center of isfahan city were studied . a multi - stage sampling method was conducted in the way that 4 health centers were randomly selected and a list of patients with hypertension was provided those had a file in the centers and were eligible to be included in the study , then , samples with equal volume were selected using systematic random sampling method from each center . inclusion criteria were as follow : 1 ) diagnosis of hypertension , 2 ) periodical follow - up of treatment for 1 year at least , 3 ) aged > 18 and 60 > years , 4 ) being literate and 5 ) consumption of a reducing hypertension drug at least . exclusion criteria were considered as : 1 ) having a special mental disease or 2 ) unmotivated to participate in the study . demographic characteristics including marital status , education , family history of hypertension , duration and type of treatment . bmq includes 18 questions in the two parts of general and specific beliefs about drug . the specific part explores patients beliefs about the medications prescribed for a specific condition ( beliefs about hypertension ) . the aforementioned part consists of two sub scales in terms of personal beliefs about need for medication to control blood pressure ( specific- necessity ) and concerns about the adverse consequences ( specific- concerns ) of drug ( 5 states ) . the general consists of two sub scales includes total belief that assess individuals belief regarding doctors procedure to use drugs ( general- overuse ) and damage scale that test the beliefs of patients about drugs harmfulness ( general- harm ) . each question was scored based on 5 point likert scale ( 1-strongly disagree , 2-disagree , 3- no comments , 4-agree , 5- completely agree ) . scale of specific beliefs ( necessity & concern ) had 5 questions and ranged 5 to 25 . higher scores of specific beliefs sub scale of need indicated the stronger perception to use drug to maintain health compared to personal need . higher scores of specific beliefs sub scale of specific- concerns indicated more concern about adverse effect of drug consumption . in general beliefs , sub scales of general - overuse and general- harm had 5 questions and scored 4 to 20 . higher scores of general beliefs sub scale of general- harm demonstrated more negative perspective to all drugs , and considering drugs as an addictive and toxic substance , moreover , higher scores in general - overuse sub scales indicated more negative opinions in terms of drug prescription methods . morisky medication adherence scale ( mmas ) was used to study adherence to treatment in the current survey . this scale is one of the most common structured self - report instruments to study medication adherence in chronic diseases ( 11 - 14 ) . mmas is a reliable instrument ( alpha = 0.83 ) in the evaluation of adherence to treatment that is significantly associated with blood pressure control ( 13 ) . mmas scored as follow : 2 < low adherence , 1 or 2 average adherences and 0 high adherences ( 15 ) . in this study , this questionnaire was developed by hornet and et al . by using of collecting and reviewing all available tools in the related field . standard method was considered in translation and back translation process for the cultural adaptation of questionnaires ( 19 ) . at first , questionnaire translation process was implemented by the two bilingual translators from english to persian , independently . face validity was evaluated in both quantitative and qualitative phases . in the qualitative stage , face validity measurement , relevancy , ambiguous and difficulty researcher used specialist s view of points to correct items . in the second phase ( quantitative stage ) , impact score was calculated . at this stage , the experts were asked to categorize items of questionnaire based on a 5 point likert scale ranged from very important ( score 5 ) to never important ( score 1 ) . questions with impact score higher than 1.5 were suitable for further analysis and were also retained . to evaluate content validity , qualitative and quantitative methods were used ( 16 ) . in the qualitative method some of specialists were interviewed and asked to present their comments in terms of compliance with grammar , use of appropriate and understandable words , proper scoring for the completion of tool , proportionality of selected scales and items placement in their proper place . after collecting expert s comments , researcher provided final version of instrument with regards to the collected data . to determine content validity , content validity rate ( cvr ( and content validity index ( cvi ) comments of 13 experts and scholars in the related fields were taken . to assess the content validity rate , experts were asked to comment on items related to structures in the form essential , useful but non - essential and non - essential . in final , cvr was estimated with regards to the experts numbers and the formula of cvr= ne(n/2 ) /(n/2 ) for each items . in this formula n means the total number of professionals who was participated in this stage to assess the validity and ne means number of specialists who voted the necessary options . in the current survey , exploratory factor analysis with principal components analysis and varimax rotation was used to assess construct validity . to assess the adequacy of sample size and the correlation between the variables , kaiser - meyer - olkin ( kmo ) and bartlett s test of sphericity was used . in this study , turning point of 0.4 was considered as the minimum of required load factor to maintain each item extracted from factor analysis . maximum likelihood analysis was used to measure the structure validity and items conformity of original version of questionnaire with persian version . structure validity measurement was aimed to response to this question that to what extent the structure of the questionnaire has compliance with the primary goal of questionnaire . this model is evaluated by chi - squared test , the comparative fit index ( cfi ) , the normed fit index ( nfi ) , and the root mean square error of approximation ( rmsea ) . internal consistency means a degree that questions summarized by an index and correlated with each other that cronbach s alpha coefficient is the most common method for its calculation . according to this method , a reliable instrument has cronbach s alpha coefficient greater than or equal to 0.7 ( 17 ) . to determine the internal consistency , item - total correlation method was used that measures the correlation of each question with instrument , and also decide to eliminate some of questions . to test final scale validity , the mean score of the beliefs about medicines of patients with good medication adherence was compared with patients with poor medication adherence with assumption of higher score of beliefs in patients with good medication adherence , therefore , the mean score of medication adherence was applied based on mmas questionnaire . prior to the survey , the current study was confirmed by the ethical committee of isfahan university of medical science ( project no 393777 ) , moreover , informed consent form and data confidentiality as well as being voluntary to exclude from the study were considered by the project researcher . all parts of the questionnaire was controlled by the researcher and returned to the patients if they were incomplete . data were analyzed using spss software ( ver.21 ) through descriptive methods , cronbach s alpha , pearson correlation , kmo , bartlett s test of sphericity and varimax rotation . to investigate structure validity , amos software and chi - squared test , cfi , nfi , and rmsea were executed . in a cross - sectional survey , 612 patients with hypertension attended to health center of isfahan city were studied . a multi - stage sampling method was conducted in the way that 4 health centers were randomly selected and a list of patients with hypertension was provided those had a file in the centers and were eligible to be included in the study , then , samples with equal volume were selected using systematic random sampling method from each center . inclusion criteria were as follow : 1 ) diagnosis of hypertension , 2 ) periodical follow - up of treatment for 1 year at least , 3 ) aged > 18 and 60 > years , 4 ) being literate and 5 ) consumption of a reducing hypertension drug at least . exclusion criteria were considered as : 1 ) having a special mental disease or 2 ) unmotivated to participate in the study . demographic characteristics including marital status , education , family history of hypertension , duration and type of treatment . bmq includes 18 questions in the two parts of general and specific beliefs about drug . the specific part explores patients beliefs about the medications prescribed for a specific condition ( beliefs about hypertension ) . the aforementioned part consists of two sub scales in terms of personal beliefs about need for medication to control blood pressure ( specific- necessity ) and concerns about the adverse consequences ( specific- concerns ) of drug ( 5 states ) . the general consists of two sub scales includes total belief that assess individuals belief regarding doctors procedure to use drugs ( general- overuse ) and damage scale that test the beliefs of patients about drugs harmfulness ( general- harm ) . each question was scored based on 5 point likert scale ( 1-strongly disagree , 2-disagree , 3- no comments , 4-agree , 5- completely agree ) . scale of specific beliefs ( necessity & concern ) had 5 questions and ranged 5 to 25 . higher scores of specific beliefs sub scale of need indicated the stronger perception to use drug to maintain health compared to personal need . higher scores of specific beliefs sub scale of specific- concerns indicated more concern about adverse effect of drug consumption . in general beliefs , sub scales of general - overuse and general- harm had 5 questions and scored 4 to 20 . higher scores of general beliefs sub scale of general- harm demonstrated more negative perspective to all drugs , and considering drugs as an addictive and toxic substance , moreover , higher scores in general - overuse sub scales indicated more negative opinions in terms of drug prescription methods . morisky medication adherence scale ( mmas ) was used to study adherence to treatment in the current survey . this scale is one of the most common structured self - report instruments to study medication adherence in chronic diseases ( 11 - 14 ) . mmas is a reliable instrument ( alpha = 0.83 ) in the evaluation of adherence to treatment that is significantly associated with blood pressure control ( 13 ) . mmas scored as follow : 2 < low adherence , 1 or 2 average adherences and 0 high adherences ( 15 ) . this questionnaire was developed by hornet and et al . by using of collecting and reviewing all available tools in the related field . standard method was considered in translation and back translation process for the cultural adaptation of questionnaires ( 19 ) . at first , questionnaire translation process was implemented by the two bilingual translators from english to persian , independently . face validity was evaluated in both quantitative and qualitative phases . in the qualitative stage , face validity measurement , relevancy , ambiguous and difficulty researcher used specialist s view of points to correct items . in the second phase ( quantitative stage ) , impact score was calculated . at this stage , the experts were asked to categorize items of questionnaire based on a 5 point likert scale ranged from very important ( score 5 ) to never important ( score 1 ) . questions with impact score higher than 1.5 were suitable for further analysis and were also retained . to evaluate content validity , qualitative and quantitative methods were used ( 16 ) . in the qualitative method some of specialists were interviewed and asked to present their comments in terms of compliance with grammar , use of appropriate and understandable words , proper scoring for the completion of tool , proportionality of selected scales and items placement in their proper place . after collecting expert s comments , researcher provided final version of instrument with regards to the collected data . to determine content validity , content validity rate ( cvr ( and content validity index ( cvi ) were investigated for 18 items . to do this , comments of 13 experts and scholars in the related fields were taken . to assess the content validity rate , experts were asked to comment on items related to structures in the form essential , useful but non - essential and non - essential . in final , cvr was estimated with regards to the experts numbers and the formula of cvr= ne(n/2 ) /(n/2 ) for each items . in this formula n means the total number of professionals who was participated in this stage to assess the validity and ne means number of specialists who voted the necessary options . in the current survey , exploratory factor analysis with principal components analysis and varimax rotation was used to assess construct validity . to assess the adequacy of sample size and the correlation between the variables , kaiser - meyer - olkin ( kmo ) and bartlett s test of sphericity was used . in this study , turning point of 0.4 was considered as the minimum of required load factor to maintain each item extracted from factor analysis . maximum likelihood analysis was used to measure the structure validity and items conformity of original version of questionnaire with persian version . structure validity measurement was aimed to response to this question that to what extent the structure of the questionnaire has compliance with the primary goal of questionnaire . this model is evaluated by chi - squared test , the comparative fit index ( cfi ) , the normed fit index ( nfi ) , and the root mean square error of approximation ( rmsea ) . internal consistency means a degree that questions summarized by an index and correlated with each other that cronbach s alpha coefficient is the most common method for its calculation . according to this method , a reliable instrument has cronbach s alpha coefficient greater than or equal to 0.7 ( 17 ) . to determine the internal consistency , item - total correlation method was used that measures the correlation of each question with instrument , and also decide to eliminate some of questions . to test final scale validity , the mean score of the beliefs about medicines of patients with good medication adherence was compared with patients with poor medication adherence with assumption of higher score of beliefs in patients with good medication adherence , therefore , the mean score of medication adherence was applied based on mmas questionnaire . prior to the survey , the current study was confirmed by the ethical committee of isfahan university of medical science ( project no 393777 ) , moreover , informed consent form and data confidentiality as well as being voluntary to exclude from the study were considered by the project researcher . all parts of the questionnaire was controlled by the researcher and returned to the patients if they were incomplete . data were analyzed using spss software ( ver.21 ) through descriptive methods , cronbach s alpha , pearson correlation , kmo , bartlett s test of sphericity and varimax rotation . to investigate structure validity , amos software and chi - squared test , cfi , nfi , and rmsea were executed . the mean age of patients was 44.08 10.38 years and 314 of patients were women , 10% was single , 52% had high school degree , and , in brief , 40% of patients had good medication adherence ( table 1 ) . face validity tested for all items using impact score showed that all items had impact score higher than 1.5 . according to 18 explored items by the specialist s comments about content validity , all questionnaire items were scored higher than 0.79 . demographic characteristics of included patients kmo test was used to investigate adequate sample size before implementing exploratory factor analysis . bartlett s test of sphericity was also significant ( p < 0.001 , 8453.45 ) that reported appropriate correlation to analyze variables . exploratory factor analysis was conducted on 18 items and it found that items were collected in 6 factors with special value greater than one . according to the four factor structure of the original questionnaire with varimax rotation , structure validity was studied by limiting to 4 factors . results of the factor analysis for the developed scale in the form of four factors with 18 items showed in table 1 . one item only had a load factor greater than 0.4 in more than two different factors . in general , four factors explained 57% of the total variance ( table 1 ) . exploring indicator variables that formed bmq was done using confirmatory factor analysis and by considering four indexes of fitness that all of indexes confirmed the proposed model . chi - square was 6.425 and p= 0.45 that reported an appropriate fitness between the proposed model and collected data . cfi and nfi are the relative fitness that compare model with theoretical model associated with null model with acceptable value of 0.95 . cfi and nfi were 0.958 and 0.962 , respectively that indicated the ideal fitness of the model . rmsea measures the difference between the sample coefficients and society coefficients that suggest closer value to zero indicates the better fitness of the model . reliability was tested after conducting factor analysis using cronbach s alpha for the questionnaire and for each factor . total reliability of instrument was 0.91 and 0.85 0.93 for four factors ( table 3 ) . fl : factor loading cronbach s alpha reliability coefficients and item - total correlations of the components , a : alpha values , r : item - total correlation item s total statistics of bmq in the current study , the score of patients beliefs about medicines was compared in good adherence to poor adherence group . the results reported that patients with good adherence had higher score in bmq compared with patients with low adherence . comparison of all items between the two groups showed that patients with good adherence had higher score in 15 items ( 83% of items ) ( table 5 ) . validity and reliability of bmq was tested in patients with hypertension because of the poor adherence of patients to prescribed drugs ; moreover , with regard to the chronic and serious nature of this disease , lack of cooperation and adherence might be risky and it is so necessary to follow and scrutiny . while , many studies have been introduced their patient s believes about medication as one of the most important predictors of drug treatment and ultimate success in controlling of disease . in this study , exploratory factor analysis showed four - factor structure of the bmq in the two categorization including specific beliefs about prescribed medicines in hypertensive patients and public opinion about all drugs . , the internal consistency and correlation of each question with the measurement tool showed that mentioned instrument achieved the consistent scores over time which points to the reliability of the tool for exploring beliefs about medicines in patients with hypertension in iranian society . special beliefs in the current questionnaire were about drug requirements to keep health ( special beliefs necessity ) and concerns about the drug ( special beliefs- concerns ) . the construct of special beliefs- necessity indicates the perceived rule toward protection against deterioration of the patient s current and future health . the construct of special beliefs- concerns investigates patient concerns regarding the use of blood pressure drugs in both the emotional aspect ( need to blood pressure medication worry me ) and cognitive ( medications are a mystery to me ) . as many studies suggested , although patients believed in effective role of drugs in treating of a variety of diseases , however , when several studies have investigated the beliefs about medicines in patients , it was found that strong beliefs about the potential harms of drugs hid beliefs about benefits . it seems likely persuade researchers to make a questionnaire for exploring negative beliefs in general beliefs . in general beliefs construct , two sub scales explores the nature of drugs in general harms and general- overuse of drugs by doctors . given the nature of hypertension in various studies , poor adherence to prescribed medicines is a major cause of treatment failure ( 18 ) . criterion validity of the final questionnaire showed that this tool had a good ability to separate iranian patients adheres to drug therapy . the current study had some limitations as the same as other survey . despite the patients attending in health centers , it is not possible to generalize these samples to the entire community . researcher was not able to test predictive validity because of inaccessibility of reliable tool in terms of beliefs about medicines . due to the cross - sectional study , we were not capable to investigate causal relationship between the medication adherence and beliefs about medicines in patients with hypertension , then the current correlation indicate only to the existed relationship and prospective studies are needed to study this relationship . to increase the discriminant validity ( a limitation of current survey ) , it suggests to use classified sampling with more samples . despite of these limitations , results of face , content , construct , criterion validity and reliability of bmq was confirmed to use in patients with hypertension . the current results finally approved the usability of bmq in iranian patients with hypertension for prescribed drugs and , in general , for all drugs . this questionnaire is a flexible tool to study beliefs about medicines in various diseases as belied by the developers of the questionnaire . the researchers recommend to asses validity and reliability and to use the questionnaire for investigating other diseases as well .

purpose : despite the importance of patients believes as one of the most important predictors of treatment adherence behavior , it is rarely considered in numerous studies . then the current survey was aimed to develop and study the beliefs about medicines questionnaire ( bmq ) in patients with hypertension.materials and methods : the bmq developed by horn and et al . was used in the current study . this questionnaire included 18 questions in both public and private beliefs regarding drug . face and content validity of the tool was investigated using expert s panel . in final , aforementioned tool was tested in 612 participants . to explore structure validity and internal consistency , exploratory and confirmatory factor analysis and alpha coefficient was conducted . to evaluate the final validity of instrument , the mean score of the beliefs about medicines of patients with appropriate medication adherence were compared to patients without medication adherence.results:the mean age of patients was 44.08 10.38 years . women were 314 of participants . calculated face validity by the impact score for each item indicated that all the items of the questionnaire had the impact score of more than 1.5 . in total , all items had scored higher than 0.79 by considering 18 items for content validity . the average of content validity index for the questionnaire was 0.93 . load of factors were in the range of 0.51 to 0.79 . four items explained % 57 of the total variance . comparison of all instrument items in the two groups showed that patients with good adherence had higher score in 15 items ( % 83 of all items).conclusion : the findings suggested that the bmq in patients with hypertension as a valid and reliable instrument can be used to evaluate the patients medication adherence .

1. INTRODUCTION 2. MATERIALS AND METHODS 2.1. Participants 2.2. Instrument 2.3. Translation of BMQ 2.4. Face validity 2.5. Content validity 2.6. Construct validity 2.7. Reliability 2.8. The validity of final scale 2.9. Procedure 2.10. Analysis 3. RESULTS 4. DISCUSSION 5. CONCLUSIONS

modern approach in the management of hypertension is medication approach , and blood pressure medicines are the most commonly drugs prescribed by doctors . evidence showed that drug treatment of blood pressure can diminish diastolic blood pressure by 5 to 6 mmhg , and then it can likely increase the quality of life , and also decrease the risk of coronary heart disease by 20 to 25 percent and stroke by 35 to 40 per cent ( 3 ) . studies conducted in iran show 48.1% patients with high blood pressure are treated and 21.3% only is controlled ( 4 ) . it is estimated that only 57 percent of patients with high blood pressure have controlled blood pressure across the world ( 5 ) . in the recent years , iran statistics report provides a worse situation in the way that the prevalence of hypertension in tehran suggested that only 36% of patients with hypertension used blood pressure drugs ( 23% of men and 43% of women ) that only 40% had normal blood pressure ( 45% of men and 39% of women ) ( 6 ) . it is supposed that about 50% of patients , who have received blood pressure drugs , do not continue treatment within a year ( 7 ) . the researcher s attention focused on adherence to drug consumption in patients with hypertension due to the low number of patients consuming blood pressure medications as well as lack of control of significant amounts of blood pressure . poor adherence to drug therapy is a major obstacle to achieve afore mentioned health outcomes ; moreover , non - adherence that is one of the main factors of failures to achieve normal blood pressure , refractory blood pressure and sudden loss of control is emphasized ( 8) . numerous theories and cognitive models have been applied to explain the behavior of adherence to treatment that suggested that patient s beliefs have been regarded as one of the most important predictors of adherence behavior ( 9 , 10 ) . beliefs about medicines can be categorized by general and special beliefs that are associated with different diseases and cultural groups . despite the importance of this belief in adherence to the treatment of blood pressure , the current study showed that the role of beliefs has been rarely considered in iranian society patients that might be caused by the lack of appropriate instrument in relevance with demographic and cultural characteristics of iran society . thus the current survey was aimed to develop and study the beliefs about medicines questionnaire ( bmq ) in patients with hypertension . in a cross - sectional survey , 612 patients with hypertension attended to health center of isfahan city were studied . a multi - stage sampling method was conducted in the way that 4 health centers were randomly selected and a list of patients with hypertension was provided those had a file in the centers and were eligible to be included in the study , then , samples with equal volume were selected using systematic random sampling method from each center . inclusion criteria were as follow : 1 ) diagnosis of hypertension , 2 ) periodical follow - up of treatment for 1 year at least , 3 ) aged > 18 and 60 > years , 4 ) being literate and 5 ) consumption of a reducing hypertension drug at least . exclusion criteria were considered as : 1 ) having a special mental disease or 2 ) unmotivated to participate in the study . demographic characteristics including marital status , education , family history of hypertension , duration and type of treatment . bmq includes 18 questions in the two parts of general and specific beliefs about drug . the specific part explores patients beliefs about the medications prescribed for a specific condition ( beliefs about hypertension ) . the aforementioned part consists of two sub scales in terms of personal beliefs about need for medication to control blood pressure ( specific- necessity ) and concerns about the adverse consequences ( specific- concerns ) of drug ( 5 states ) . the general consists of two sub scales includes total belief that assess individuals belief regarding doctors procedure to use drugs ( general- overuse ) and damage scale that test the beliefs of patients about drugs harmfulness ( general- harm ) . higher scores of specific beliefs sub scale of need indicated the stronger perception to use drug to maintain health compared to personal need . morisky medication adherence scale ( mmas ) was used to study adherence to treatment in the current survey . this scale is one of the most common structured self - report instruments to study medication adherence in chronic diseases ( 11 - 14 ) . mmas is a reliable instrument ( alpha = 0.83 ) in the evaluation of adherence to treatment that is significantly associated with blood pressure control ( 13 ) . in this study , this questionnaire was developed by hornet and et al . by using of collecting and reviewing all available tools in the related field . standard method was considered in translation and back translation process for the cultural adaptation of questionnaires ( 19 ) . at first , questionnaire translation process was implemented by the two bilingual translators from english to persian , independently . face validity was evaluated in both quantitative and qualitative phases . in the qualitative stage , face validity measurement , relevancy , ambiguous and difficulty researcher used specialist s view of points to correct items . in the second phase ( quantitative stage ) , impact score was calculated . at this stage , the experts were asked to categorize items of questionnaire based on a 5 point likert scale ranged from very important ( score 5 ) to never important ( score 1 ) . questions with impact score higher than 1.5 were suitable for further analysis and were also retained . to evaluate content validity , qualitative and quantitative methods were used ( 16 ) . in the qualitative method some of specialists were interviewed and asked to present their comments in terms of compliance with grammar , use of appropriate and understandable words , proper scoring for the completion of tool , proportionality of selected scales and items placement in their proper place . after collecting expert s comments , researcher provided final version of instrument with regards to the collected data . to determine content validity , content validity rate ( cvr ( and content validity index ( cvi ) comments of 13 experts and scholars in the related fields were taken . to assess the content validity rate , experts were asked to comment on items related to structures in the form essential , useful but non - essential and non - essential . in final , cvr was estimated with regards to the experts numbers and the formula of cvr= ne(n/2 ) /(n/2 ) for each items . in this formula n means the total number of professionals who was participated in this stage to assess the validity and ne means number of specialists who voted the necessary options . in the current survey , exploratory factor analysis with principal components analysis and varimax rotation was used to assess construct validity . to assess the adequacy of sample size and the correlation between the variables , kaiser - meyer - olkin ( kmo ) and bartlett s test of sphericity was used . in this study , turning point of 0.4 was considered as the minimum of required load factor to maintain each item extracted from factor analysis . maximum likelihood analysis was used to measure the structure validity and items conformity of original version of questionnaire with persian version . structure validity measurement was aimed to response to this question that to what extent the structure of the questionnaire has compliance with the primary goal of questionnaire . this model is evaluated by chi - squared test , the comparative fit index ( cfi ) , the normed fit index ( nfi ) , and the root mean square error of approximation ( rmsea ) . internal consistency means a degree that questions summarized by an index and correlated with each other that cronbach s alpha coefficient is the most common method for its calculation . according to this method , a reliable instrument has cronbach s alpha coefficient greater than or equal to 0.7 ( 17 ) . to determine the internal consistency , item - total correlation method was used that measures the correlation of each question with instrument , and also decide to eliminate some of questions . to test final scale validity , the mean score of the beliefs about medicines of patients with good medication adherence was compared with patients with poor medication adherence with assumption of higher score of beliefs in patients with good medication adherence , therefore , the mean score of medication adherence was applied based on mmas questionnaire . prior to the survey , the current study was confirmed by the ethical committee of isfahan university of medical science ( project no 393777 ) , moreover , informed consent form and data confidentiality as well as being voluntary to exclude from the study were considered by the project researcher . all parts of the questionnaire was controlled by the researcher and returned to the patients if they were incomplete . to investigate structure validity , amos software and chi - squared test , cfi , nfi , and rmsea were executed . in a cross - sectional survey , 612 patients with hypertension attended to health center of isfahan city were studied . a multi - stage sampling method was conducted in the way that 4 health centers were randomly selected and a list of patients with hypertension was provided those had a file in the centers and were eligible to be included in the study , then , samples with equal volume were selected using systematic random sampling method from each center . inclusion criteria were as follow : 1 ) diagnosis of hypertension , 2 ) periodical follow - up of treatment for 1 year at least , 3 ) aged > 18 and 60 > years , 4 ) being literate and 5 ) consumption of a reducing hypertension drug at least . exclusion criteria were considered as : 1 ) having a special mental disease or 2 ) unmotivated to participate in the study . demographic characteristics including marital status , education , family history of hypertension , duration and type of treatment . bmq includes 18 questions in the two parts of general and specific beliefs about drug . the specific part explores patients beliefs about the medications prescribed for a specific condition ( beliefs about hypertension ) . the aforementioned part consists of two sub scales in terms of personal beliefs about need for medication to control blood pressure ( specific- necessity ) and concerns about the adverse consequences ( specific- concerns ) of drug ( 5 states ) . the general consists of two sub scales includes total belief that assess individuals belief regarding doctors procedure to use drugs ( general- overuse ) and damage scale that test the beliefs of patients about drugs harmfulness ( general- harm ) . higher scores of specific beliefs sub scale of need indicated the stronger perception to use drug to maintain health compared to personal need . morisky medication adherence scale ( mmas ) was used to study adherence to treatment in the current survey . this scale is one of the most common structured self - report instruments to study medication adherence in chronic diseases ( 11 - 14 ) . mmas is a reliable instrument ( alpha = 0.83 ) in the evaluation of adherence to treatment that is significantly associated with blood pressure control ( 13 ) . this questionnaire was developed by hornet and et al . standard method was considered in translation and back translation process for the cultural adaptation of questionnaires ( 19 ) . at first , questionnaire translation process was implemented by the two bilingual translators from english to persian , independently . face validity was evaluated in both quantitative and qualitative phases . in the qualitative stage , face validity measurement , relevancy , ambiguous and difficulty researcher used specialist s view of points to correct items . in the second phase ( quantitative stage ) , impact score was calculated . at this stage , the experts were asked to categorize items of questionnaire based on a 5 point likert scale ranged from very important ( score 5 ) to never important ( score 1 ) . questions with impact score higher than 1.5 were suitable for further analysis and were also retained . to evaluate content validity , qualitative and quantitative methods were used ( 16 ) . in the qualitative method some of specialists were interviewed and asked to present their comments in terms of compliance with grammar , use of appropriate and understandable words , proper scoring for the completion of tool , proportionality of selected scales and items placement in their proper place . after collecting expert s comments , researcher provided final version of instrument with regards to the collected data . to determine content validity , content validity rate ( cvr ( and content validity index ( cvi ) were investigated for 18 items . to do this , comments of 13 experts and scholars in the related fields were taken . to assess the content validity rate , experts were asked to comment on items related to structures in the form essential , useful but non - essential and non - essential . in final , cvr was estimated with regards to the experts numbers and the formula of cvr= ne(n/2 ) /(n/2 ) for each items . in this formula n means the total number of professionals who was participated in this stage to assess the validity and ne means number of specialists who voted the necessary options . in the current survey , exploratory factor analysis with principal components analysis and varimax rotation was used to assess construct validity . to assess the adequacy of sample size and the correlation between the variables , kaiser - meyer - olkin ( kmo ) and bartlett s test of sphericity was used . in this study , turning point of 0.4 was considered as the minimum of required load factor to maintain each item extracted from factor analysis . maximum likelihood analysis was used to measure the structure validity and items conformity of original version of questionnaire with persian version . structure validity measurement was aimed to response to this question that to what extent the structure of the questionnaire has compliance with the primary goal of questionnaire . this model is evaluated by chi - squared test , the comparative fit index ( cfi ) , the normed fit index ( nfi ) , and the root mean square error of approximation ( rmsea ) . internal consistency means a degree that questions summarized by an index and correlated with each other that cronbach s alpha coefficient is the most common method for its calculation . according to this method , a reliable instrument has cronbach s alpha coefficient greater than or equal to 0.7 ( 17 ) . to determine the internal consistency , item - total correlation method was used that measures the correlation of each question with instrument , and also decide to eliminate some of questions . to test final scale validity , the mean score of the beliefs about medicines of patients with good medication adherence was compared with patients with poor medication adherence with assumption of higher score of beliefs in patients with good medication adherence , therefore , the mean score of medication adherence was applied based on mmas questionnaire . prior to the survey , the current study was confirmed by the ethical committee of isfahan university of medical science ( project no 393777 ) , moreover , informed consent form and data confidentiality as well as being voluntary to exclude from the study were considered by the project researcher . all parts of the questionnaire was controlled by the researcher and returned to the patients if they were incomplete . to investigate structure validity , amos software and chi - squared test , cfi , nfi , and rmsea were executed . the mean age of patients was 44.08 10.38 years and 314 of patients were women , 10% was single , 52% had high school degree , and , in brief , 40% of patients had good medication adherence ( table 1 ) . face validity tested for all items using impact score showed that all items had impact score higher than 1.5 . according to 18 explored items by the specialist s comments about content validity , all questionnaire items were scored higher than 0.79 . demographic characteristics of included patients kmo test was used to investigate adequate sample size before implementing exploratory factor analysis . exploratory factor analysis was conducted on 18 items and it found that items were collected in 6 factors with special value greater than one . according to the four factor structure of the original questionnaire with varimax rotation , structure validity was studied by limiting to 4 factors . results of the factor analysis for the developed scale in the form of four factors with 18 items showed in table 1 . one item only had a load factor greater than 0.4 in more than two different factors . in general , four factors explained 57% of the total variance ( table 1 ) . exploring indicator variables that formed bmq was done using confirmatory factor analysis and by considering four indexes of fitness that all of indexes confirmed the proposed model . cfi and nfi are the relative fitness that compare model with theoretical model associated with null model with acceptable value of 0.95 . cfi and nfi were 0.958 and 0.962 , respectively that indicated the ideal fitness of the model . reliability was tested after conducting factor analysis using cronbach s alpha for the questionnaire and for each factor . total reliability of instrument was 0.91 and 0.85 0.93 for four factors ( table 3 ) . fl : factor loading cronbach s alpha reliability coefficients and item - total correlations of the components , a : alpha values , r : item - total correlation item s total statistics of bmq in the current study , the score of patients beliefs about medicines was compared in good adherence to poor adherence group . the results reported that patients with good adherence had higher score in bmq compared with patients with low adherence . comparison of all items between the two groups showed that patients with good adherence had higher score in 15 items ( 83% of items ) ( table 5 ) . validity and reliability of bmq was tested in patients with hypertension because of the poor adherence of patients to prescribed drugs ; moreover , with regard to the chronic and serious nature of this disease , lack of cooperation and adherence might be risky and it is so necessary to follow and scrutiny . while , many studies have been introduced their patient s believes about medication as one of the most important predictors of drug treatment and ultimate success in controlling of disease . in this study , exploratory factor analysis showed four - factor structure of the bmq in the two categorization including specific beliefs about prescribed medicines in hypertensive patients and public opinion about all drugs . , the internal consistency and correlation of each question with the measurement tool showed that mentioned instrument achieved the consistent scores over time which points to the reliability of the tool for exploring beliefs about medicines in patients with hypertension in iranian society . special beliefs in the current questionnaire were about drug requirements to keep health ( special beliefs necessity ) and concerns about the drug ( special beliefs- concerns ) . the construct of special beliefs- necessity indicates the perceived rule toward protection against deterioration of the patient s current and future health . the construct of special beliefs- concerns investigates patient concerns regarding the use of blood pressure drugs in both the emotional aspect ( need to blood pressure medication worry me ) and cognitive ( medications are a mystery to me ) . as many studies suggested , although patients believed in effective role of drugs in treating of a variety of diseases , however , when several studies have investigated the beliefs about medicines in patients , it was found that strong beliefs about the potential harms of drugs hid beliefs about benefits . given the nature of hypertension in various studies , poor adherence to prescribed medicines is a major cause of treatment failure ( 18 ) . criterion validity of the final questionnaire showed that this tool had a good ability to separate iranian patients adheres to drug therapy . the current study had some limitations as the same as other survey . despite the patients attending in health centers , it is not possible to generalize these samples to the entire community . researcher was not able to test predictive validity because of inaccessibility of reliable tool in terms of beliefs about medicines . due to the cross - sectional study , we were not capable to investigate causal relationship between the medication adherence and beliefs about medicines in patients with hypertension , then the current correlation indicate only to the existed relationship and prospective studies are needed to study this relationship . to increase the discriminant validity ( a limitation of current survey ) , it suggests to use classified sampling with more samples . despite of these limitations , results of face , content , construct , criterion validity and reliability of bmq was confirmed to use in patients with hypertension . the current results finally approved the usability of bmq in iranian patients with hypertension for prescribed drugs and , in general , for all drugs . this questionnaire is a flexible tool to study beliefs about medicines in various diseases as belied by the developers of the questionnaire . the researchers recommend to asses validity and reliability and to use the questionnaire for investigating other diseases as well .

since the discovery of carbon nanotubes ( cnts ) , a number of applications have been studied to take advantage of their superior thermal,1 mechanical,2 and electronic properties.3 cnts have been highlighted as promising materials for applications such as composites,4 supercapacitors,5 and gas and toxin sensors in the food industry , military , and environmental fields;6 for these applications , cnts have been mass produced by chemical vapor deposition ( cvd).7,8 increasing the production yield of cnts requires a combination of catalysts and supports,7,9 and iron ( fe ) catalysts on alumina supports have been used successfully for mass production.10 however , the presence of fe catalysts in as - produced cnts may limit performance in some applications , such as chemical sensors11 and semiconducting composites for high - voltage power cables.12 even trace concentrations of metal ions can cause data misinterpretation in the former and electrical breakdown in the latter . this significant catalyst involvement in redox activity has been detected through hydrogen peroxide electrocatalysis examination13 and electrochemical activity demonstrations using fe - rich cnts.14 high - purity cnts have been created by investigating the synthesis15 or purification process,16 and a simple and precise analytical method is needed to determine the often very low concentrations of metals in these cnts . several methods have been used to measure metal concentrations , such as thermogravimetric analysis ( tga ) , energy - dispersive x - ray spectroscopy ( edx ) , x - ray fluorescence spectrometry ( xrf ) , inductive coupled plasma optical emission spectroscopy ( icp - oes ) , inductive coupled plasma mass spectrometry ( icp - ms ) , and neutron activation analysis ( naa).17 tga is a simple and widely available method , providing a total ash content consisting of metal carbides or oxides.18 when the residue obtained from tga was analyzed by x - ray diffraction , the metallic impurities were identifiable in the form of carbides or oxides as well.19 edx is able to identify elements , but its measurement of concentration is inaccurate due to the small scanning area . xrf is simple and quick and does not require sample preparation , but it does require a large sample volume ( 0.1 g).20 among the available analytical tools , naa is the most advanced and accurate tool for determining metals at low concentrations , is highly sensitive , and requires no sample preparation . ge et al.21,22 used icp - ms to analyze metal impurities in cnts with high detection sensitivity . yang et al.23 used both icp - ms and icp - oes for the same purpose . however , this sophisticated method is expensive , time - consuming , and not widely available . ultraviolet - visible ( uv / vis ) spectroscopy can be used as an alternative tool for measuring low metal concentrations , and has the advantages of low cost , ease in handling , wide availability , and high sensitivity . uv / vis spectroscopy detects the light absorption of molecules with non - bonding electrons or -electrons.24 fe , a major catalyst for cnt production , is a transition metal unsuitable for detection by uv / vis spectroscopy . therefore , chemical modification is required to measure fe concentrations using a uv / vis spectrometer . one of the most prominent ligands for fe complexes is phenanthroline ( phen).25 this bidentate chelating agent has two nitrogen atoms at the ortho positions of the rigid half - ring structure . the resulting fe phen complex has an absorbance at a specific wavelength , with the peak intensity proportional to the fe cation concentration in the sample . the use of phen as a chelating agent has advantages due to its wide commercial availability and low cost relative to other fe - coordinating spectrometric ligands such as n , n-ethylenebis(ethanesulfonamide),26 ferrozine , and ammonium pyrrolidinedithiocarbamate ( apdc ) . furthermore , quantitative analysis of fe using phen has been conducted in waste water and tissue homogenates.27 this method has also been proposed for environmentally friendly portable lab - on - paper devices,28 fe recycling in fe - rich sludge,29 and biological applications such as chelation to prevent fe toxicity during hemorrhaging.30 the interaction between phen and cnts has also been suggested for selective recognition of copper and hydrogen peroxide sensing.31 this study assessed a simple and precise method for quantitative measurement of fe concentrations in cnts using uv / vis spectroscopy . a procedure for sample preparation and fe analysis using uv / vis spectroscopy was developed by modifying the established procedure to form a fe phen complex from fe catalyst residues in cnts which displays a red orange color in aqueous solution.32 a calibration relationship between optical absorbance and fe concentration was constructed by using a series of standard fe solutions of known concentrations . the absorption of the fe phen complex at a visible wavelength increased linearly with increasing fe phen concentration in solution;24 hence , fe concentrations in an unknown solution may be evaluated easily and accurately . this study also assessed optimized acid reflux conditions for complete extraction of fe from cnts for accurate measurement of fe concentrations . the colorimetric method using uv / vis spectroscopy was verified by confirming fe concentrations using icp - oes . to our knowledge , this study is the first to report that the fe content of cnts can be accurately measured with a simple , reliable , and readily available uv / vis spectroscopic technique . figure 1 shows the cnt - a used to develop the fe content determination method . scanning electron microscopy ( sem ) shows non - bundled and individually separated tubes , but some degree of entanglement appeared between the structures ( figure 1 a ) . transmission electron microscopy ( tem ) reveals multi - layered tubes with an average diameter of 15.54.79 nm , which was calculated from 90 nanotubes ( figure 1 b ) . metal catalyst particles were usually enclosed by thick graphitic layers ( figure 1 c ) ; the graphitic layer should be removed or at least cracked so that etchants may access and eliminate these metallic impurities . raman spectra ( not shown ) of the cnts had an average intensity ratio of the g and d bands ( ig / id ) of 0.760.04 , indicating that they possess quite low crystallinity . in a derivative of the tga curve ( not shown ) , the oxidation peak temperature of the cnts was 650 c . a 3-g sample of ap - cnts was burned at 900 c in air to completely remove all carbonaceous material , and the ashes obtained were 3.51 wt . % of the cnts . as shown in figure 1 d , the ashes were red in color , indicating that iron oxide was formed while burning the cnts in air . tem images show their multilayered structure and an average diameter of 15.5 nm , calculated from 90 nanotubes . d ) red - colored ash formed by oxidizing cnts at 900 c , 3.51 % by weight , taken by a digital camera . in this spectrometric study , phen complex with a tricyclic organic ligand , 1,10-phenanthroline.25 for stable formation of the fe phen complex , known as ferroin,33 the acidity of the aqueous solution should be kept in the ph 47 range by adding a sodium acetate buffer solution.3436 at acidities below ph 3 , the red color of the solution vanishes ; above ph 7 , fe ions precipitate as iron hydroxide.33 under atmospheric conditions , stable trivalent fe ions ( fe ) are usually produced . hydroxylamine as a reducing agent converts the valence state of fe ions from trivalent ( fe ) to divalent ( fe ) . the reduction mechanism for fe ions has been suggested as [ equation ( 1)]:37 1 phen then reacts with fe to form the red orange colored fe phen complex , by the reaction shown in figure 2 a. a ) scheme of fe phen complex formation ; coordination of an fe ion with three phen molecules via lone - pair electrons on the n atoms of phen . b ) standard solutions of fe phen complex used for calibration , containing fe concentrations from 09 ppm , taken by a digital camera . the red orange color of the solutions becomes stronger as the fe concentration increases . c ) absorption spectra of the standard solutions in the visible spectral range , measured by uv / vis spectroscopy . d ) linear relationship between optical absorbance and fe concentration for the standard solutions . the absorbance was measured at the maximum peak position of 510 nm for the absorption spectra shown in panel c. linear fitting : a=0.20423 c+0.00716 , r=0.99994 . the coefficient of determination to designate linearity is calculated using r=. here , yi is the observed absorbance value , is the mean absorbance , and 1 is the fitted absorbance value . e ) fe concentrations measured from the constructed solutions containing both al and fe ions , using uv / vis spectroscopy . a series of standard fe phen complex solutions with fe concentrations of 19 ppm were made , and a photograph of these solutions is presented in figure 2 b. the solution visibly reddened with increasing fe concentration . absorption spectra for these solutions , measured in the visible spectral range of =400600 nm , exhibited absorption peaks at =510 nm ( figure 2 c).38 according to the lambert beer law,24 a linear relationship was drawn between the fe concentrations of the solutions and the corresponding maximum absorbance values , as shown in figure 2 d , in which the linearity , indicated by the r value , is very close to 1 . the equation obtained from linear regression was used to calculate the fe concentration of an unknown solution by measuring the maximum absorbance value in its absorption spectrum . our ap - cnts were synthesized by cvd with an fe catalyst embedded on alumina supports . because the solutions prepared for analysis of fe impurities may therefore also contain al impurities , it was necessary to confirm whether the presence of al ions in the solution has any influence on the formation of fe phen complexes , to ensure the assessment of fe concentrations is accurate . solutions were prepared by mixing the pure fe and al solutions and then by following the procedure for formation of fe phen complexes . fe concentrations were measured in these solutions by uv / vis spectroscopy . figure 2 e shows that the constructed and measured fe concentrations were a nearly exact match for each other , with negligible deviation . this indicates that the fe concentration can be precisely detected without disturbance from al ions in solution , a finding that is consistent with previous studies.39,40 figure 2 d shows the calibration equation for fe concentrations from 0 to 9 ppm . if the prepared solution has a higher fe concentration , it must be diluted to within this detection range . actual fe concentrations [ fe ] in the ap - cnts can be simply calculated from fe concentrations measured for the diluted solution prepared using cnts or ash , as follows : 2 here , mcnt and mfe are the masses of the ap - cnts , including metallic impurities and fe , respectively , expressed in grams . df stands for a dilution factor , which indicates how many times the solution is diluted to fall within the fe detection range of 09 ppm . in our experiment , dilution was conducted in two steps : diluting a 10 ml ( vi ) acid solution in a 100 ml volumetric flask ( v1 ) followed by a second dilution obtained by sampling a 5 ml ( v2 ) volume taken from v1 in a 50 ml volumetric flask ( vf ) . cfe is the fe concentration of the diluted solution expressed in ppm ( g ml ) , which is calculated by inputting the absorbance value of an unknown solution into the calibration equation given in figure 2 d. rash is the ratio of the ash mass ( mash ) of the ap - cnts to the ap - cnt mass ( mcnt ) . for icp - oes measurement , the fe concentration in the acid solution may vary depending on how completely the metal impurities are dissolved . the dissolution of metal impurities can also be affected by various factors such as the morphology and volume of cnts dissolved , acid type , reflux temperature , and time , so the acid reflux conditions should be optimized for accurate fe analysis . aqua regia , hcl / hno3=3:1 , was used in this study to dissolve metal catalysts in the cnts because it is a strong oxidant that is frequently used to purify cnts through liquid oxidation.41 figure 3 a shows fe concentrations extracted by aqua regia from the cnts and ashes as a function of reflux time . for these same solutions , the fe concentrations were measured by uv / vis spectroscopy ( solid lines in figure 3 a ) and icp - oes ( dashed lines in figure 3 a ) , which are in good agreement with each other ( figure 3 a ) . the colorimetric method using uv / vis spectroscopy is reliable and applicable for assessing fe content in cnts and ashes , as confirmed by icp - oes . comparison of fe concentrations measured by uv / vis spectroscopy and icp - oes for the solutions prepared by treating a ) cnts and ash in the hcl / hno3 ( 3:1 ) mixture as a function of reflux time and b ) cnts in various single and mixed acid solvents for 6 h. solvent numbers are denoted as follows ( mixture ratios as v / v ) : c)f ) images of solutions taken by digital camera after holding cnts at reflux for 6 h in acid mixtures of c ) hcl / hno3 ( 3:1 ) , d ) h2so4/hno3 ( 3:1 ) , e ) hclo4/hno3 ( 3:1 ) , and f ) hclo4/ f - hno3 ( 3:1 ) . in figure 3 a , the fe concentration in the cnts was threefold lower than that in the ashes . this considerable difference is probably because the fe catalysts in the cnts were not completely dissolved during reflux in acid . figure 3 c shows an image of the solution taken after the cnts were held at reflux in aqua regia for 6 h , where carbon precipitates were clearly observed at the bottom of the cell due to incomplete dissolution . this indicates that aqua regia has insufficient oxidizing power to dissolve all cnt materials , and thus fe catalysts within the cnts or encapsulated in graphitic layers ( figure 1 c ) were not dissolved during the acid reflux . aqua regia is a strong oxidant , but gradually loses its strength by releasing oxidative compounds in the form of nitrosyl chlorides and chlorine gas.42 stronger oxidant acids that are capable of etching away further cnt materials and graphitic layers are required to completely dissolve fe catalysts that may be encapsulated by cnts or graphitic layers . five single acids and six acid mixtures were examined to compare their extraction powers for fe catalysts from the ap - cnts ( figure 3 b ) . the acid reflux was performed for 6 h in all cases , as the fe concentration increased rapidly for a reflux time of 02 h and was observed to nearly plateau at 6 h in aqua regia ( figure 3 a ) . the fe concentrations of the refluxed solutions were analyzed using both uv / vis spectroscopy and icp - oes . the five bar graphs , 1 to 5 in figure 3 b , show the solution fe concentrations after reflux by single concentrated acids : h2so4 , hcl , hno3 , fumed - hno3 ( f - hno3 ) , and hclo4 . from the uv / vis spectrometric measurements , fe concentrations , stated with their uncertainty43 from measurement process , were 3.130.12 and 3.790.13 kppm for the h2so4- and hcl - refluxed solutions , respectively , but reached 10.090.13 and 12.140.14 kppm for the hno3- and hclo4-refluxed solutions , respectively . fe concentrations for hno3 ( 70 % ) or f - hno3 ( 93 % ) were observed at similar levels , 10.300.13 kppm , for the 6 h reflux , but the reflux appeared to progress more rapidly for f - hno3 . hclo4 , a strong oxidizing agent with oxidation power originating from the perchlorate ion ( clo4 ) , was capable of extracting an fe concentration of 12.140.13 kppm from the cnts . in treatments with single acids , hcl and h2so4 were less oxidative in digesting the cnts and graphitic layers . the ability to fully etch carbon materials seems to be closely related to the oxidizing nature of an acid rather than its acidic strength.44 among the single acids tested , hno3 and hclo4 are considered to be powerful oxidants , and effective for extracting fe catalysts from cnts . nevertheless , none of the single acids were able to completely digest the cnts , because black precipitates were observed in all solutions after reflux with single acids . because hclo4 and hno3 exhibited the strongest extraction capacity among the single acids , they were mixed together to enhance their oxidizing power . hclo4 and hno3 mixtures were prepared at three different ratios of 1:3 , 1:1 , and 3:1 . as shown in figure 3 b , the hclo4/hno3 mixtures resulted in fe concentrations of 12.620.14 , 14.380.14 , and 14.680.14 kppm for ratios of 1:3 , 1:1 , and 3:1 in the 6 h reflux experiments , respectively . more fe catalyst was extracted as the hclo4 fraction was increased in the hclo4/hno3 mixture . among the three hclo4/hno3 mixtures , the strongest acid mixture , 3:1 , produced a semitransparent brown solution upon reflux of the cnts for 6 h ( figure 3 e ) , indicating that the cnts were almost dissolved in this mixture , but not completely . for further dissolution of the cnts , hno3 was replaced with f - hno3 in a hclo4/f - hno3 ratio of 3:1 . as shown in figure 3 f , a transparent solution was obtained without any trace of the cnts for this acid mixture after 6 h , suggesting that the ap - cnts were completely dissolved , and all fe catalysts were extracted . in this acid mixture , the fe concentration was 14.520.14 kppm . we also investigated a h2so4/hno3 mixture ( 3:1 ) , which has been widely used for the oxidation of cnts.45 the refluxed solution contained fe concentrations as high as 14.560.14 kppm , but showed a dark - brown color , as depicted in figure 3 d. h2so4 alone had the weakest fe extraction capacity among the single acids examined , but exhibited a synergetic effect when mixed with hno3 in a 3:1 ratio . as shown in figure 3 b , the h2so4/hno3 ( 3:1 ) , hclo4/hno3 ( 1:1 ) , hclo4/hno3 ( 3:1 ) , and hclo4/f - hno3 ( 3:1 ) mixtures yielded similar fe concentrations of 14.560.14 , 14.380.14 , 14.670.14 , and 14.520.14 kppm , respectively , but only the hclo4/f - hno3 ( 3:1 ) mixture produced a transparent and clear solution . during acid reflux , complete fe extraction depends on the destruction of cnts or graphitic layers enclosing the fe catalysts . it seems that h2so4/hno3 ( 3:1 ) , hclo4/hno3 ( 1:1 ) , and hclo4/hno3 ( 3:1 ) mixtures destroyed or cracked all cnts or graphitic layers such that the encapsulated fe catalysts could be extracted , whereas the hclo4/f - hno3 ( 3:1 ) mixture totally destroyed all carbon materials . hence , the four mixtures tested are almost equally powerful for fe extraction from cnts , but hclo4/f - hno3 ( 3:1 ) is superior to the others in consuming the carbon materials during reflux . nitric acid is thought to digest carbon materials via a nitration process with nitronium ( no2 ) ions . according to a computational study by gerber et al.,46 nitronium ions first attack the most reactive carbon atoms on surface defects , and produce and then enlarge the vacancies for a prolonged reaction time . in concentrated nitric acid , nitronium ions form through equilibrium self - dissociation according to the following reaction : 2 hno3no2+no3+h2o.47 the formation of these particular ions can be accelerated by adding a strong acid such as sulfuric or perchloric acid.48 the reactions are hno3 + 2 h2so4no2+h3o+2 hso4 for sulfuric acid and hno3 + 2 hclo4no2+h3o+2 clo4 for perchloric acid.49 importantly , the experiment by gerber et al.46 revealed that more hno3 is dissociated to form more no2 ions if a greater quantity of its complementary acid is added . this may explain the increased consumption of cnts as the fraction of hclo4 was increased during reflux with the hclo4/hno3 mixture . furthermore , involvement of water in the reaction suppresses no2 formation : greater than 50 mol % water causes zero dissociation in hno3.47 thus , the hclo4/f - hno3 ( 3:1 ) mixture consumes cnts completely by formation of a greater quantity of nitronium ions . for all 11 reflux solutions listed in figure 3 b , the fe concentrations measured by uv / vis spectroscopy were in good agreement with those measured using icp - oes within an average difference of 6.0 % . therefore , the colorimetric method developed in this study using uv / vis spectroscopy appears to be a reliable tool for accurate measurement of fe concentrations in acid refluxed solutions of cnts . in an attempt to demonstrate its reliability , this method was applied to other cnt samples , namely cnt - b and cnt - c . both cnts were synthesized using fe and co catalysts on alumina supports by catalytic chemical vapor deposition ( ccvd ) . in figure 4 a d , cnts occurred entangled , and metal catalysts were enclosed by cnts for both cnt - b and -c . the ashes , obtained by burning at 900 c in air , appeared grey because fe and co were used as catalysts ( figure 4 e , f ) . fe measurements were made three times for each cnt sample using uv / vis spectroscopy and icp - oes ( figure 4 g ) . fe concentrations measured by both methods closely match each other within the average differences of 1.66 and 0.38 % for cnt - b and -c , respectively , supporting that the uv / vis spectroscopic method is comparable to the widely used icp - oes technique in assessing the fe content of cnts . measurement precision , as indicated by the small error bars , illustrates the reliability of this method . sem images of a ) cnt - b and b ) cnt - c . tem images of c ) cnt - b and d ) cnt - c show that multiple graphitic layers encase catalyst particles . the ashes , produced by oxidizing cnts at 900 c , have a grey color for both e ) cnt - b and f ) cnt - c . g ) fe concentrations measured by uv / vis spectroscopy and icp - oes for cnt - b and cnt - c . easy - to - use and widely available uv / vis spectroscopy was used to determine the concentration of fe catalysts in cnts by formation of colored complexes with the 1,10-phenanthroline ( phen ) ligand . this colorimetric method revealed a linear relationship between fe concentration and optical absorbance of the cnt refluxed solution . an unknown concentration of fe was calculated by fitting its corresponding absorbance to the linear equation . cnts were held at reflux in a variety of single and mixed acids to compare their capacity to extract fe from cnts , so that fe catalysts encapsulated by cnts or graphitic layers could be fully dissolved . among the single or mixed acids considered , mixtures of h2so4/hno3 ( 3:1 ) , hclo4/hno3 ( 1:1 ) , hclo4/hno3 ( 3:1 ) , and hclo4/f - hno3 ( 3:1 ) exhibited the strongest dissolution capacity for fe . with regard to the refluxed solutions , fe concentrations measured by uv / vis spectroscopy and icp - oes were in good agreement with each other , indicating that our colorimetric method using uv / vis spectroscopy is reliable and applicable to the assessment of fe content in cnts . materials : cnts provided by jeio company ( korea ) were multi - walled synthesized by ccvd . reagents used for fe determination were purchased from sigma aldrich : hydroxylamine hydrochloride ( nh2ohhcl , 99 % ) , 1,10-phenanthroline ( c12h8n2 , 99 % ) , ammonium iron(ii ) sulfate hexahydrate ( [ fe(nh4)2(so4)26 h2o ) ] , 99.997 % ) , acetic acid ( ch3cooh , 99.9 % ) , and sodium hydroxide ( naoh , 97 % ) . fe flakes ( 99.99 % ) and aluminum ( al ) pellets ( 99.999 % ) purchased from lts chemicals ( chestnut ridge , ny , usa ) were used for validation . the concentrated acids used to extract fe components from cnts and ashes were hydrochloric acid ( hcl , 35 % , daejung ) , nitric acid ( hno3 , 70 % , daejung ) , fumed nitric acid ( f - hno3 , 93 % , matsunoen ) , sulfuric acid ( h2so4 , 9598 % , sigma aldrich ) , and perchloric acid ( hclo4 , 70 % , sigma aldrich ) . an fe standard solution with an fe concentration of 500 ppm was made by dissolving [ fe(nh4)2(so4)26 h2o ) ] ( 0.3509 g ) in h2so4 ( 0.25 ml ) in a 100 ml volumetric flask and then diluting with distilled water . a sodium acetate ( ch3coona ) buffer solution was prepared by mixing 6 m ch3cooh ( 100 ml ) with 5 m naoh ( 100 ml ) . aqueous solutions of hydroxylamine ( 10 wt % ) and o - phen ( 0.1 wt % ) were prepared at room temperature and 60 c , respectively . a classical colorimetric method for fe determination has been described elsewhere,34,35 and the procedure was modified here for sample preparation . a series of fe solutions was obtained by diluting the 500 ppm fe standard solution : 0 , 0.2 , 0.6 , 1 , 1.4 , and 1.8 ml standard solutions of 500 ppm were added to 100 ml volumetric flasks to produce calibration solutions with fe concentrations of 0 , 1 , 3 , 5 , 7 , and 9 ppm , respectively . subsequently , sodium acetate buffer ( 8 ml ) , hydroxylamine solution ( 1 ml ) , and o - phen solution ( 10 ml ) in turn were added at intervals of 10 min between each solution . distilled water was added to make up 100 ml calibration solutions . to determine whether the presence of al in solution influenced the accuracy of fe determination , we prepared five solutions at various fe and al ratios while maintaining the total concentration of these ions at 10 ppm , as described below . individual stock solutions for fe and al were separately produced by dissolving fe flakes ( 0.0525 g ) and al pellets ( 0.0560 g ) in concentrated hcl , respectively . mixtures with different ratios of fe and al stock solutions were prepared , and buffer solution , hydroxylamine solution , and o - phen solution were subsequently added . to prepare solution samples for fe determination , the cnts ( 60 mg ) or ashes ( 15 mg ) were held at reflux with acid ( 10 ml ) in an oil bath maintained at 130 c . next , the acid mixture was filtered , and the filtrate was collected in a 100 ml volumetric flask . the filtrates , with volumes of 5 and 1 ml for cnts and ashes , respectively , were transferred to a 50 ml volumetric flask . buffer solution , hydroxylamine solution , and o - phen solution were added , and distilled water was finally added to bring the volume to 50 ml , developing red orange colored solutions for spectroscopic measurement of fe content . the optical absorbance used to determine fe content was assessed not only based on the red orange colored fe phen complexes , but also by the reagents contained in the solution , such as cnts . blank solutions for correction of spectroscopic measurements were prepared by following the same procedure as that followed for calibration samples , mixing the same amounts of all reagents including the cnts , but omitting phen to prohibit the formation of red orange colored fe characterization : cnts were characterized using field emission scanning electron microscopy ( fe - sem , hitachi s-4700 ) and high - resolution transmission electron microscopy ( hrtem , tecnai g2 f20 ) to observe their morphologies and structures . raman spectroscopy ( renishaw system 3000 , laser =633 nm ) was used to characterize the crystalline nature and structural defects in cnts . inductively coupled plasma optical emission spectrometry ( icp - oes , perkinelmer optima 4300 dv ) was used to measure metal impurity content in the cnt or ash samples by examining the specific wavelengths of =238.204 and 396.153 nm , corresponding to fe and al , respectively . the solution for icp - oes analysis was typically prepared by boiling the sample in a closed teflon vessel with aqua regia ( hcl / hno3=3:1 ) at 200 c for 1 day and then cooling to room temperature . after the solvent was evaporated , the residue was diluted with 2 % nitric acid solution before measurement . the absorbance values of the solutions prepared for fe determination were measured by uv / vis near infrared spectroscopy ( uv / vis - nir spectrophotometer , agilent , cary 5000 ) in the spectral range of =400600 nm . the peak absorbance value was recorded at =510 nm for the fe phen complexes . a fitting equation was obtained by plotting the peak absorbance values versus fe concentrations , and was used to determine the fe concentration for an unknown sample .

iron catalysts have been used widely for the mass production of carbon nanotubes ( cnts ) with high yield . in this study , uv / visible spectroscopy was used to determine the fe catalyst content in cnts using a colorimetric technique . fe ions in solution form red orange complexes with 1,10-phenanthroline , producing an absorption peak at =510 nm , the intensity of which is proportional to the solution fe concentration . a series of standard fe solutions were formulated to establish the relationship between optical absorbance and fe concentration . many fe catalysts were microscopically observed to be encased by graphitic layers , thus preventing their extraction . fe catalyst dissolution from cnts was investigated with various single and mixed acids , and fe concentration was found to be highest with cnts being held at reflux in hclo4/hno3 and h2so4/hno3 mixtures . this novel colorimetric method to measure fe concentrations by uv / vis spectroscopy was validated by inductively coupled plasma optical emission spectroscopy , indicating its reliability and applicability to asses fe content in cnts .

Introduction Results and Discussion Conclusions Experimental Section

since the discovery of carbon nanotubes ( cnts ) , a number of applications have been studied to take advantage of their superior thermal,1 mechanical,2 and electronic properties.3 cnts have been highlighted as promising materials for applications such as composites,4 supercapacitors,5 and gas and toxin sensors in the food industry , military , and environmental fields;6 for these applications , cnts have been mass produced by chemical vapor deposition ( cvd).7,8 increasing the production yield of cnts requires a combination of catalysts and supports,7,9 and iron ( fe ) catalysts on alumina supports have been used successfully for mass production.10 however , the presence of fe catalysts in as - produced cnts may limit performance in some applications , such as chemical sensors11 and semiconducting composites for high - voltage power cables.12 even trace concentrations of metal ions can cause data misinterpretation in the former and electrical breakdown in the latter . this significant catalyst involvement in redox activity has been detected through hydrogen peroxide electrocatalysis examination13 and electrochemical activity demonstrations using fe - rich cnts.14 high - purity cnts have been created by investigating the synthesis15 or purification process,16 and a simple and precise analytical method is needed to determine the often very low concentrations of metals in these cnts . several methods have been used to measure metal concentrations , such as thermogravimetric analysis ( tga ) , energy - dispersive x - ray spectroscopy ( edx ) , x - ray fluorescence spectrometry ( xrf ) , inductive coupled plasma optical emission spectroscopy ( icp - oes ) , inductive coupled plasma mass spectrometry ( icp - ms ) , and neutron activation analysis ( naa).17 tga is a simple and widely available method , providing a total ash content consisting of metal carbides or oxides.18 when the residue obtained from tga was analyzed by x - ray diffraction , the metallic impurities were identifiable in the form of carbides or oxides as well.19 edx is able to identify elements , but its measurement of concentration is inaccurate due to the small scanning area . ge et al.21,22 used icp - ms to analyze metal impurities in cnts with high detection sensitivity . ultraviolet - visible ( uv / vis ) spectroscopy can be used as an alternative tool for measuring low metal concentrations , and has the advantages of low cost , ease in handling , wide availability , and high sensitivity . uv / vis spectroscopy detects the light absorption of molecules with non - bonding electrons or -electrons.24 fe , a major catalyst for cnt production , is a transition metal unsuitable for detection by uv / vis spectroscopy . therefore , chemical modification is required to measure fe concentrations using a uv / vis spectrometer . the resulting fe phen complex has an absorbance at a specific wavelength , with the peak intensity proportional to the fe cation concentration in the sample . furthermore , quantitative analysis of fe using phen has been conducted in waste water and tissue homogenates.27 this method has also been proposed for environmentally friendly portable lab - on - paper devices,28 fe recycling in fe - rich sludge,29 and biological applications such as chelation to prevent fe toxicity during hemorrhaging.30 the interaction between phen and cnts has also been suggested for selective recognition of copper and hydrogen peroxide sensing.31 this study assessed a simple and precise method for quantitative measurement of fe concentrations in cnts using uv / vis spectroscopy . a procedure for sample preparation and fe analysis using uv / vis spectroscopy was developed by modifying the established procedure to form a fe phen complex from fe catalyst residues in cnts which displays a red orange color in aqueous solution.32 a calibration relationship between optical absorbance and fe concentration was constructed by using a series of standard fe solutions of known concentrations . this study also assessed optimized acid reflux conditions for complete extraction of fe from cnts for accurate measurement of fe concentrations . the colorimetric method using uv / vis spectroscopy was verified by confirming fe concentrations using icp - oes . to our knowledge , this study is the first to report that the fe content of cnts can be accurately measured with a simple , reliable , and readily available uv / vis spectroscopic technique . figure 1 shows the cnt - a used to develop the fe content determination method . transmission electron microscopy ( tem ) reveals multi - layered tubes with an average diameter of 15.54.79 nm , which was calculated from 90 nanotubes ( figure 1 b ) . in this spectrometric study , phen complex with a tricyclic organic ligand , 1,10-phenanthroline.25 for stable formation of the fe phen complex , known as ferroin,33 the acidity of the aqueous solution should be kept in the ph 47 range by adding a sodium acetate buffer solution.3436 at acidities below ph 3 , the red color of the solution vanishes ; above ph 7 , fe ions precipitate as iron hydroxide.33 under atmospheric conditions , stable trivalent fe ions ( fe ) are usually produced . the red orange color of the solutions becomes stronger as the fe concentration increases . c ) absorption spectra of the standard solutions in the visible spectral range , measured by uv / vis spectroscopy . d ) linear relationship between optical absorbance and fe concentration for the standard solutions . e ) fe concentrations measured from the constructed solutions containing both al and fe ions , using uv / vis spectroscopy . a series of standard fe phen complex solutions with fe concentrations of 19 ppm were made , and a photograph of these solutions is presented in figure 2 b. the solution visibly reddened with increasing fe concentration . absorption spectra for these solutions , measured in the visible spectral range of =400600 nm , exhibited absorption peaks at =510 nm ( figure 2 c).38 according to the lambert beer law,24 a linear relationship was drawn between the fe concentrations of the solutions and the corresponding maximum absorbance values , as shown in figure 2 d , in which the linearity , indicated by the r value , is very close to 1 . the equation obtained from linear regression was used to calculate the fe concentration of an unknown solution by measuring the maximum absorbance value in its absorption spectrum . because the solutions prepared for analysis of fe impurities may therefore also contain al impurities , it was necessary to confirm whether the presence of al ions in the solution has any influence on the formation of fe phen complexes , to ensure the assessment of fe concentrations is accurate . fe concentrations were measured in these solutions by uv / vis spectroscopy . this indicates that the fe concentration can be precisely detected without disturbance from al ions in solution , a finding that is consistent with previous studies.39,40 figure 2 d shows the calibration equation for fe concentrations from 0 to 9 ppm . actual fe concentrations [ fe ] in the ap - cnts can be simply calculated from fe concentrations measured for the diluted solution prepared using cnts or ash , as follows : 2 here , mcnt and mfe are the masses of the ap - cnts , including metallic impurities and fe , respectively , expressed in grams . df stands for a dilution factor , which indicates how many times the solution is diluted to fall within the fe detection range of 09 ppm . cfe is the fe concentration of the diluted solution expressed in ppm ( g ml ) , which is calculated by inputting the absorbance value of an unknown solution into the calibration equation given in figure 2 d. rash is the ratio of the ash mass ( mash ) of the ap - cnts to the ap - cnt mass ( mcnt ) . for icp - oes measurement , the fe concentration in the acid solution may vary depending on how completely the metal impurities are dissolved . aqua regia , hcl / hno3=3:1 , was used in this study to dissolve metal catalysts in the cnts because it is a strong oxidant that is frequently used to purify cnts through liquid oxidation.41 figure 3 a shows fe concentrations extracted by aqua regia from the cnts and ashes as a function of reflux time . for these same solutions , the fe concentrations were measured by uv / vis spectroscopy ( solid lines in figure 3 a ) and icp - oes ( dashed lines in figure 3 a ) , which are in good agreement with each other ( figure 3 a ) . the colorimetric method using uv / vis spectroscopy is reliable and applicable for assessing fe content in cnts and ashes , as confirmed by icp - oes . comparison of fe concentrations measured by uv / vis spectroscopy and icp - oes for the solutions prepared by treating a ) cnts and ash in the hcl / hno3 ( 3:1 ) mixture as a function of reflux time and b ) cnts in various single and mixed acid solvents for 6 h. solvent numbers are denoted as follows ( mixture ratios as v / v ) : c)f ) images of solutions taken by digital camera after holding cnts at reflux for 6 h in acid mixtures of c ) hcl / hno3 ( 3:1 ) , d ) h2so4/hno3 ( 3:1 ) , e ) hclo4/hno3 ( 3:1 ) , and f ) hclo4/ f - hno3 ( 3:1 ) . in figure 3 a , the fe concentration in the cnts was threefold lower than that in the ashes . this considerable difference is probably because the fe catalysts in the cnts were not completely dissolved during reflux in acid . figure 3 c shows an image of the solution taken after the cnts were held at reflux in aqua regia for 6 h , where carbon precipitates were clearly observed at the bottom of the cell due to incomplete dissolution . this indicates that aqua regia has insufficient oxidizing power to dissolve all cnt materials , and thus fe catalysts within the cnts or encapsulated in graphitic layers ( figure 1 c ) were not dissolved during the acid reflux . five single acids and six acid mixtures were examined to compare their extraction powers for fe catalysts from the ap - cnts ( figure 3 b ) . the acid reflux was performed for 6 h in all cases , as the fe concentration increased rapidly for a reflux time of 02 h and was observed to nearly plateau at 6 h in aqua regia ( figure 3 a ) . the fe concentrations of the refluxed solutions were analyzed using both uv / vis spectroscopy and icp - oes . the five bar graphs , 1 to 5 in figure 3 b , show the solution fe concentrations after reflux by single concentrated acids : h2so4 , hcl , hno3 , fumed - hno3 ( f - hno3 ) , and hclo4 . from the uv / vis spectrometric measurements , fe concentrations , stated with their uncertainty43 from measurement process , were 3.130.12 and 3.790.13 kppm for the h2so4- and hcl - refluxed solutions , respectively , but reached 10.090.13 and 12.140.14 kppm for the hno3- and hclo4-refluxed solutions , respectively . in treatments with single acids , hcl and h2so4 were less oxidative in digesting the cnts and graphitic layers . the ability to fully etch carbon materials seems to be closely related to the oxidizing nature of an acid rather than its acidic strength.44 among the single acids tested , hno3 and hclo4 are considered to be powerful oxidants , and effective for extracting fe catalysts from cnts . as shown in figure 3 b , the hclo4/hno3 mixtures resulted in fe concentrations of 12.620.14 , 14.380.14 , and 14.680.14 kppm for ratios of 1:3 , 1:1 , and 3:1 in the 6 h reflux experiments , respectively . among the three hclo4/hno3 mixtures , the strongest acid mixture , 3:1 , produced a semitransparent brown solution upon reflux of the cnts for 6 h ( figure 3 e ) , indicating that the cnts were almost dissolved in this mixture , but not completely . as shown in figure 3 f , a transparent solution was obtained without any trace of the cnts for this acid mixture after 6 h , suggesting that the ap - cnts were completely dissolved , and all fe catalysts were extracted . in this acid mixture , the fe concentration was 14.520.14 kppm . as shown in figure 3 b , the h2so4/hno3 ( 3:1 ) , hclo4/hno3 ( 1:1 ) , hclo4/hno3 ( 3:1 ) , and hclo4/f - hno3 ( 3:1 ) mixtures yielded similar fe concentrations of 14.560.14 , 14.380.14 , 14.670.14 , and 14.520.14 kppm , respectively , but only the hclo4/f - hno3 ( 3:1 ) mixture produced a transparent and clear solution . during acid reflux , complete fe extraction depends on the destruction of cnts or graphitic layers enclosing the fe catalysts . it seems that h2so4/hno3 ( 3:1 ) , hclo4/hno3 ( 1:1 ) , and hclo4/hno3 ( 3:1 ) mixtures destroyed or cracked all cnts or graphitic layers such that the encapsulated fe catalysts could be extracted , whereas the hclo4/f - hno3 ( 3:1 ) mixture totally destroyed all carbon materials . hence , the four mixtures tested are almost equally powerful for fe extraction from cnts , but hclo4/f - hno3 ( 3:1 ) is superior to the others in consuming the carbon materials during reflux . in concentrated nitric acid , nitronium ions form through equilibrium self - dissociation according to the following reaction : 2 hno3no2+no3+h2o.47 the formation of these particular ions can be accelerated by adding a strong acid such as sulfuric or perchloric acid.48 the reactions are hno3 + 2 h2so4no2+h3o+2 hso4 for sulfuric acid and hno3 + 2 hclo4no2+h3o+2 clo4 for perchloric acid.49 importantly , the experiment by gerber et al.46 revealed that more hno3 is dissociated to form more no2 ions if a greater quantity of its complementary acid is added . for all 11 reflux solutions listed in figure 3 b , the fe concentrations measured by uv / vis spectroscopy were in good agreement with those measured using icp - oes within an average difference of 6.0 % . therefore , the colorimetric method developed in this study using uv / vis spectroscopy appears to be a reliable tool for accurate measurement of fe concentrations in acid refluxed solutions of cnts . fe measurements were made three times for each cnt sample using uv / vis spectroscopy and icp - oes ( figure 4 g ) . fe concentrations measured by both methods closely match each other within the average differences of 1.66 and 0.38 % for cnt - b and -c , respectively , supporting that the uv / vis spectroscopic method is comparable to the widely used icp - oes technique in assessing the fe content of cnts . g ) fe concentrations measured by uv / vis spectroscopy and icp - oes for cnt - b and cnt - c . easy - to - use and widely available uv / vis spectroscopy was used to determine the concentration of fe catalysts in cnts by formation of colored complexes with the 1,10-phenanthroline ( phen ) ligand . this colorimetric method revealed a linear relationship between fe concentration and optical absorbance of the cnt refluxed solution . cnts were held at reflux in a variety of single and mixed acids to compare their capacity to extract fe from cnts , so that fe catalysts encapsulated by cnts or graphitic layers could be fully dissolved . with regard to the refluxed solutions , fe concentrations measured by uv / vis spectroscopy and icp - oes were in good agreement with each other , indicating that our colorimetric method using uv / vis spectroscopy is reliable and applicable to the assessment of fe content in cnts . the concentrated acids used to extract fe components from cnts and ashes were hydrochloric acid ( hcl , 35 % , daejung ) , nitric acid ( hno3 , 70 % , daejung ) , fumed nitric acid ( f - hno3 , 93 % , matsunoen ) , sulfuric acid ( h2so4 , 9598 % , sigma aldrich ) , and perchloric acid ( hclo4 , 70 % , sigma aldrich ) . a series of fe solutions was obtained by diluting the 500 ppm fe standard solution : 0 , 0.2 , 0.6 , 1 , 1.4 , and 1.8 ml standard solutions of 500 ppm were added to 100 ml volumetric flasks to produce calibration solutions with fe concentrations of 0 , 1 , 3 , 5 , 7 , and 9 ppm , respectively . to prepare solution samples for fe determination , the cnts ( 60 mg ) or ashes ( 15 mg ) were held at reflux with acid ( 10 ml ) in an oil bath maintained at 130 c . buffer solution , hydroxylamine solution , and o - phen solution were added , and distilled water was finally added to bring the volume to 50 ml , developing red orange colored solutions for spectroscopic measurement of fe content . the optical absorbance used to determine fe content was assessed not only based on the red orange colored fe phen complexes , but also by the reagents contained in the solution , such as cnts . raman spectroscopy ( renishaw system 3000 , laser =633 nm ) was used to characterize the crystalline nature and structural defects in cnts . inductively coupled plasma optical emission spectrometry ( icp - oes , perkinelmer optima 4300 dv ) was used to measure metal impurity content in the cnt or ash samples by examining the specific wavelengths of =238.204 and 396.153 nm , corresponding to fe and al , respectively . the absorbance values of the solutions prepared for fe determination were measured by uv / vis near infrared spectroscopy ( uv / vis - nir spectrophotometer , agilent , cary 5000 ) in the spectral range of =400600 nm . the peak absorbance value was recorded at =510 nm for the fe phen complexes . a fitting equation was obtained by plotting the peak absorbance values versus fe concentrations , and was used to determine the fe concentration for an unknown sample .

major drug discovery efforts for novel targeted therapies of various human malignancies focus on the human kinome . this is not surprising because protein kinases , being the largest family of enzymes in mammals , play pivotal roles in many cellular processes including proliferation , differentiation , maintenance , apoptosis , and human disease . indeed , modulation of phosphorylation signaling pathways by small molecules has demonstrated considerable clinical efficacy in the treatment of devastating disorders such as cancer . the introduction of imatinib for the therapy of chronic myelogenous leukemia ( cml ) and more recently the use of crizotinib in anaplastic lymphoma kinase ( alk ) dependent tumors constitute milestones in the development of modern therapeutics . however , as most kinase inhibitors compete with atp for binding to highly conserved residues in the enzyme s active site , achieving potent inhibition while preserving selectivity is an issue that substantially contributes to unwanted side effects and thus high failure rates in drug development programs . consequently , non - atp - mimetic kinase inhibitors that are anchored to more diverse regions of the atp binding site may result in more selective inhibitors . as natural compounds provide a rich source of bioactive chemical scaffolds , we recently investigated the total syntheses of the -carboline alkaloids bauerines a , b , and c. bauerine c ( 1 , chart 1 ) , isolated from the blue - green alga dichotrix baueriana , features a distinct 6,7-dichloroindole substructure and has been shown to exhibit both antiproliferative and antiviral properties . in addition , related 1-oxo- -carbolines such as 2 ( chart 1 ) have been reported as ret kinase inhibitors and it has been suggested those compounds are able to interact with hinge backbone residues in a canonical way via the pyridone nh and carbonyl , respectively . recently , we reported a novel cdc2-like - kinase ( clk ) inhibitor , kh - cb19 ( 3 ) , which we designed based on the peculiar 6,7-dichloroindole motif of natural compound 1 ( chart 1 ) . because of methylation at the indole nitrogen , instead , cocrystal structures of 3 with clk3 revealed an unusual , non - atp mimetic binding mode . selectivity screening against a comprehensive panel of kinases showed that the inhibitor is highly specific for clk kinases , allowing pharmacologic modulation of alternative pre - mrna splicing . notably , we found that compound 3 forms an unusual halogen bond between the 6-chloro substituent and kinase hinge residues . although those interactions are considered weak in comparison to classical hydrogen bonds , they benefit from a lower desolvation penalty and may very well contribute to inhibitor specificity . other well - known examples of carbonyl our observations with our novel inhibitor 3 prompted us to further explore the chemical and biological space around this scaffold , focusing on diversely functionalized derivatives of alkaloid 1 , e.g. , 3-aminobauerine c ( 4 ) ( chart 1 ) , and in particular substitution patterns that will not allow canonical atp - mimetic hydrogen bonds with the kinase hinge backbone . we hypothesized that derivatives of 1 either bearing bulky substituents at position 3 or genuine tetracyclic spiro analogues will most likely prevent the classical atp - mimetic binding mode mediated by the pyridone nh and carbonyl group . in the course of these efforts we incidentally discovered a novel synthetic procedure for the preparation of 3-substituted 4-cyano-1,2,3,4-tetrahydro-1-oxo--carbolines . starting from substituted ethyl 3-cyanomethylindole-2-carboxylates , this approach allows for the convenient synthesis of highly substituted tricyclic ( 58 ) and tetracyclic spiro ( 911 ) analogues of bauerine c ( 1 ) as depicted in table 1 . to enable an in - depth investigation of sar , we expanded our initial set of compounds with a variety of structurally modified analogues 1624 . herein , we present the activity of this novel class of compounds as potent and specific inhibitors of a number of serine / threonine kinases including the disease - related kinases pim1 and dapk3 . dapk3 , also known as zip kinase ( zipk ) or dap - like kinase ( dlk ) , belongs to a family of kinases that are part of the camk group and that have been implicated in modulation of cell death signaling cascades . furthermore , it has been shown that dapk3 is involved in inflammatory processes and smooth muscle myosin phosphorylation , which suggests dapk3 as an attractive target for related disorders such as hypertension and asthma . cocrystal structures of optimized compounds with pim1 and dapk3 ( zipk ) revealed an atp - competitive but not atp - mimetic binding mode that lacks classical hydrogen bond interactions with the kinase hinge backbone . instead the chlorine atoms are oriented toward the hinge region and , similar to our clk inhibitor 3 , form in some cases halogen bonds with backbone carbonyls . because of their distinct binding modes , the screened inhibitors showed a limited number of cross reactivities with other kinases over a comprehensive kinase selectivity panel . furthermore , selected compounds display interesting differential kinase affinities , indicating that this scaffold may represent a versatile design template for the development of novel bmp2k ( bike ) , clk , and drak1 inhibitors which might find clinical applications outside the oncology target area alkaloid 1 was prepared starting from diazotated 2,3-dichloroaniline and 3-(ethoxycarbonyl)piperidone using a japp the initial set of 1-oxo--carbolines including 4 and compounds 511 ( table 1 ) was synthesized by reaction of substituted ethyl 3-cyanomethyl-1h - indole-2-carboxylates such as 12 with gaseous ammonia / ammonium chloride and the corresponding ketones following our previously reported procedure . on the basis of our hypothesis discussed above , four sections of the core scaffold were selected for further modifications in order to establish sar : ( i ) the pyridone ring of natural compound 1 , ( ii ) the spirocycle attached to position 3 , ( iii ) the 9-methyl substituent at the indole nitrogen , and ( iv ) the nitrile group at position 4 . for the preparation of 3-azabauerine c ( 16 ) in which the pyridone ring of alkaloid 1 is replaced by a pyridazinone , ethyl 6,7-dichloroindole-2-carboxylate ( 13 ) was alkylated with methyl iodide in dmf after deprotonation with sodium hydride to give n - methylated indole 14 ( scheme 1a ) . formylation of 14 using vilsmeier conditions afforded carbaldehyde 15 which was cyclized with hydrazine , yielding 3-azabauerine c ( 16 ) . reagents and conditions for part a : ( i ) nah , dmf , 40 c , 1 h , then ch3i , room temp , 5 h ; ( ii ) pocl3 , dmf , room temp , 40 min , then 60 c , 4 h ; ( iii ) hydrazine , 1,4-dioxane , reflux , 3 h. percentages given correspond to relative yields . reagents and conditions for part b : ( i ) nh3 , nh4cl , 100 c , 1 h , sealed tube ; ( ii ) tfa , ch2cl2 , room temp , 35 min . percentages given correspond to relative yields . to elucidate the effect of heteroatoms within the saturated spiro ring at c-3 of the -carboline core , nitrogen - containing n - methyl- and n - ethyl - spiropiperidine derivatives 17 and 18were prepared in the known manner using 12 and appropriate n - alkyl-4-piperidone building blocks ( scheme 1b ) . synthesis of n - unsubstituted analogue 20 was achieved via the n - boc - protected intermediate 19 by cyclization of 12 with n - boc-4-piperidone , followed by mild deprotection with tfa in dichloromethane . we decided to use the n - boc - protected intermediate , as the boc group should be stable toward the strong basic conditions applied during the cyclization step . furthermore the intermediate was considered to be of possible interest for sar because of the bulky substituent at the piperidine nitrogen . in order to address the importance of the 9-methyl substituent in the tetracyclic spiro series , 22 was prepared from 21 and cyclohexanone using our general procedure ( scheme 2a ) . by employment of triethylbenzylammonium chloride ( tebac ) mediated phase - transfer catalysis,22 can be alkylated with dimethyl sulfate and potassium carbonate in acetonitrile , yielding 9-methyl derivative 9 . this protocol allows for the selective alkylation of the indole nitrogen without affecting the lactam nitrogen and constitutes a feasible alternative to our previously reported procedure for selective n - methylation of 1-oxo--carbolines . to determine the importance of the cyano group at position 4 , we chose to prepare primary amine 23 from our original lead compound 5 . the reduction of nitriles in the presence of amides is usually quite challenging , since both carbon atoms are at the same oxidation level . furthermore , in the case of 5 , catalytic hydrogenation using raney nickel , palladium , or platinum on carbon catalysts is not promising because these methods bear the risk of removing halogen substituents from aromatic rings . initial attempts using sodium borohydride in combination with cobalt or nickel salts were unsuccessful , yet we were able to selectively reduce the cyano group of 5 with tetra - n - butylammonium borohydride ( scheme 2b ) . finally , amine 23 was acetylated with acetic anhydride in toluene to give acetamide 24 . reagents and conditions for part a : ( i ) cyclohexanone , nh3 , nh4cl , 100 c , 1 h , sealed tube ; ( ii ) tebac , k2co3 , ch3cn , room temp , 15 min , then 22 and ( ch3o)2so2 , room temp , 12 h. percentages given correspond to relative yields . reagents and conditions for part b : ( i ) bu4nbh4 , ch2cl2 , reflux , 24 h ; ( ii ) ac2o , toluene , 80 c , 1 h. percentages given correspond to relative yields . all compounds including alkaloid 1 were screened against a kinase panel using a thermal stability shift assay . this assay depicts a fast and reliable method to determine a ligand affinity ranking , and the obtained results correlate well with previously published kinetic data . kinase target hits for active compounds are summarized in table 2 , applying a tm shift value of 4.0 c as cutoff ( for complete tm shift screening results , see table s1 in supporting information ) . comparison of hits identified in the screen with the kinome tree revealed a distinct and specific pattern of kinase targets ( figure 1 ) . overall we found that compounds bearing a spiropiperidine moiety such as 17 , 18 , and 20 are highly potent inhibitors of pim1/3 and dapk3 , whereas bauerine c ( 1 ) and derivatives 4 and 16 as well as primary amine 23 exhibited exciting activities against so far largely unexplored kinases such as bmp2k and drak1 . kinome tree visualization of inhibitor selectivity for compound 20 combining both tm shift and kinetic assay results ( illustration reproduced courtesy of cell signaling technology , inc . targets with significant tm shift have been colored in red ( > 8 c ) and yellow ( > 4 c ) , respectively . from investigation of the sar around parent compound 1 a bit more closely and with particular regard to pim kinases , the tm shift assay revealed 3,3-dimethyl derivative 5 and 3-aminobauerine c ( 4 ) to exert reasonable affinity toward pim1 , both superior to bauerine c ( 1 ) ( table s1 , supporting information ) . notably , compound 6 , which does not have a methyl substituent at the indole nitrogen , and the halogen - free analogue 8 showed significantly reduced activity against pim1 in comparison to 5 , suggesting beneficial effects of both the 9-methyl and 7,8-dichloro substituents . prolongation of alkyl chains at position 3 as in 7 also led to weaker binding , whereas among the initial group of spiro compounds 9 , 10 , and 11 , expansion of ring size and introduction of heteroatoms to the saturated spirocarbocycle seemed to favor activity . remarkably , removal of the 9-methyl group in the spirocyclohexyl series as in 22 compared to 9 did not result in such a dramatic loss in activity as observed for the tricyclic compounds ( 5 vs 6 ) , probably indicating a different mode of binding . reduction of the nitrile group at c-4 again led to a slight decrease in affinity for primary amine 23 in comparison to lead compound 5 with regard to pim1 . n - acetylation of amine 23 to give acetamide 24 resulted in complete loss of activity . strikingly , investigation of the newly prepared spiropiperidine compounds revealed 17 , 18 , and 20 as highly potent inhibitors of pim1 kinase . the top compound in this series was found to be 20 , which is not substituted at the spiropiperidine nitrogen . introduction of an n - methyl substituent as in 17 was well - tolerated ; however , an n - ethyl group as in 18 resulted in marginally diminished activity . notably , n - boc - protected intermediate 19 did not exhibit any inhibitory potency . to further examine the selectivity of the newly identified inhibitors , kinase profiling using the thermal shift assay was extended for up to more than 100 kinases ( table s1 , supporting information ) . for 20 , we observed temperature shifts of > 8 c only for pim1 , pim3 , and dapk3 , emphasizing that compound 20 not only is a highly potent inhibitor of the pim family of kinases but also displays good selectivity . among the pim kinase family , tm shift data showed a significant lower tm shift for pim2 , suggesting lower activity of 20 on this target . similar selectivity patterns were detected for n - methyl- and n - ethylspiropiperidine derivatives 17 and 18 , yet these were slightly less potent against all three pim kinases and inhibition of dapk3 was significantly reduced . in general , both tri- and tetracyclic compounds exhibited a comparable trend in activity for pim1 and clk1 , yet the spiropiperidines 17 , 18 , and 20 did give considerably higher tm shift values for pim1 compared to clk1 . among the other compounds , especially 5 and 3-aminobauerine c ( 4 ) demonstrated reasonable activity against pim1 . interestingly , only parent alkaloid 1 and its closely related derivatives 4 and 16 as well as 5 inhibited tgf receptor kinase ( tgfbr2 ) with good potency . we also observed moderate activity for 1 , 4 , and 5 against drak1 , whereas 3-azabauerine c ( 16 ) seems to be quite selective for tgfbr2 . however , the most potent compound with regard to drak1 was found to be primary amine 23 . it is noteworthy that among all evaluated compounds only bauerine c ( 1 ) gave a tm shift of > 8 c for bmp2k . in order to revalidate the results obtained in the thermal shift assay , we also determined ic50 values for our most potent compound 20 . in a kinetic enzyme assay using 100 m atp , we determined ic50 values of 60 and 90 nm for pim1 and pim3 , respectively ( data not shown ) . to further corroborate the data obtained in the tm shift assay , we also screened our top inhibitor 20 against a comprehensive kinase selectivity panel ( table 3 ) . in general , the kinetic data correlated well with the results from the tm shift assay , although we identified camk2 and pka as additional targets that were inhibited by 20 with ic50 values below 100 nm . this may be related to the activation state of these two kinases , since inactive enzymes were used for tm screening . in summary , the main cross - reactivities of compound 20 are within the agc family , in particular with pka , rock , and pkn1/2 . outside the agc family however , in contrast to the clinically evaluated imidazo[1,2-b]pyridazine - based pim inhibitor sgi-1776 , compounds 17 and 20 did not exhibit any significant activity against pdgfr , flt3 , or kit . to gain insights on the binding mode and thus be able to rationalize sar , we first determined the crystal structures of 17 and 20 in complex with pim1 ( pdb codes 3cxw and 3cy2 ) ( figure 2a and supporting information figure s1 ) . the structures revealed that both inhibitors do not establish any classical hydrogen bond interactions with the hinge region . interestingly , the lactam moiety does not contribute to inhibitor binding , as the hydrophobic dichlorobenzene ring is oriented toward the hinge while the pyridone and spiropiperidine rings are pointing toward the back of the binding pocket . this allows formation of bidentate h - bonds between the spiropiperidine nitrogen and asn172 as well as asp186 . the observed binding mode for 17 and 20 is therefore in contrast to the one described for other 1-oxo--carbolines reported as cdk2 or ret inhibitors . as shown in figure 3 , compounds that are not substituted at positions 3 and 4 are likely to establish classical , atp - mimetic interactions between the pyridone ring and kinase hinge backbone residues . these can be mediated via formation of a hydrogen bond between the inhibitor s lactam carbonyl and the backbone nh of leu83 ( cdk2 numbering ) and a second one between the pyridone - nh and glu81 ( figure 3a ) . alternatively , all interactions can be established between the pyridone moiety and leu83 exclusively ( figure 3b ) . however , either of those binding modes is incompatible with 17 and 20 , probably because of the bulky nature of the spiro ring which prevents the novel inhibitors from assuming an atp - mimetic orientation . ( a ) crystal structure of 17 bound to pim1 ( pdb code 3cxw ) . ( b ) cocrystal structure of 17 in complex with human dapk3 ( pdb code 3bhy ) . ( c ) comparison of electron density maps of 17 bound to pim1 ( top ) or dapk3 ( bottom ) , respectively . ( d ) superimposition of hinge areas of pim1 and dapk3 structures with bound inhibitor 17 . in contrast to the spirocyclic compound 17 ( figure 2 ) main interactions are mediated between the inhibitor s lactam ring and kinase hinge backbone residues glu81 and leu83 ( a ) or leu83 exclusively ( b ) . looking back at our pim1 complex , we surprisingly found that the cyano group is not involved in a hydrogen bond interaction with lys67 , which is in contrast to comparably substituted 3-cyanopyridones reported as pim inhibitors by cheney et al . various other pim inhibitor scaffolds , e.g. , pyrrolo[2,3-a]carbazoles and 3h - benzothieno[3,2-d]pyrimidin-4-ones , also form hydrogen bonds with this highly conserved residue . the dfg motif assumes an active in conformation , and two hydrophobic residues , the gatekeeper leu120 and the dfg - adjacent ile185 , enclose the tricyclic core ring system ( not shown ) . as in several other reported pim inhibitor complexes,17 and 20 also induce a conformational change in the glycine - rich p - loop by which phe49 flips into the active site ( not shown ) . this structural rearrangement on the one hand enables additional stacking effects with the inhibitor but also shifts the kinase in an inactive conformation which is incompatible with substrate binding . regarding the difference in activity observed for different pim isoforms , despite the high sequence homology among all three isoforms , kinetic studies suggest that atp km values for pim1/pim3 and pim2 differ remarkably . this may explain the so far unsuccessful efforts to target all three kinases with an atp - competitive inhibitor . pim2 has a leucine residue instead of glutamate at position 124 in the hinge region , which disrupts the polar interactions with arg122 observed in pim1.as a result , the arginine side chain is disordered , which in combination with another amino acid change ( val126ala ) might influence hinge dynamics and hence inhibitor binding due to reduced availability of hydrophobic interactions and higher desolvation energy . in pim3 the glutamate residue at position 124 is conserved but like pim2 val126 is replaced by alanine which may cause slightly weaker binding of the inhibitors . interestingly , the dapk3 cocrystal structure with 17 ( pdb code 3bhy ) revealed a different mode of binding compared to the pim1 structure ( figure 2b ) . in the dapk3 complex , the inhibitor s lactam carbonyl establishes a hydrogen bond with a water molecule in the active site which itself is involved in a hydrogen bond network with ser21 ( not shown ) . the spiropiperidine ring appears a little more twisted compared to the pim1 structure ( figure 2c ) and does not seem to participate in any polar interactions . however , the electron density for the piperidine in the obtained cocrystal structure was weak , possibly indicating multiple conformations for this moiety . similar to pim1 , 17 benefits from hydrophobic interactions with the gatekeeper ( leu93 ) and an isoleucine ( ile160 ) residue n - frontal to the dfg motif ( not shown ) . more interestingly , in this structure we observed establishment of halogen bonds between the 7,8-dichloro substituents of 17 and the backbone carbonyl of glu94 and val96 , respectively ( figure 2d ) , similar to the interactions we had discovered for our recently reported novel clk inhibitor 3 ( pdb code 2vag ) . the expanded nature of the hinge region in pim1 due to insertion of two proline residues ( pro123 and pro125 ) prohibits formation of such a halogen bond as illustrated in figure 2d . in the dapk3 complex the halogen in position 8 was partially radiolyzed . additional electron density observed in the vicinity of the 7,8-dichloro substituents was interpreted as a chloride ion with partial occupancy . we also observed complete radiolysis of the c cl bond at this position in the data set collected on the pim1 complex with 20 ( pdb code 3cy2 , supporting information figure s1 ) . as aberrant expression of pim kinases has been observed for numerous types of cancer , e.g. , leukemia , lymphoma , and solid tumors such as prostate , colon , and pancreatic cancer , we evaluated the antiproliferative potential of our most potent inhibitor 20 and its effects on pim downstream signaling . for this purpose , 20 was screened against five human leukemia cell lines and ic50 values were determined after 48 h of treatment , showing low micromolar potency comparable to data previously reported for imidazo[1,2-b]pyridazine pim inhibitors ( table 4 ) . however , consistent with our observation that 20 is selective for pim1 with regard to flt3 in contrast to imidazo[1,2-b]pyridazine - based pim inhibitors , cytotoxic activity for cell lines harboring the flt3-itd mutation such as mv4;11 and molm13 was reduced . to further confirm the biological activity of our lead compound , we also tested 20 for its ability to inhibit phosphorylation of a known pim downstream target . using the human mv4;11 acute myeloid leukemia ( aml ) cell line , we found a dose - dependent decrease in phosphorylation of 4e - bp1 as a result of pim inhibition by 20 ( figure 4 ) . mv4;11 cells were incubated with increasing concentrations of 20 for indicated times and harvested , and protein extracts were separated by sds page . the effect of 20 on the pim endogenous target 4e - bp1 was followed by western blotting with the indicated phosphospecific antibody . membranes were stripped and reprobed with nonphosphospecific and anti - actin antibodies to check for equal loading . in this study we present substituted 7,8-dichloro-1-oxo--carbolines as a novel and versatile scaffold for the development of non - atp - mimetic kinase inhibitors . sars around the core scaffold which we derived from the natural compound bauerine c ( 1 ) were established , and optimized compounds of the 4-cyano-1,2,3,4-tetrahydro-1-oxo--carboline type such as the spiropiperidine 20 are highly potent inhibitors of the oncogenic pim kinases and dapk3 ( zipk ) . because of their unusual binding mode revealed by x - ray crystallography , the inhibitors display good specificity among a comprehensive kinase selectivity panel with only a few off - targets within and outside the agc family . upon binding to pim1 the inhibitors induce a conformational change in the p - loop , which shifts the kinase into an inactive state . the novel compounds also exhibit efficacy in cellular assays , as 20 was found to deplete pim - dependent phosphorylation of downstream effector proteins and demonstrated antiproliferative activity at low micromolar concentrations in related cell viability screens . importantly , we found an intriguing binding mode for the n - methyl analogue 17 in complex with dapk3 which revealed formation of halogen bonds with the kinase hinge region . these interactions may contribute to inhibitor selectivity and further underscore the interesting features and versatility of our 6,7-dichloroindole scaffold . because of active site similarities , this novel class of compounds may also serve well as template for the preparation of new clk inhibitors . moreover , activities observed for some of the other analogues presented herein indicate that selected compounds such as alkaloid 1 and primary amine 23 could also be optimized for the development of new targeted inhibitors of bmp2k ( bike ) and drak1 . consequently , efforts to further improve potency and selectivity of those compounds are under way . nmr spectra were recorded on a jeol jnmr - gsx 400 and a jeol jnmr - gsx 500 ( jeol , peabody , ma , u.s . ) , using tms as internal standard . chemical shifts are given in ppm , and coupling constants are given in hertz . mass spectra ( electronic ionization , ei , 70 ev ) were recorded using a hewlett - packard 5989 a mass spectrometer with a 59980 b particle beam lc / ms interface ( agilent technologies , palo alto , ca , u.s . ) . ir spectra were recorded as kbr disks on a perkin - elmer ft - ir paragon 1000 ( perkin - elmer , waltham , ma , u.s . ) or jasco ft / ir-410 ( jasco , easton , md , u.s . ) . melting points were determined with a bchi b-540 apparatus ( bchi , flawil , switzerland ) and are uncorrected . elemental analyses were performed using a chn - elementaranalysator rapid ( heraeus , hanau , germany ) or elementaranalysator vario el ( elementar , hanau , germany ) . purification by flash column chromatography ( fcc ) was done using silica gel 60 ( merck , darmstadt , germany ) . all chemicals were purchased from sigma - aldrich , fluka , and acros and used without further purification . bauerine c ( 1 ) was prepared as reported previously , and procedures and spectral data for compounds 412 and 21 are provided in ref ( 10 ) . for procedures and experimental data for compounds 14 and 15 as well as general additional spectroscopic data see supporting information . compound 15 ( 474 mg , 1.6 mmol ) and hydrazine hydrate ( 192 mg , 4.8 mmol ) were dissolved in 1,4-dioxane ( 50 ml ) , and the mixture was heated to reflux for 3 h. after the mixture was cooled , the crude precipitated product was filtered off and recrystallized from ethyl acetate to give 241 mg ( 56% ) of 16 as a yellow solid . mp 307 c ; h nmr ( 400 mhz , dmso - d6 ) 12.96 ( br s , 1 h , n h ) , 8.78 ( s , 1 h , 1-h ) , 8.19 ( d , j = 8.5 hz , 1 h , 9-h ) , 7.56 ( d , j = 8.5 hz , 1 h , 8-h ) , 4.63 ( s , 3 h , n ch3 ) ; ms ei m / z ( relative intensity , % ) 270 [ m + 4 ] ( 14 ) , 268 [ m + 2 ] ( 67 ) , 266 [ m ] ( 100 ) ( c11h7cl2n3o0.5h2o ) c , h , n. synthesis of compounds 1719 and 22 was achieved by using the previously reported procedure . briefly , the appropriate ethyl 3-(cyanomethyl)indole-2-carboxylate ( 12 or 21 , 3.21 mmol ) and ammonium chloride ( 6.42 mmol ) were suspended in the given amount of ketone in a glass tube . after the mixture was cooled to 80 c , gaseous ammonia was introduced into the tube until an amount of 10 ml was condensed . the tube was closed tightly and heated to 100 c for 16 h in an autoclave . the mixture was allowed to reach room temperature and after evaporation of excess ammonia extracted with ethyl acetate ( 3 30 ml ) . the combined organic layers were dried over sodium sulfate , filtered , and the solvent was removed . 17 was prepared from 12 ( 1.00 g , 3.21 mmol ) and n - methyl-4-piperidone ( 5 ml ) following the general procedure for preparation of spiro 4-cyano-1-oxo--carbolines . the crude product was recrystallized from ethanol to give 568 mg ( 47% ) of 17 as pale pink crystals . mp 284 c ; h nmr ( 400 mhz , dmso - d6 , 50 c ) 8.12 ( br s , 1 h , n h ) , 7.88 ( d , j = 8.6 hz , 1 h , 5-h ) , 7.43 ( d , j = 8.6 hz , 1 h , 6-h ) , 4.97 ( s , 1 h , 4-h ) , 4.46 ( s , 3 h , 9-ch3 ) , 2.66 ( m , 1 h , 2-/6-h ) , 2.47 ( m , 1 h , 2-/6-h ) , 2.33 ( br t , j = 5.5 hz , 2 h , 2-/6-h ) , 2.22 ( s , 3 h , 1-ch3 ) , 2.06 ( m , 1 h , 3-/5-h ) , 1.97 ( m , 1 h , 3-/5-h ) , 1.70 ( br t , j = 5.5 hz , 2 h , 3-/5-h ) ; ms ei m / z ( relative intensity , % ) 380 [ m + 4 ] ( 8) , 378 [ m + 2 ] ( 35 ) , 376 [ m ] ( 53 ) , 317 ( 100 ) , 236 ( 30 ) , 195 ( 23 ) . ( c18h18cl2n4o ) c , h , n. 18 was prepared from 12 ( 1.00 g , 3.21 mmol ) and n - ethyl-4-piperidone ( 5 ml ) following the general procedure for preparation of spiro 4-cyano-1-oxo--carbolines . the crude product was recrystallized from ethanol to give 914 mg ( 73% ) of 18 as white crystals . mp 276 c ; h nmr ( 500 mhz , dmso - d6 , 50 c ) 8.15 ( br s , 1 h , n h ) , 7.89 ( d , j = 8.5 hz , 1 h , 5-h ) , 7.43 ( d , j = 8.5 hz , 1 h , 6-h ) , 4.97 ( s , 1 h , 4-h ) , 4.46 ( s , 3 h , 9-ch3 ) , 2.70 ( m , 1 h , 2-/6-h ) , 2.48 ( m , 1 h , 2-/6-h ) , 2.38 ( q , j = 7.1 hz , 2 h , ch2ch3 ) , 2.36 ( br t , j = 5.5 hz , 2 h , 2-/6-h ) , 2.05 ( m , 1 h , 3-/5-h ) , 1.97 ( m , 1 h , 3-/5-h ) , 1.70 ( br t , j = 5.5 hz , 2 h , 3-/5-h ) , 1.00 ( t , j = 7.1 hz , 3 h , ch2ch3 ) ; ms ei m / z ( relative intensity , % ) 394 [ m + 4 ] ( 8) , 392 [ m + 2 ] ( 44 ) , 390 [ m ] ( 69 ) , 375 ( 18 ) , 317 ( 100 ) , 236 ( 21 ) , 195 ( 20 ) ( c19h20cl2n4o ) c , h , n. 19 was prepared from 12 ( 1.00 g , 3.21 mmol ) and n - boc-4-piperidone ( 4.50 g , 22.58 mmol ) following the general procedure for preparation of spiro-4-cyano-1-oxo--carbolines . the crude product was recrystallized from ethanol to give 722 mg ( 49% ) of 19 as white crystals . mp 250 c ; h nmr ( 500 mhz , cdcl3 , 50 c ) 7.58 ( d , j = 8.5 hz , 1 h , 5-h ) , 7.31 ( d , j = 8.5 hz , 1 h , 6-h ) , 6.28 ( br s , 1 h , n h ) , 4.55 ( s , 3 h , 9-ch3 ) , 4.15 ( s , 1 h , 4-h ) , 3.95 ( d , j = 14.3 hz , 1 h , 2-/6-h ) , 3.83 ( d , j = 14.0 hz , 1 h , 2-/6-h ) , 3.32 ( dd , j = 12.0 hz , 2.5 hz , 1 h , 2-/6-h ) , 3.23 ( dd , j = 11.8 hz , 2.0 hz , 1 h , 2-/6-h ) , 2.15 2.02 ( m , 2 h , 3-/5-h ) , 1.91 ( d , j = 14.0 hz , 1 h , 3-/5-h ) , 1.81 ( dd , j = 12.0 hz , 2.5 hz , 2 h , 3-/5-h ) , 1.47 ( s , 9 h , c ( ch3)3 ) ; ms ei m / z ( relative intensity , % ) 466 [ m + 4 ] ( 2 ) , 464 [ m + 2 ] ( 14 ) , 462 [ m ] ( 20 ) , 389 ( 31 ) , 362 ( 75 ) , 333 ( 32 ) , 317 ( 100 ) , 305 ( 88 ) , 264 ( 59 ) , 236 ( 54 ) , 195 ( 35 ) . anal . ( c22h24cl2n4o3 ) c , h , n. to a solution of 19 ( 270 mg , 0.58 mmol ) in dichloromethane ( 30 ml ) was added trifluoroacetic acid ( 7.5 ml ) , and the mixture was stirred at room temperature for 35 min . the volatile compounds were removed by azeotropic rotary evaporation with toluene ( 10 ml ) , and the residue was resuspended in 2 m naoh solution ( 10 ml ) . after extraction with ethyl acetate ( 3 10 ml ) , the combined organic layers were dried over mgso4 and the solvent was evaporated . the crude product was recrystallized from ethanol to give 124 mg ( 59% ) of 20 as white crystals . mp 289 c ; h nmr ( 500 mhz , dmso - d6 ) 8.32 ( br s , 1 h , n h ) , 7.91 ( d , j = 8.5 hz , 1 h , 5-h ) , 7.44 ( d , j = 8.5 hz , 1 h , 6-h ) , 5.08 ( s , 1 h , 4-h ) , 4.46 ( s , 3 h , 9-ch3 ) , 2.99 ( m , 1 h , 2-/6-h ) , 2.78 ( m , 1 h , 2-/6-h ) , 2.64 ( m , 2 h , 2-/6-h ) , 2.23 ( br s , 1 h , 1-h ) , 1.89 ( m , 2 h , 3-/5-h ) , 1.59 ( m , 2 h , 3-/5-h ) ; ms ei m / z ( relative intensity , % ) 366 [ m + 4 ] ( 4 ) , 364 [ m + 2 ] ( 22 ) , 362 [ m ] ( 36 ) , 317 ( 39 ) , 305 ( 100 ) , 236 ( 48 ) , 195 ( 25 ) . anal . ( c17h16cl2n4o ) c , h , n. 22 was prepared from 21(10 ) ( 1.00 g , 3.21 mmol ) and cyclohexanone ( 5 ml ) following the general procedure for preparation of spiro 4-cyano-1-oxo--carbolines . the crude product was recrystallized from ethanol to give 220 mg ( 19% ) of 22 as white crystals . mp 281 c ; h nmr ( 400 mhz , dmso - d6 ) 12.71 ( br s , 1 h , 9-h ) , 8.17 ( br s , 1 h , 2-h ) , 7.87 ( d , j = 8.3 hz , 1 h , 5-h ) , 7.41 ( d , j = 8.3 hz , 1 h , 6-h ) , 5.06 ( s , 1 h , 4-h ) , 1.951.35 ( m , 10 h , 2-/3-/4-/5-/6-h ) ; ms ei m / z ( relative intensity , % ) 351 [ m + 4 ] ( 16 ) , 349 [ m + 2 ] ( 62 ) , 347 [ m ] ( 100 ) , 222 ( 34 ) , 195 ( 43 ) . anal . ( c17h15cl2n3o ) c , h , n. triethylbenzylammonium chloride ( tebac ; 200 mg , 0.877 mmol ) and k2co3 ( 138 mg , 1.00 mmol ) were suspended in acetonitrile ( 30 ml ) , and the mixture was stirred for 15 min at room temperature . a solution of 22 ( 348 mg , 1.00 mmol ) in acetonitrile ( 20 ml ) was added , followed by slow addition of dimethyl sulfate ( 126 mg , 1.00 mmol ) , and the mixture was stirred at room temperature for 12 h. the solvent was removed by evaporation . the residue was redissolved in ethyl acetate ( 50 ml ) , and the mixture was washed with water ( 2 20 ml ) and brine ( 2 20 ml ) . the combined organic layers were dried over mgso4 , and the solvent was evaporated . the crude product was recrystallized from ethanol to give 202 mg ( 56% ) of 9 as white crystals . ( c18h17cl2n3o ) c , h , n. tetra - n - butylammonium borohydride ( 2.40 g , 9.31 mmol ) and 5 ( 1.00 g , 3.10 mmol ) were dissolved in dichloromethane ( 100 ml ) , and the mixture was refluxed for 24 h. after the mixture was cooled , the solvent was evaporated and the residue carefully resuspended in 2 m hcl solution ( 20 ml ) followed by refluxing for 30 min . the mixture was poured onto ice , basified with 2 m naoh , and extracted with ethyl acetate ( 3 10 ml ) . the combined organic layers were dried over mgso4 , and the solvent was evaporated . the crude product was purified by fcc ( eluent , dichloromethane / ethanol ( 5:1 ) ) . the product was recrystallized from toluene to give 400 mg ( 40% ) of 23 as white needles . mp 202 c ; h nmr ( 400 mhz , cd2cl2-d2 ) 7.54 ( d , j = 8.5 hz , 1 h , 5-h ) , 7.21 ( d , j = 8.5 hz , 1 h , 6-h ) , 5.47 ( br s , 1 h , 2-h ) , 4.53 ( s , 3 h , n ch3 ) , 3.08 ( dd , j = 17.3 hz , 8.0 hz , 1 h , ch2 ) , 2.94 ( dd , j = 17.3 hz , 8.0 hz , 1 h , ch2 ) , 2.93 ( t , j = 8.0 hz , 1 h , 4-h ) , 1.42 ( s , 3 h , 3-ch3 ) , 1.29 ( s , 3 h , 3-ch3 ) , 1.13 ( s , 2 h , nh2 ) ; ms ei m / z ( relative intensity , % ) 329 [ m + 4 ] ( 1 ) , 327 [ m + 2 ] ( 4 ) , 325 [ m ] ( 6 ) , 296 ( 44 ) , 281 ( 100 ) . anal . ( c15h17cl2n3o ) c , h , n. to a solution of 23 ( 388 mg , 1.19 mmol ) ) in toluene ( 50 ml ) was added acetic anhydride ( 123 mg , 1.20 mmol ) , and the mixture was stirred for 1 h at 80 c . after the mixture was cooled , the solvent was removed by rotary evaporation and the residue purified by fcc . mp 299 c ; h nmr ( 400 mhz , dmso - d6 ) 7.74 ( br t , j = 5.7 hz , 2 h , n h ) , 7.58 ( d , j = 8.6 hz , 1 h , 5-h ) , 7.32 ( d , j = 8.6 hz , 1 h , 6-h ) , 4.45 ( s , 3 h , n ch3 ) , 3.47 ( ddd , j = 13 hz , 7.0 hz , 5.7 hz , 1 h , ch2 ) , 3.12 ( br t , j = 7.0 hz , 1 h , 4-h ) , 2.97 ( ddd , j = 13 hz , 7.0 hz , 5.7 hz , 1 h , ch2 ) , 1.62 ( s , 3 h , co ch3 ) , 1.34 ( s , 3 h , 3-ch3 ) , 1.17 ( s , 3 h , 3-ch3 ) ; ms ei m / z ( relative intensity , % ) 371 [ m + 4 ] ( 1 ) , 369 [ m + 2 ] ( 7 ) , 367 [ m ] ( 11 ) , 308 ( 64 ) , 295 ( 100 ) , 281 ( 38 ) , 260 ( 33 ) . anal . ( c17h19cl2n3o2 ) c , h , n. pim1 was purified as described previously . dapk3 ( coding for residues v9 to g288 ) was cloned into the t7 expression vector pnic28-bsa4 and expressed as a tev ( tobacco etch virus protease ) cleavable n - terminal his6 fusion protein in the phage resistant strain bl21 ( de3 ) r3 co - transformed with a chloramphenicol resistant coexpression plasmid ( pcoex ) expressing -phosphatase . an amount of 1 ml of an overnight culture was used to inoculate 2 l of tb medium containing 50 g / ml kanamycin and 40 g / ml chloramphenicol . e. coli cells were grown in 2.5 l baffled flasks at 37 c until od600 reached 2.0 . the cultures were cooled to 25 c , and expression of dapk3 was induced by adding 0.5 mm iptg at an od600 of 2.2 . cells were resuspended in lysis buffer ( 50 mm hepes buffer , ph 7.5 , 500 mm nacl , 5% glycerol ) and lysed by sonication . the cell lysate was loaded onto a ni affinity , histrap , 5 ml column ( ge / amersham biosciences ) equilibrated in lysis buffer . the column was washed using 10 column volumes of 50 mm hepes buffer , ph 7.5 , 500 mm nacl , 20 mm imidazole , and 5% glycerol , and dapk3 was eluted using 50 mm hepes buffer , ph 7.5 , 500 mm nacl , 150 mm imidazole , and 5% glycerol . dtt was added to a final concentration of 5 mm , and the his - tag was removed by the addition of 200 g of tev protease ( 14 h at 4 c ) . the protein was loaded onto a hiload 16/60 superdex 200 prep grade , 120 ml column ( ge / amersham bioscience ) equilibrated in 50 mm hepes , ph 7.5 , 500 mm nacl , 5% glycerol , 5 mm dtt . after size exclusion chromatography the recombinant protein was more than 95% pure as judged by sds aliquots of the purified proteins were set up for crystallization using a mosquito crystallization robot ( ttp labtech , royston u.k . ) . coarse screens were typically set up onto greiner three - well plates using three different drop ratios of precipitant to protein per condition ( 100 + 50 nl , 75 + 75 nl , and 50 + 100 nl ) . initial hits were optimized further using greiner three - well plates and scaling up the drop sizes in steps . crystallizations were carried out using the sitting drop vapor diffusion method at 4 c . pim1 ( 6 mg / ml ) was concentrated in the presence of 17 m -carboline ( 17 or 20 ) and 17 m consensus peptide ( arkrrrhpsgppta - amide ) . crystals with 17 were grown at 4 c in 1.0 l sitting drops , mixing 0.75 l of the solution with 0.25 l of a reservoir solution containing 0.14 m sodium malonate , 0.07 m bis - tris propane , ph 7.5 , 14% peg 3350 , 7% ethylene glycol . crystals of pim1 with 20 were grown at 4 c in 1.0 l sitting drops , mixing 0.8 l of protein solution with 0.2 l of a reservoir solution containing 0.56 m sodium succinate , ph 7.0 . crystals of dapk3 in complex with 20 were grown by mixing 50 l of the protein ( 12.6 mg / ml in the presence of 1 mm 20 ) with 100 l of reservoir solution containing 0.1 m spg , ph 6.0 , 30.0% peg 1k . crystals were directly flash frozen in liquid nitrogen in the case of the dapk3 complex or were supplemented by 20% ethylene glycol ( in the case of the pim1 complexes ) and flash frozen . data were collected on an fr - e superbright source using an raxis iv plate detector at 1.542 ( pim1a/20 ) or at the swiss light source beamline px10 at 0.97912 ( pim1a/17 ) or 0.9807 ( dapk3/20 ) . indexing and integration were carried out using hkl2000 or mosflm , and scaling was performed with scalepack or scala . initial phases were calculated by molecular replacement with phaser using the known models of pim1 and dap . initial models were built by arp / warp , and building was completed manually with coot . thermal motions were analyzed using tlsmd , and hydrogen atoms were included in late refinement cycles . data collection and refinement statistics can be found in table s2 ( supporting information ) . the models and structure factors have been deposited with pdb accession codes 3cxw ( pim1/17 ) , 3cy2 ( pim1/20 ) , and 3bhy ( dapk3/17 ) . human leukemia cell lines mv4;11 , rs4;11 , molm13 , sem , and k562 were purchased from dsmz and maintained in rpmi 1640 with 10% fcs and 1% penicillin / streptomycin at 37 c in 5% co2 . to evaluate the effect on the cell proliferation , 20 ( 010 m ) was added to the culture medium and the cultures were incubated for 48 h. cell proliferation and viability were assayed using cell proliferation reagent wst-1 from roche diagnostics according to the manufacturer s instructions . cell survival was calculated as a percentage normalized to control cultures , and ic50 values were calculated using graphpad prism . mv4;11 cells were grown at 5 10/ml , treated with 20 at 010 m for various times , harvested , and rinsed with ice - cold pbs . ice - cold lysis buffer [ 10 mm tris - hcl ( ph 7.4 ) , 150 mm nacl , 1% triton x-100 , 0.5 mm edta , 10% glycerol , 10 mm naf , 1 mm na3vo4 , protease inhibitor cocktail ] was added to the cells , and the mixture was incubated for 20 min on ice followed by 15 min of centrifugation at 12000 g . cleared lysates were assayed for protein concentration using the bradford protein assay system ( bio - rad ) . an amount of 50 g of protein was subjected to 15% sds page and transferred onto a pvdf membrane . membranes were blotted with primary antibodies ( diluted according to the manufacturer s recommendations ) , followed by horseradish peroxidase - conjugated secondary antibodies , and the proteins were detected by supersignal west femto maximum sensitivity substrate ( pierce ) . the same blots were stripped and reprobed with desired antibodies to confirm equal loading with the following antibodies : anti - phospho-4e - bp1 , anti-4e - bp1 ( cell signaling technology ) , and anti--actin ( sigma ) .

development of both potent and selective kinase inhibitors is a challenging task in modern drug discovery . the innate promiscuity of kinase inhibitors largely results from atp - mimetic binding to the kinase hinge region . we present a novel class of substituted 7,8-dichloro-1-oxo--carbolines based on the distinct structural features of the alkaloid bauerine c whose kinase inhibitory activity does not rely on canonical atp - mimetic hinge interactions . intriguingly , cocrystal structures revealed an unexpected inverted binding mode and the presence of halogen bonds with kinase backbone residues . the compounds exhibit excellent selectivity over a comprehensive panel of human protein kinases while inhibiting selected kinases such as the oncogenic pim1 at low nanomolar concentrations . together , our biochemical and structural data suggest that this scaffold may serve as a valuable template for the design and development of specific inhibitors of various kinases including the pim family of kinases , clks , dapk3 ( zipk ) , bmp2k ( bike ) , and others .

Introduction Results and Discussion Conclusion Experimental Section

major drug discovery efforts for novel targeted therapies of various human malignancies focus on the human kinome . this is not surprising because protein kinases , being the largest family of enzymes in mammals , play pivotal roles in many cellular processes including proliferation , differentiation , maintenance , apoptosis , and human disease . the introduction of imatinib for the therapy of chronic myelogenous leukemia ( cml ) and more recently the use of crizotinib in anaplastic lymphoma kinase ( alk ) dependent tumors constitute milestones in the development of modern therapeutics . however , as most kinase inhibitors compete with atp for binding to highly conserved residues in the enzyme s active site , achieving potent inhibition while preserving selectivity is an issue that substantially contributes to unwanted side effects and thus high failure rates in drug development programs . consequently , non - atp - mimetic kinase inhibitors that are anchored to more diverse regions of the atp binding site may result in more selective inhibitors . as natural compounds provide a rich source of bioactive chemical scaffolds , we recently investigated the total syntheses of the -carboline alkaloids bauerines a , b , and c. bauerine c ( 1 , chart 1 ) , isolated from the blue - green alga dichotrix baueriana , features a distinct 6,7-dichloroindole substructure and has been shown to exhibit both antiproliferative and antiviral properties . in addition , related 1-oxo- -carbolines such as 2 ( chart 1 ) have been reported as ret kinase inhibitors and it has been suggested those compounds are able to interact with hinge backbone residues in a canonical way via the pyridone nh and carbonyl , respectively . recently , we reported a novel cdc2-like - kinase ( clk ) inhibitor , kh - cb19 ( 3 ) , which we designed based on the peculiar 6,7-dichloroindole motif of natural compound 1 ( chart 1 ) . because of methylation at the indole nitrogen , instead , cocrystal structures of 3 with clk3 revealed an unusual , non - atp mimetic binding mode . selectivity screening against a comprehensive panel of kinases showed that the inhibitor is highly specific for clk kinases , allowing pharmacologic modulation of alternative pre - mrna splicing . , 3-aminobauerine c ( 4 ) ( chart 1 ) , and in particular substitution patterns that will not allow canonical atp - mimetic hydrogen bonds with the kinase hinge backbone . we hypothesized that derivatives of 1 either bearing bulky substituents at position 3 or genuine tetracyclic spiro analogues will most likely prevent the classical atp - mimetic binding mode mediated by the pyridone nh and carbonyl group . in the course of these efforts we incidentally discovered a novel synthetic procedure for the preparation of 3-substituted 4-cyano-1,2,3,4-tetrahydro-1-oxo--carbolines . herein , we present the activity of this novel class of compounds as potent and specific inhibitors of a number of serine / threonine kinases including the disease - related kinases pim1 and dapk3 . dapk3 , also known as zip kinase ( zipk ) or dap - like kinase ( dlk ) , belongs to a family of kinases that are part of the camk group and that have been implicated in modulation of cell death signaling cascades . cocrystal structures of optimized compounds with pim1 and dapk3 ( zipk ) revealed an atp - competitive but not atp - mimetic binding mode that lacks classical hydrogen bond interactions with the kinase hinge backbone . instead the chlorine atoms are oriented toward the hinge region and , similar to our clk inhibitor 3 , form in some cases halogen bonds with backbone carbonyls . because of their distinct binding modes , the screened inhibitors showed a limited number of cross reactivities with other kinases over a comprehensive kinase selectivity panel . furthermore , selected compounds display interesting differential kinase affinities , indicating that this scaffold may represent a versatile design template for the development of novel bmp2k ( bike ) , clk , and drak1 inhibitors which might find clinical applications outside the oncology target area alkaloid 1 was prepared starting from diazotated 2,3-dichloroaniline and 3-(ethoxycarbonyl)piperidone using a japp the initial set of 1-oxo--carbolines including 4 and compounds 511 ( table 1 ) was synthesized by reaction of substituted ethyl 3-cyanomethyl-1h - indole-2-carboxylates such as 12 with gaseous ammonia / ammonium chloride and the corresponding ketones following our previously reported procedure . on the basis of our hypothesis discussed above , four sections of the core scaffold were selected for further modifications in order to establish sar : ( i ) the pyridone ring of natural compound 1 , ( ii ) the spirocycle attached to position 3 , ( iii ) the 9-methyl substituent at the indole nitrogen , and ( iv ) the nitrile group at position 4 . this protocol allows for the selective alkylation of the indole nitrogen without affecting the lactam nitrogen and constitutes a feasible alternative to our previously reported procedure for selective n - methylation of 1-oxo--carbolines . the reduction of nitriles in the presence of amides is usually quite challenging , since both carbon atoms are at the same oxidation level . this assay depicts a fast and reliable method to determine a ligand affinity ranking , and the obtained results correlate well with previously published kinetic data . overall we found that compounds bearing a spiropiperidine moiety such as 17 , 18 , and 20 are highly potent inhibitors of pim1/3 and dapk3 , whereas bauerine c ( 1 ) and derivatives 4 and 16 as well as primary amine 23 exhibited exciting activities against so far largely unexplored kinases such as bmp2k and drak1 . from investigation of the sar around parent compound 1 a bit more closely and with particular regard to pim kinases , the tm shift assay revealed 3,3-dimethyl derivative 5 and 3-aminobauerine c ( 4 ) to exert reasonable affinity toward pim1 , both superior to bauerine c ( 1 ) ( table s1 , supporting information ) . notably , compound 6 , which does not have a methyl substituent at the indole nitrogen , and the halogen - free analogue 8 showed significantly reduced activity against pim1 in comparison to 5 , suggesting beneficial effects of both the 9-methyl and 7,8-dichloro substituents . prolongation of alkyl chains at position 3 as in 7 also led to weaker binding , whereas among the initial group of spiro compounds 9 , 10 , and 11 , expansion of ring size and introduction of heteroatoms to the saturated spirocarbocycle seemed to favor activity . remarkably , removal of the 9-methyl group in the spirocyclohexyl series as in 22 compared to 9 did not result in such a dramatic loss in activity as observed for the tricyclic compounds ( 5 vs 6 ) , probably indicating a different mode of binding . strikingly , investigation of the newly prepared spiropiperidine compounds revealed 17 , 18 , and 20 as highly potent inhibitors of pim1 kinase . for 20 , we observed temperature shifts of > 8 c only for pim1 , pim3 , and dapk3 , emphasizing that compound 20 not only is a highly potent inhibitor of the pim family of kinases but also displays good selectivity . this may be related to the activation state of these two kinases , since inactive enzymes were used for tm screening . to gain insights on the binding mode and thus be able to rationalize sar , we first determined the crystal structures of 17 and 20 in complex with pim1 ( pdb codes 3cxw and 3cy2 ) ( figure 2a and supporting information figure s1 ) . the structures revealed that both inhibitors do not establish any classical hydrogen bond interactions with the hinge region . interestingly , the lactam moiety does not contribute to inhibitor binding , as the hydrophobic dichlorobenzene ring is oriented toward the hinge while the pyridone and spiropiperidine rings are pointing toward the back of the binding pocket . as shown in figure 3 , compounds that are not substituted at positions 3 and 4 are likely to establish classical , atp - mimetic interactions between the pyridone ring and kinase hinge backbone residues . however , either of those binding modes is incompatible with 17 and 20 , probably because of the bulky nature of the spiro ring which prevents the novel inhibitors from assuming an atp - mimetic orientation . ( c ) comparison of electron density maps of 17 bound to pim1 ( top ) or dapk3 ( bottom ) , respectively . in contrast to the spirocyclic compound 17 ( figure 2 ) main interactions are mediated between the inhibitor s lactam ring and kinase hinge backbone residues glu81 and leu83 ( a ) or leu83 exclusively ( b ) . this structural rearrangement on the one hand enables additional stacking effects with the inhibitor but also shifts the kinase in an inactive conformation which is incompatible with substrate binding . pim2 has a leucine residue instead of glutamate at position 124 in the hinge region , which disrupts the polar interactions with arg122 observed in pim1.as a result , the arginine side chain is disordered , which in combination with another amino acid change ( val126ala ) might influence hinge dynamics and hence inhibitor binding due to reduced availability of hydrophobic interactions and higher desolvation energy . the spiropiperidine ring appears a little more twisted compared to the pim1 structure ( figure 2c ) and does not seem to participate in any polar interactions . more interestingly , in this structure we observed establishment of halogen bonds between the 7,8-dichloro substituents of 17 and the backbone carbonyl of glu94 and val96 , respectively ( figure 2d ) , similar to the interactions we had discovered for our recently reported novel clk inhibitor 3 ( pdb code 2vag ) . we also observed complete radiolysis of the c cl bond at this position in the data set collected on the pim1 complex with 20 ( pdb code 3cy2 , supporting information figure s1 ) . , leukemia , lymphoma , and solid tumors such as prostate , colon , and pancreatic cancer , we evaluated the antiproliferative potential of our most potent inhibitor 20 and its effects on pim downstream signaling . in this study we present substituted 7,8-dichloro-1-oxo--carbolines as a novel and versatile scaffold for the development of non - atp - mimetic kinase inhibitors . sars around the core scaffold which we derived from the natural compound bauerine c ( 1 ) were established , and optimized compounds of the 4-cyano-1,2,3,4-tetrahydro-1-oxo--carboline type such as the spiropiperidine 20 are highly potent inhibitors of the oncogenic pim kinases and dapk3 ( zipk ) . because of their unusual binding mode revealed by x - ray crystallography , the inhibitors display good specificity among a comprehensive kinase selectivity panel with only a few off - targets within and outside the agc family . upon binding to pim1 the inhibitors induce a conformational change in the p - loop , which shifts the kinase into an inactive state . importantly , we found an intriguing binding mode for the n - methyl analogue 17 in complex with dapk3 which revealed formation of halogen bonds with the kinase hinge region . because of active site similarities , this novel class of compounds may also serve well as template for the preparation of new clk inhibitors . moreover , activities observed for some of the other analogues presented herein indicate that selected compounds such as alkaloid 1 and primary amine 23 could also be optimized for the development of new targeted inhibitors of bmp2k ( bike ) and drak1 . compound 15 ( 474 mg , 1.6 mmol ) and hydrazine hydrate ( 192 mg , 4.8 mmol ) were dissolved in 1,4-dioxane ( 50 ml ) , and the mixture was heated to reflux for 3 h. after the mixture was cooled , the crude precipitated product was filtered off and recrystallized from ethyl acetate to give 241 mg ( 56% ) of 16 as a yellow solid . the combined organic layers were dried over sodium sulfate , filtered , and the solvent was removed . ( c22h24cl2n4o3 ) c , h , n. to a solution of 19 ( 270 mg , 0.58 mmol ) in dichloromethane ( 30 ml ) was added trifluoroacetic acid ( 7.5 ml ) , and the mixture was stirred at room temperature for 35 min . the volatile compounds were removed by azeotropic rotary evaporation with toluene ( 10 ml ) , and the residue was resuspended in 2 m naoh solution ( 10 ml ) . after extraction with ethyl acetate ( 3 10 ml ) , the combined organic layers were dried over mgso4 and the solvent was evaporated . ( c17h15cl2n3o ) c , h , n. triethylbenzylammonium chloride ( tebac ; 200 mg , 0.877 mmol ) and k2co3 ( 138 mg , 1.00 mmol ) were suspended in acetonitrile ( 30 ml ) , and the mixture was stirred for 15 min at room temperature . a solution of 22 ( 348 mg , 1.00 mmol ) in acetonitrile ( 20 ml ) was added , followed by slow addition of dimethyl sulfate ( 126 mg , 1.00 mmol ) , and the mixture was stirred at room temperature for 12 h. the solvent was removed by evaporation . the residue was redissolved in ethyl acetate ( 50 ml ) , and the mixture was washed with water ( 2 20 ml ) and brine ( 2 20 ml ) . ( c18h17cl2n3o ) c , h , n. tetra - n - butylammonium borohydride ( 2.40 g , 9.31 mmol ) and 5 ( 1.00 g , 3.10 mmol ) were dissolved in dichloromethane ( 100 ml ) , and the mixture was refluxed for 24 h. after the mixture was cooled , the solvent was evaporated and the residue carefully resuspended in 2 m hcl solution ( 20 ml ) followed by refluxing for 30 min . the combined organic layers were dried over mgso4 , and the solvent was evaporated . ( c15h17cl2n3o ) c , h , n. to a solution of 23 ( 388 mg , 1.19 mmol ) ) in toluene ( 50 ml ) was added acetic anhydride ( 123 mg , 1.20 mmol ) , and the mixture was stirred for 1 h at 80 c . dapk3 ( coding for residues v9 to g288 ) was cloned into the t7 expression vector pnic28-bsa4 and expressed as a tev ( tobacco etch virus protease ) cleavable n - terminal his6 fusion protein in the phage resistant strain bl21 ( de3 ) r3 co - transformed with a chloramphenicol resistant coexpression plasmid ( pcoex ) expressing -phosphatase . dtt was added to a final concentration of 5 mm , and the his - tag was removed by the addition of 200 g of tev protease ( 14 h at 4 c ) . pim1 ( 6 mg / ml ) was concentrated in the presence of 17 m -carboline ( 17 or 20 ) and 17 m consensus peptide ( arkrrrhpsgppta - amide ) . crystals of dapk3 in complex with 20 were grown by mixing 50 l of the protein ( 12.6 mg / ml in the presence of 1 mm 20 ) with 100 l of reservoir solution containing 0.1 m spg , ph 6.0 , 30.0% peg 1k . to evaluate the effect on the cell proliferation , 20 ( 010 m ) was added to the culture medium and the cultures were incubated for 48 h. cell proliferation and viability were assayed using cell proliferation reagent wst-1 from roche diagnostics according to the manufacturer s instructions . cell survival was calculated as a percentage normalized to control cultures , and ic50 values were calculated using graphpad prism . ice - cold lysis buffer [ 10 mm tris - hcl ( ph 7.4 ) , 150 mm nacl , 1% triton x-100 , 0.5 mm edta , 10% glycerol , 10 mm naf , 1 mm na3vo4 , protease inhibitor cocktail ] was added to the cells , and the mixture was incubated for 20 min on ice followed by 15 min of centrifugation at 12000 g . membranes were blotted with primary antibodies ( diluted according to the manufacturer s recommendations ) , followed by horseradish peroxidase - conjugated secondary antibodies , and the proteins were detected by supersignal west femto maximum sensitivity substrate ( pierce ) . the same blots were stripped and reprobed with desired antibodies to confirm equal loading with the following antibodies : anti - phospho-4e - bp1 , anti-4e - bp1 ( cell signaling technology ) , and anti--actin ( sigma ) .

in the eyes of several marine mollusks , a distinct class of photoreceptors is found , with properties that markedly diverge from those of classical invertebrate visual receptors : the light - sensing structure is ciliary , rather than microvillar ( miller , 1958 ; barber et al . , 1967 ) , the receptor potential is hyperpolarizing ( mcreynolds and gorman , 1970 ; mpitsos , 1973 ) , and the transduction cascade involves mobilization of cgmp instead of signaling via the phosphoinositide pathway ( gomez and nasi , 1995 ) . the ion channels that underlie the photocurrent in these sensory cells have been characterized ( gomez and nasi , 1994a ) , and are of special interest because they share functional properties with two superfamilies of ion channel proteins believed to have evolved from a common ancestor , namely , cyclic nucleotide gated channels ( cngcs ) and voltage - dependent potassium channels ( guy et al . , 1991 ; kaupp , 1991 ; yau , 1994 ) . on the one hand , they are activated by cgmp and , like other members of the cngc family , are blocked by the organic antagonists l - cis - diltiazem and 3,4-dichlorobenzamil ( gomez and nasi , 1997 ) and exhibit a pronounced outward rectification arising from voltage - dependent blockade by extracellular ca and mg ( nasi and gomez , 1999 ) . on the other hand , ; gomez and nasi , 1994a ) , and they are susceptible to blockade by k channel antagonists , especially 4-aminopyridine ( 4-ap ) ( gomez and nasi , 1994b ) . as such , these channels seemingly bridge the gap between these two canonical classes of proteins . the evolutionary kinship of cngcs to voltage - dependent channels naturally raises the question of how a chemically controlled mechanism for channel opening was acquired , and whether the gating structures implicated are homologous to those operated by transmembrane potential in their ancestral cousins . in voltage - dependent potassium channels , seminal experiments by armstrong ( 1971 ) on pore - blocker accessibility had indicated that the main gate seems to be located near the inner vestibule of the permeation pathway , a notion subsequently corroborated in cloned shaker channels using cysteine scanning mutagenesis ( liu et al . , 1997 ; holmgren et al . , 1998 this structural arrangement is consistent with more recent crystallographic data on a voltage - gated bacterial k channel ( jiang et al . , 2003 ) , which show a constriction formed by bundling together of multiple -helices near the intracellular side , and has been suggested to be a design feature of considerable generality , though subject to some variations ( for review see swartz , 2004 ) . in addition , other structural motifs participate in modulating ionic fluxes through the pore of voltage - gated channels : these include a cytosolically located tethered ( armstrong et al . , 1973 ) , comprising the initial stretch of residues at the amino terminus of the polypeptide that mediates the rapid inactivation observed in a variety of channels known as n - type inactivation ( hoshi et al . , moreover , molecular motions involving residues in the pore loop can collapse the external entrance to the pore and quench ionic current , and are responsible for the slower c - type inactivation ( liu et al . , 1996 ) . a tantalizing scheme has been proposed for the gating mechanism of the photocurrent in ciliary photoreceptors ( shimatani and katagiri , 1995 ) : the light - sensitive k conductance would be comprised of transient voltage - gated k channels akin to ia , and illumination would generate the photoresponse by removing their steady - state inactivation . the seminal observation was that membrane depolarization in the dark elicits an inactivating outward current , but if a similar voltage stimulation is applied during illumination the current becomes sustained . moreover , application of 4-ap at millimolar concentrations suppresses both the depolarization - activated current and the photocurrent ( shimatani and katagiri , 1995 ) . the appeal of this conjecture is twofold : first , the explicit identity of the allosteric transitions triggered by the internal messenger for light transduction and of the proposed moving parts of the gating machinery ; second , the evolutionary thread linking homologous functions in distantly related ion channels that have come to subserve entirely disparate functions . in the present report we examined whether a common signaling pathway is responsible for the modulation of the voltage - gated currents and the activation of the light - dependent current , and addressed in a systematic way the hypothesis that the same population of ion channels is implicated in the two cases . preliminary aspects of this work were previously presented in abstract form ( gomez and nasi , 2000b ) . pecten irradians ( bay scallop ) were obtained from the aquatic resources division of the marine biological laboratory . the techniques for enzymatically isolating viable ciliary photoreceptors and performing whole - cell patch - clamp recording have been described in detail previously ( gomez and nasi , 1994a ) . cells plated in a flow chamber were continuously perfused with artificial sea water ( asw ) containing ( in mm ) 480 nacl , 10 kcl , 10 cacl2 , 49 mgcl2 , 10 hepes , 5.5 d - glucose , ph 7.75 . intracellular solution used to fill thin - wall borosilicate patch pipettes contained 100 mm kcl , 200 mm k - glutamate , 22 mm nacl , 5 mm mg atp , 10 mm hepes , 1 mm egta , 100 m gtp , and 300 mm sucrose , ph 7.3 ( electrode resistance 24 m , in asw ) . series resistance was routinely compensated , and current signals were low - pass filtered at 1.52 khz ( 3 db ) with a bessel 4-pole filter , before digitizing at 35 khz sampling rate with 12-bit resolution . the slowly hydrolyzable cyclic nucleotide analogue 8-bromo cyclic guanosine monophosphate ( 8-br - cgmp ) was purchased from alexis and from sigma - aldrich . apamin , guanosine 5-o-[3-thiotriphosphate ] ( gtp--s ) , and 4-ap ( > 99% pure ) were from sigma - aldrich . intracellular application of test substances was performed by dissolving them in the internal solution and dialyzing them via the patch pipette . rapid extracellular application entailed pressure ejection from a glass micropipette ( 34 m tip diameter ) positioned 50 m from the target cell . the puffer pipette was connected to a solenoid - operated valve and a precision regulator to apply pressurized nitrogen . alternatively , the solution in the entire recording chamber was exchanged with a flowthrough system . the optical stimulators consisted of a 100-w tungsten - halogen light source ( oriel corporation ) , a condenser , an infrared absorbing filter , an electromechanical shutter ( vincent associates ) , and collimating and field lenses and filters . an adjustable pinhole or an iris diaphragm placed in a conjugated image plane restricted the illuminated region on the recording chamber to a disc 200 m in diameter . the output beam was combined with that of the microscope illuminator via a beam splitter prism placed above the condenser , as previously described ( nasi , 1991 ) . broadband light was used in all the experiments ( 515670 nm ) , determined the combination of a heat - absorbing filter and an edge filter ( schott glass technologies ) interposed in the light path . effective light intensity was calibrated in vivo by matching the photocurrent amplitude to that obtained with monochromatic light ( 500 nm , 3-cavity interference filter ; ditric optics ) , the intensity of which was measured with a radiometer ( udt hawthorne ; model 370 ) . light intensity is expressed in terms of equivalent photon flux at 500 nm ( gomez and nasi , 1994a , 1995 ) . calibrated neutral - density filters ( melles griot ) provided controlled light attenuation . during experimental manipulations , the cells were viewed with a newvicon tv camera ( model wv-1550 ; panasonic ) using a near - infrared long - pass filter for illumination ( > 780 nm ; andover corporation ) . the slowly hydrolyzable cyclic nucleotide analogue 8-bromo cyclic guanosine monophosphate ( 8-br - cgmp ) was purchased from alexis and from sigma - aldrich . apamin , guanosine 5-o-[3-thiotriphosphate ] ( gtp--s ) , and 4-ap ( > 99% pure ) were from sigma - aldrich . intracellular application of test substances was performed by dissolving them in the internal solution and dialyzing them via the patch pipette . rapid extracellular application entailed pressure ejection from a glass micropipette ( 34 m tip diameter ) positioned 50 m from the target cell . the puffer pipette was connected to a solenoid - operated valve and a precision regulator to apply pressurized nitrogen . alternatively , the solution in the entire recording chamber was exchanged with a flowthrough system . the optical stimulators consisted of a 100-w tungsten - halogen light source ( oriel corporation ) , a condenser , an infrared absorbing filter , an electromechanical shutter ( vincent associates ) , and collimating and field lenses and filters . an adjustable pinhole or an iris diaphragm placed in a conjugated image plane restricted the illuminated region on the recording chamber to a disc 200 m in diameter . the output beam was combined with that of the microscope illuminator via a beam splitter prism placed above the condenser , as previously described ( nasi , 1991 ) . broadband light was used in all the experiments ( 515670 nm ) , determined the combination of a heat - absorbing filter and an edge filter ( schott glass technologies ) interposed in the light path . effective light intensity was calibrated in vivo by matching the photocurrent amplitude to that obtained with monochromatic light ( 500 nm , 3-cavity interference filter ; ditric optics ) , the intensity of which was measured with a radiometer ( udt hawthorne ; model 370 ) . light intensity is expressed in terms of equivalent photon flux at 500 nm ( gomez and nasi , 1994a , 1995 ) . calibrated neutral - density filters ( melles griot ) provided controlled light attenuation . during experimental manipulations , the cells were viewed with a newvicon tv camera ( model wv-1550 ; panasonic ) using a near - infrared long - pass filter for illumination ( > 780 nm ; andover corporation ) . we first set out to replicate in our experimental system ( namely , enzymatically isolated photoreceptors from pecten irradians ) the results that shimatani and katagiri ( 1995 ) had obtained in the intact retina of the japanese scallop patinopecten yessoensis . dark - adapted ciliary pecten photoreceptors also exhibit a prominent voltage - activated outward current , a component of which ( from now on referred to , noncommittally , as ik ) exhibits a distinct inactivation , as well as a sustained component . 1 a ( left ) illustrates a typical family of traces evoked by 100-ms - long depolarizing steps at 10-mv increments , delivered in the dark from a holding potential of 70 mv . the current peaks in 510 ms and decays to a plateau with a time constant in the range of 1525 ms , both parameters inversely related to the size of the depolarizing voltage step . repetition of the same stimulation protocol during application of a steady background light of near - saturating intensity ( 3.47 10 photonscms ) results in currents completely devoid of decay kinetics , presenting instead a near - rectangular appearance ( fig . 1 a , middle ) ; after a 1-min period of dark adaptation , the original time course was fully restored ( right ) . the phenomenon proved highly reproducible ( n = 17 ) . a simple criterion to compare quantitatively the extent of the falling phase of the response in the dark vs. in the light is the ratio plateau : peak of the current , measured at some fixed depolarization within the range that produces a prominent current decay ( e.g. , 10 to 0 mv ) . 1 b , were 0.45 0.13 ( standard deviation ) and 0.92 0.07 , respectively ; the difference was highly significant ( p < 10 , t test for paired measurements ) . 1 c illustrates the dependency of this modulatory effect on light intensity . a voltage step of fixed amplitude ( from 70 to 0 mv ) the loss of the decay phase of the current was graded with photostimulation , ( n = 3 ) . the intensity range over which light modulates the time course of the voltage - gated outward current is the same as the operating range for the photocurrent . in summary , the basic observations made in intact retinas of patinopecten are fully corroborated in isolated photoreceptors of pecten . a ) depolarization - activated outward currents were measured in the dark from a holding potential of 70 mv . the time course of this current is dramatically altered by applying steady illumination ( middle ) , which virtually eliminates the decay kinetics leaving a sustained current . after a brief ( 1-min ) period of dark adaptation , repetition of the voltage stimulation produced again the characteristic inactivating time course . ( b ) comparison of the extent of the decay ( ratio of plateau amplitude : peak amplitude ) for the current elicited by a depolarization to 10 mv in the dark and in the light , pooled for 17 cells . ( c ) dependency of the modulation of current decay on the intensity of stimulating light . a depolarizing step of fixed amplitude ( 70 to 0 mv ) was applied in the dark and during sustained illumination of different intensities ; light attenuation is indicated in logarithmic units ( io , the unattenuated beam , was 13.8 10 photonscms ) . since the original phenomenon was described , a handful of studies have outlined the basic characteristics of the light - transduction pathway of ciliary visual receptors , suggesting that a go is the likely downstream effector of rhodopsin photoisomerization , and that the light - sensitive conductance is activated via mobilization of cgmp , possibly involving regulation of a guanylate cyclase ( gomez and nasi , 1995 ; kojima et al . , 1997 ; gomez and nasi , 2000a ) . if the apparent loss of inactivation of ik is the mechanism that is also responsible for the activation of il , the signaling cascade must also be common , and therefore chemical stimulation of the phototransduction pathway should mimic the modulatory effects of light on depolarization - activated currents . the poorly hydrolyzable gtp analogue , gtp--s , is known to interfere with g protein shutoff , so that stimulation of the corresponding receptor produces a persistent activation of the signaling cascade . in the case of ciliary photoreceptor cells , we had previously demonstrated that after intracellular dialysis with gtp--s , brief light stimulation evokes a response that does not deactivate normally ; instead , a sustained photocurrent remains long after the flash ( gomez and nasi , 2000a ) . 2 a shows the effects of 100 m gtp--s on voltage - activated outward currents : the first family of traces , recorded after several minutes of dialysis in the dark , exhibits the characteristic inactivating time course . subsequently , the photoreceptor was stimulated with a single 500-ms flash of light ( 7 10 photonsscm ) . after a 3-min period of dark adaptation , the voltage clamp protocol was repeated , but the outward currents had a noticeably attenuated falling phase . the data from a total of six cells are summarized in the bar graph in fig . 2 b : the values of the plateau : peak ratio measured at 10 mv depolarization were 0.45 0.22 ( sd ) for the initial voltage stimulation , and 0.73 0.27 after brief light exposure and subsequent dark adaptation . a t test for paired measurements confirmed statistical significance of the difference ( p < 0.001 ) . ( a ) depolarizing voltage - clamp steps delivered at 10-mv increments from a holding potential of 80 mv in the dark , in a photoreceptor subjected to intracellular perfusion with 100 m gtp--s via the patch electrode . the first family of traces ( left ) was recorded after several minutes of dialysis in the dark . the cell was then briefly illuminated ( 500 ms ; light monitor trace not drawn to scale ) and then dark adapted again for 3 min . the voltage stimulation series was repeated ( right ) , producing outward currents that exhibited a noticeably reduced decay . ( b ) pooled values of the plateau : peak ratio measured with a depolarizing step to 10 mv depolarization in the dark , in gtp--s treated photoreceptors before and after exposure to a brief light stimulation ( n = 6 ) . 3 a , intracellular dialysis with 50 m 8-br - cgmp evoked an outward current , which was accompanied by an increase in membrane conductance ; previous work had established that the properties of this conductance coincide with those of the light - sensitive conductance ( gomez and nasi , 1995 , 1997 ) . in fig . 3 b , the same cell was stimulated with depolarizing voltage steps delivered at 10-mv increments from a holding potential of 80 mv , in the dark ; outward currents exhibited a much attenuated falling phase ( compare with fig . 1 a ) . the average extent of the decay of the current in control and in 8-br - cgmp treated photoreceptors the mean values obtained were 0.45 0.13 ( n = 17 ) and 0.69 0.22 ( n = 11 ) , respectively ; a t test for independent samples established that the difference was highly significant ( p < 0.001 ) . moreover , as illustrated in fig . 3 d , the 8-br - cgmp treated photoreceptor lost its susceptibility to any further modulation by bright sustained illumination ( 7 10 photonscms ) ( plateau : peak ratio at 10 mv = 0.71 0.24 , p > 0.35 , t test for paired measurements ) . internal perfusion with 200 m 8-br - camp did not antagonize the falling phase of the depolarization - activated current ( unpublished data ; n = 3 ) , thus indicating that such modulatory effect has a similar nucleotide specificity as the activation of the light - sensitive conductance ( gomez and nasi , 1995 ) . ( a ) effect of intracellular perfusion of 50 m 8-br - cgmp via the patch pipette . the holding potential was 30 mv , and a repetitive rectangular perturbation ( 10 mv , 10 hz ) was applied to monitor the input resistance of the cell . ( insets ) expanded segments of recording , to illustrate the increase in membrane conductance that accompanied the development of the outward current evoked by the cgmp analogue . ( b ) outward currents elicited by voltage steps from 80 to 20 mv in the dark , after the cgmp - dependent current stabilized . ( c ) comparison of the plateau : peak ratio for voltage steps to 10 mv in control photoreceptors dialyzed with standard intracellular solution and cells treated with 50 m 8-br - cgmp . ( d ) repetition of the voltage steps in the presence of steady light , same cell as in a and b. we assessed whether the action of cgmp analogs is likely to be direct , or may implicate additional effectors ; channel phosphorylation by a cgmp - dependent protein kinase ( pkg ) is a plausible mechanism by which illumination could exert its modulatory influence on voltage - activated currents . to test this possibility , we examined the effect of a potent ( k1/2 0.23 m ) and selective membrane - permeable pkg inhibitor , kt-5823 . illustrate the photostimulation - induced reduction of the decay of ik in a photoreceptor under control conditions , measured in the dark ( left ) and during illumination ( right ) . the same cell was subsequently locally superfused with 3 m kt-5823 via a puffer pipette , and the voltage clamp protocol was repeated after 4 min of continuous application of the drug . the time course of the outward current recorded in the dark was completely unaffected by the pkg antagonist , as was the ability of light ( 3.47 10 photonscms ) to attenuate the decay of ik . the outcome of this test is therefore not consistent with a downstream phosphorylation step , and suggests instead a direct effect of cgmp . effect of a cgmp - dependent protein kinase inhibitor on modulation by light of the voltage - dependent k current . ( a ) outward current elicited by membrane depolarization in the dark and in the presence of steady light , under control conditions . the characteristic photostimulation - induced reduction of the falling phase of ik in a photoreceptor is observed . ( b ) the same cell was subsequently locally superfused with 3 m kt-5823 , a potent and selective pkg inhibitor , and subjected to the same regime of voltage and light stimulation . the next goal was to test in a systematic way the conjecture of the identity of the il and ik . important clues can be obtained by comparing the effect of pharmacological blockers ; the hypothesis that identifies the activation of the photocurrent as the removal of steady - state inactivation of the voltage - dependent current makes the explicit prediction that an antagonist effective on the latter should also suppress the light - evoked current . the shaker - type k channel antagonist 4-ap is also a powerful blocker of the light response of ciliary photoreceptors ( gomez and nasi , 1994b ) , and a key argument in support of the contention that il is the same as ik was the demonstration that the latter was also blocked by 2 mm 4-ap ( shimatani and katagiri , 1995 ) . those effects proved fully reproducible in isolated pecten ciliary photoreceptors : superfusion with 2 mm 4-ap markedly suppressed the depolarization - elicited transient outward current ( average block 70 10% for a step from 60 to 0 mv , n = 4 ) , as well as the plateau ( 62 10% ; unpublished data ) . although this observation is qualitatively in agreement with the working hypothesis , the single dose used ( 2 mm ) is much too high to permit a useful comparison of the susceptibility of the two currents to this antagonist , since in a previous study we had reported that 4-ap suppression of the light response occurs in the micromolar range ( gomez and nasi , 1994b ) . we therefore reexamined the effects of the drug at a concentration range more relevant to its action on light responsiveness , to ascertain whether the dose dependency of 4-ap effects is similar on both currents . 5 a shows a family of voltage - activated currents measured in the dark ; the chamber was then superfused with 100 m 4-ap ( fig . 5 b ) while a flash of light ( 1.7 10 photonscms ) was repeatedly presented ; the drug caused swift suppression of the photocurrent to near undetectable levels . however , administration of voltage steps during exposure to 4-ap ( fig . 5 c ) revealed that the peak outward current was completely unaffected , and only a reduction in the plateau phase of the current is visible ( n = 5 ) . similar effects were obtained upon further reducing the 4-ap concentration to 20 m ( n = 4 ) or to 10 m ( n = 3 ) . the discrepancy , by over two orders of magnitude , of the 4-ap concentrations required to suppress ik vs. those effective on il poses a significant hurdle for the working hypothesis , if 4-ap antagonism derived from a direct occlusion of the permeation pore of the channels , which are hypothesized to be the same . the notion could be rescued , however , if at these low concentrations 4-ap acted allosterically , preventing the removal of inactivation . in such case , the activation of ik could occur normally , but , according to the model , the photocurrent would not . this interpretation may also be in line with the observation that , in cells exposed to 4-ap , light ( 2.76 10 photonscms ) failed to antagonize the falling phase of the voltage - activated current , as illustrated in fig . 5 d. therefore , the differential effects of 4-ap do not rule out the possibility that ik and il may be comprised by the same set of ion channels . ( a ) control family of traces in response to 100-ms voltage steps at 10-mv increments , from a holding voltage of 70 mv . ( b ) time course of the peak amplitude of the photocurrent evoked by a flash of fixed intensity in asw and during administration of 100 m 4-ap by local superfusion via a puffer pipette ( indicated by the horizontal bar ) ; holding potential 30 mv . same cell as in a. ( inset ) raw traces of the light - evoked currents . ( c ) repetition of the depolarizing voltage steps during superfusion with 100 m 4-ap . at this concentration , the reduction in the peak of ik is negligible , and only a modest depression of the plateau level is observed . ( d ) voltage steps administered to the same cell during steady illumination in the presence of the antagonist . a differential effect of a channel antagonist would be immediately informative if the blockade affected only the transient voltage - dependent k current , irrespective of mechanism , without compromising the photocurrent , because in such case the notion that il stems from removal of ik inactivation would become untenable . we screened a wide variety of toxins known to target different types of voltage - gated k channels ; the high specificity of action , at least in some cases , and blocking affinities that can reach into the nanomolar range made this class of antagonists particularly appealing . the list included agitoxin 2 , dendrotoxin - k , and margatoxin , especially promising because they affect some transient voltage - gated k channels ( of the kv 1 family ) ; in addition , we examined the effects of apamin , bds - i , bds - ii , charybdotoxin , e4031 , and tertiapin . all proved inert on both voltage- and light - evoked currents , except for apamin , a toxin known for its powerful effects on small - conductance ca - activated k channels . as shown in fig . 6 a , local superfusion with 100 nm apamin reversibly depressed the current evoked by light flashes of constant intensity ( 1.1 10 photonscms ) . the changes in the peak amplitude of the light response before , during , and after application of apamin are plotted as a function of time in fig . 6 b. by contrast , the toxin was completely inert on the voltage - activated currents . c shows families of traces in asw and during application of the same concentration of apamin . the i v relations for the peak and the plateau amplitude of the depolarization - activated current in the two conditions are plotted in fig . 6 d. inhibition by apamin , therefore , is qualitatively akin to that of 4-ap , though more modest , and thus provides no additional insight . it is interesting to note that an apamin - sensitive , cgmp - dependent k channel has been previously described ( mule et al . , 1999 ) . ( a ) superimposed responses to flashes of constant intensity administered in asw , during local superfusion with 100 nm apamin , and after washing the toxin . the peak amplitude of the photocurrent is plotted as a function of time , as the cell was stimulated with one flash every minute . ( c ) families of outward currents evoked in the dark in asw and during application of apamin . ( d ) i v plot for the peak ( squares ) and plateau ( triangles ) ik in control conditions ( filled symbols ) and in the presence of apamin ( open symbols ) . having failed to identify suitable toxin blockers of the voltage - gated transient k current , we turned to inorganic blockers , even though their affinity is typically orders of magnitude lower and their action is seldom as selective . barium is known to occlude the permeation pore in a variety of voltage - dependent potassium channels , including delayed rectifiers ( eaton and brodwick , 1980 ) , calcium - activated maxi - k channels ( miller et al . , 1987 ) , inward rectifiers ( hagiwara et al . , 1978 ) , and transient k channels such as shaker ( hurst et al . , 1995 ) . 7 a shows the outward currents evoked by repetitive depolarizing 100-ms voltage steps to 0 mv , administered every 30 s. upon switching to ba - asw ( replacing ca on an equimolar basis ) the transient outward current was virtually abolished ; the sustained component of the voltage - activated outward current appears to be less susceptible , and was depressed by 40% . the changes in peak amplitude of the current are plotted on the right ( n = 6 ) . by contrast , no detectable effect of replacing calcium with barium was observed on the amplitude of the response elicited by a repetitive flash of fixed intensity , as shown by fig . these results unambiguously indicate that a completely normal photocurrent can be obtained under conditions that suppress the transient outward current , and thus strongly argue against the possibility that the light - dependent current may arise from the removal of steady - state inactivation of the voltage - dependent conductance , and against the notion that the two currents share a common permeation pathway . finally , we also evaluated the effect of illumination in the time course of the depolarization - evoked outward currents in the presence of barium . 8 shows two families of depolarization - activated currents measured in the same cell superfused with 10 mm ba , in the dark and during steady light . clearly , light was still capable of modulating the kinetics of the currents , but under these conditions , the time course changed from nearly rectangular in the dark ( owing to the ba - induced suppression of the transient phase ) , to a sluggish outward relaxation reaching a larger plateau when the photoreceptor was illuminated ( n = 3 ) . in conclusion , there appears to be little doubt that the light - induced loss of decay kinetics in depolarization - activated current is not a reflection of removal of inactivation , but rather the development of an outward current that compensates for the inactivation of ik . ( a ) a repetitive depolarizing step to 0 mv was administered every 30 s. after two control stimuli in asw , the solution was switched to 10 mm ba ( replacing ca on an equimolar basis ) ; the transient outward current was gradually eliminated , and the sustained component suffered a significant reduction . on the right , ( b ) response to a repetitive 100-ms flash of fixed intensity delivered every minute . the amplitude of the photocurrent during the course of the experiment is plotted on the right . a slowly developing outward current is unmasked by barium . a photoreceptor was tested with depolarizing voltage steps while exposed to 10 mm ba ; under these conditions , the outward current virtually lacked the transient component . camp and cgmp have long been implicated in the gating of a variety of ion channels that subserve disparate functions , such as visual transduction ( fesenko et al . , 1985 ; yau and baylor , 1989 ) , olfactory transduction ( nakamura and gold , 1987 ) , and chemotaxis ( galindo et al . , 2000 ) . cyclic nucleotide - activated channels are genetically related to the family of voltage - gated k channels ( kaupp , 1991 ; guy et al . , 1991 ; yau , 1994 ) , and even possess an s4 domain , the putative voltage sensor , although their gating is only weakly voltage dependent . it is therefore plausible that in the course of the evolutionary modifications that may have given rise to the family of cngcs , some structural elements implicated in regulating the flow of ions in their ancestral voltage - dependent cousins may have been retained as the chemically operated gate . in the case of ciliary visual receptors , an explicit proposal was that the photoresponse reflects light - induced removal of the steady - state inactivation in the transient depolarization - activated k conductance ( shimatani and katagiri , 1995 ) . our observations confirmed and extended the basic phenomenology , demonstrating that direct chemical manipulations of the light - transduction cascade at the level of both the g protein and the messenger , cgmp , mimic the effect of light on the decay of ik . generation of the photocurrent and modulation of voltage - triggered currents can thus be ascribed to a common internal messenger system . to test the hypothesis that underlying conductances are the same unfortunately , even if the permeation pathway was indeed the same , the predictions would be inextricably linked to the assumptions one makes about the gating scheme : if light removed steady - state inactivation , as in the original proposal , the population of channels available for activation by photostimulation would be the same that had been opened by voltage and had undergone inactivation ; therefore the larger the transient voltage - activated current , the larger the photocurrent that could be subsequently evoked . by contrast , if light and voltage acted on the activation gate , then opening a certain fraction of the channels by either stimulus would decrease ( by the same amount ) the fraction of channels susceptible to be opened using the other stimulus , i.e. , the interaction would be occlusive . this model dependency prompted us to use an alternative strategy to test the working hypothesis . support for the notion of a shared ionic pathway can be garnered by demonstrating similar inhibitory effects of pharmacological agents on the light - dependent and the voltage - dependent currents . the observed blockade of both conductances by millimolar concentrations of 4-ap is in line with that reasoning ; moreover , we have observed a similar , although more modest , effect with l - cis - diltiazem ( unpublished data ) . nonetheless , these results are not conclusive ; by contrast , differential effects on the two currents could be strongly indicative of separate underlying conductance systems , provided that certain requirements are met . micromolar concentrations of 4-ap , a range far closer to the previously reported ic50 for the light response ( gomez and nasi , 1994b ) , nearly abolished the light response without any detectable antagonism on the transient voltage - dependent current . the interpretation of such an effect , however , depends on the mechanism of block . should the antagonism entail occlusion of the permeation pore , then clearly it would become untenable to propose that the two currents flow through the same ion channels . the mechanisms of block of 4-ap are complex and varied ; there is some general consensus that its main mode of action involves permeation into the cytoplasm in its uncharged form , and subsequent blockage from the intracellular side in its charged form ( kirsch and narahashi , 1983 ; howe and ritchie , 1991 ; choquet and korn , 1992 ) . however , the effectiveness of 4-ap blockade varies dramatically and exhibits opposite state dependencies across different k channel types , suggesting that various targets may be implicated , including gating ( kirsch and drewe , 1993 ; mccormack et al . , 1994 ) . it is thus conceivable that 4-ap at low concentration could impair the proposed mechanism of removal of steady - state inactivation , without hampering ionic conduction . an ad hoc scenario , for example , would be some interference with the action of cgmp . the working hypothesis is therefore not necessarily invalidated by the observed effect of micromolar 4-ap , or the qualitatively similar action of apamin . on the other hand , a differential effect in the opposite direction ( namely , antagonism of the transient voltage - gated k current without interference with the photocurrent ) would be far less ambiguous . our observations with barium are of that nature , and strongly argue against the idea that the mechanism of the light response consists in the control by light of an inactivation gate in a voltage - sensitive k conductance . the proposed mechanism predicts a strong outward rectification of the photocurrent , because at negative potentials , the probability of opening the voltage - dependent channels is low and hence removal of inactivation would not be relevant . although such nonlinear i v relation has indeed been described ( gomez and nasi , 1994a ) , it was shown to arise from voltage - dependent block by extracellular divalent cations ( gomez and nasi , 1994b ; nasi and gomez , 1999 ) ; in fact , in ca- and mg - free media , conduction becomes ohmic , and sizable photocurrents are measured as negative as 110 mv ( i.e. , > 70 mv more negative than the activation threshold for ik ) , which can not simply reflect a shift in surface potential . the question remains of the nature of the striking differences in the voltage step elicited currents measured in the dark or in the presence of light . clearly the phenomenon parallels the activation of the transduction cascade , and represents the gradual increase of a conductance separate from ik . we have previously reported that when the membrane potential is stepped abruptly during illumination , the current through light - dependent channels shows a distinct relaxation : with depolarizing steps , the conductance shows a time - dependent increase , while it decreases with hyperpolarizing steps . such relaxations were shown to arise from the equilibration of voltage - dependent blockade by divalent cations ( nasi and gomez , 1999 ) . the time constant of this process was measured in the range of 1520 ms , becoming faster with depolarization . these values are quite similar to those of the inactivation kinetics of the depolarization - activated k current and have a similar voltage dependency ; the amplitudes were also in the same range . in the presence of steady illumination , therefore , the falling phase of the voltage - gated current would be compensated by the growing current through the photoconductance . a perfect match between the two processes , which would be quite coincidental , is not required to give the appearance of loss of inactivation , as long as the of the inactivation is not much shorter than that of the compensating raising current . otherwise , an inflection would appear on the trace , as we have occasionally observed in the presence of light . our results therefore refute the intriguing hypothesis on the gating mechanism of the light - sensitive conductance in ciliary photoreceptors ; nonetheless , future investigations might still reveal that these light - controlled channels , while being distinct from the voltage - dependent k conductances expressed in the same cells , employ some common elements as mechanisms to regulate the ionic flux .

the hyperpolarizing receptor potential of ciliary photoreceptors of scallop and other mollusks is mediated by a cgmp - activated k conductance ; these cells also express a transient potassium current triggered by depolarization . during steady illumination , the outward currents elicited by voltage steps lose their decay kinetics . one interesting conjecture that has been proposed is that the currents triggered by light and by depolarization are mediated by the same population of channels , and that illumination evokes the receptor potential by removing their steady - state inactivation . exploiting the information that has become available on the phototransduction cascade of ciliary photoreceptors , we demonstrated that the same downstream signaling elements are implicated in the modulation of voltage - elicited currents : direct chemical stimulation both at the level of the g protein and of the final messenger that controls the light - dependent channels ( cgmp ) also attenuate the falling phase of the voltage - activated current . application of a protein kinase g antagonist was ineffective , suggesting that a cgmp - initiated phosphorylation step is not implicated . to ascertain the commonality of ionic pathways we used pharmacological blockers . although millimolar 4-aminopyridine ( 4-ap ) suppressed both currents , at micromolar concentrations only the photocurrent was blocked . conversely , barium completely and reversibly antagonized the transient voltage - activated current with no detectable effect on the light - evoked current . these results rule out that the same ionic pores mediate both currents ; the mechanism of light modulation of the depolarization - evoked k current was elucidated as a time - dependent increase in the light - sensitive conductance that is superimposed on the inactivating k current .

INTRODUCTION MATERIALS AND METHODS Chemical Stimulation Light Stimulation RESULTS DISCUSSION

in the eyes of several marine mollusks , a distinct class of photoreceptors is found , with properties that markedly diverge from those of classical invertebrate visual receptors : the light - sensing structure is ciliary , rather than microvillar ( miller , 1958 ; barber et al . , 1967 ) , the receptor potential is hyperpolarizing ( mcreynolds and gorman , 1970 ; mpitsos , 1973 ) , and the transduction cascade involves mobilization of cgmp instead of signaling via the phosphoinositide pathway ( gomez and nasi , 1995 ) . the ion channels that underlie the photocurrent in these sensory cells have been characterized ( gomez and nasi , 1994a ) , and are of special interest because they share functional properties with two superfamilies of ion channel proteins believed to have evolved from a common ancestor , namely , cyclic nucleotide gated channels ( cngcs ) and voltage - dependent potassium channels ( guy et al . on the one hand , they are activated by cgmp and , like other members of the cngc family , are blocked by the organic antagonists l - cis - diltiazem and 3,4-dichlorobenzamil ( gomez and nasi , 1997 ) and exhibit a pronounced outward rectification arising from voltage - dependent blockade by extracellular ca and mg ( nasi and gomez , 1999 ) . on the other hand , ; gomez and nasi , 1994a ) , and they are susceptible to blockade by k channel antagonists , especially 4-aminopyridine ( 4-ap ) ( gomez and nasi , 1994b ) . the evolutionary kinship of cngcs to voltage - dependent channels naturally raises the question of how a chemically controlled mechanism for channel opening was acquired , and whether the gating structures implicated are homologous to those operated by transmembrane potential in their ancestral cousins . in voltage - dependent potassium channels , seminal experiments by armstrong ( 1971 ) on pore - blocker accessibility had indicated that the main gate seems to be located near the inner vestibule of the permeation pathway , a notion subsequently corroborated in cloned shaker channels using cysteine scanning mutagenesis ( liu et al . , 1973 ) , comprising the initial stretch of residues at the amino terminus of the polypeptide that mediates the rapid inactivation observed in a variety of channels known as n - type inactivation ( hoshi et al . a tantalizing scheme has been proposed for the gating mechanism of the photocurrent in ciliary photoreceptors ( shimatani and katagiri , 1995 ) : the light - sensitive k conductance would be comprised of transient voltage - gated k channels akin to ia , and illumination would generate the photoresponse by removing their steady - state inactivation . moreover , application of 4-ap at millimolar concentrations suppresses both the depolarization - activated current and the photocurrent ( shimatani and katagiri , 1995 ) . the appeal of this conjecture is twofold : first , the explicit identity of the allosteric transitions triggered by the internal messenger for light transduction and of the proposed moving parts of the gating machinery ; second , the evolutionary thread linking homologous functions in distantly related ion channels that have come to subserve entirely disparate functions . in the present report we examined whether a common signaling pathway is responsible for the modulation of the voltage - gated currents and the activation of the light - dependent current , and addressed in a systematic way the hypothesis that the same population of ion channels is implicated in the two cases . repetition of the same stimulation protocol during application of a steady background light of near - saturating intensity ( 3.47 10 photonscms ) results in currents completely devoid of decay kinetics , presenting instead a near - rectangular appearance ( fig . a simple criterion to compare quantitatively the extent of the falling phase of the response in the dark vs. in the light is the ratio plateau : peak of the current , measured at some fixed depolarization within the range that produces a prominent current decay ( e.g. the intensity range over which light modulates the time course of the voltage - gated outward current is the same as the operating range for the photocurrent . a ) depolarization - activated outward currents were measured in the dark from a holding potential of 70 mv . ( b ) comparison of the extent of the decay ( ratio of plateau amplitude : peak amplitude ) for the current elicited by a depolarization to 10 mv in the dark and in the light , pooled for 17 cells . ( c ) dependency of the modulation of current decay on the intensity of stimulating light . since the original phenomenon was described , a handful of studies have outlined the basic characteristics of the light - transduction pathway of ciliary visual receptors , suggesting that a go is the likely downstream effector of rhodopsin photoisomerization , and that the light - sensitive conductance is activated via mobilization of cgmp , possibly involving regulation of a guanylate cyclase ( gomez and nasi , 1995 ; kojima et al . if the apparent loss of inactivation of ik is the mechanism that is also responsible for the activation of il , the signaling cascade must also be common , and therefore chemical stimulation of the phototransduction pathway should mimic the modulatory effects of light on depolarization - activated currents . in the case of ciliary photoreceptor cells , we had previously demonstrated that after intracellular dialysis with gtp--s , brief light stimulation evokes a response that does not deactivate normally ; instead , a sustained photocurrent remains long after the flash ( gomez and nasi , 2000a ) . 2 a shows the effects of 100 m gtp--s on voltage - activated outward currents : the first family of traces , recorded after several minutes of dialysis in the dark , exhibits the characteristic inactivating time course . after a 3-min period of dark adaptation , the voltage clamp protocol was repeated , but the outward currents had a noticeably attenuated falling phase . 3 a , intracellular dialysis with 50 m 8-br - cgmp evoked an outward current , which was accompanied by an increase in membrane conductance ; previous work had established that the properties of this conductance coincide with those of the light - sensitive conductance ( gomez and nasi , 1995 , 1997 ) . 3 b , the same cell was stimulated with depolarizing voltage steps delivered at 10-mv increments from a holding potential of 80 mv , in the dark ; outward currents exhibited a much attenuated falling phase ( compare with fig . internal perfusion with 200 m 8-br - camp did not antagonize the falling phase of the depolarization - activated current ( unpublished data ; n = 3 ) , thus indicating that such modulatory effect has a similar nucleotide specificity as the activation of the light - sensitive conductance ( gomez and nasi , 1995 ) . ( insets ) expanded segments of recording , to illustrate the increase in membrane conductance that accompanied the development of the outward current evoked by the cgmp analogue . ( b ) outward currents elicited by voltage steps from 80 to 20 mv in the dark , after the cgmp - dependent current stabilized . ( d ) repetition of the voltage steps in the presence of steady light , same cell as in a and b. we assessed whether the action of cgmp analogs is likely to be direct , or may implicate additional effectors ; channel phosphorylation by a cgmp - dependent protein kinase ( pkg ) is a plausible mechanism by which illumination could exert its modulatory influence on voltage - activated currents . the same cell was subsequently locally superfused with 3 m kt-5823 via a puffer pipette , and the voltage clamp protocol was repeated after 4 min of continuous application of the drug . the time course of the outward current recorded in the dark was completely unaffected by the pkg antagonist , as was the ability of light ( 3.47 10 photonscms ) to attenuate the decay of ik . effect of a cgmp - dependent protein kinase inhibitor on modulation by light of the voltage - dependent k current . the characteristic photostimulation - induced reduction of the falling phase of ik in a photoreceptor is observed . important clues can be obtained by comparing the effect of pharmacological blockers ; the hypothesis that identifies the activation of the photocurrent as the removal of steady - state inactivation of the voltage - dependent current makes the explicit prediction that an antagonist effective on the latter should also suppress the light - evoked current . the shaker - type k channel antagonist 4-ap is also a powerful blocker of the light response of ciliary photoreceptors ( gomez and nasi , 1994b ) , and a key argument in support of the contention that il is the same as ik was the demonstration that the latter was also blocked by 2 mm 4-ap ( shimatani and katagiri , 1995 ) . those effects proved fully reproducible in isolated pecten ciliary photoreceptors : superfusion with 2 mm 4-ap markedly suppressed the depolarization - elicited transient outward current ( average block 70 10% for a step from 60 to 0 mv , n = 4 ) , as well as the plateau ( 62 10% ; unpublished data ) . although this observation is qualitatively in agreement with the working hypothesis , the single dose used ( 2 mm ) is much too high to permit a useful comparison of the susceptibility of the two currents to this antagonist , since in a previous study we had reported that 4-ap suppression of the light response occurs in the micromolar range ( gomez and nasi , 1994b ) . 5 a shows a family of voltage - activated currents measured in the dark ; the chamber was then superfused with 100 m 4-ap ( fig . 5 b ) while a flash of light ( 1.7 10 photonscms ) was repeatedly presented ; the drug caused swift suppression of the photocurrent to near undetectable levels . 5 c ) revealed that the peak outward current was completely unaffected , and only a reduction in the plateau phase of the current is visible ( n = 5 ) . this interpretation may also be in line with the observation that , in cells exposed to 4-ap , light ( 2.76 10 photonscms ) failed to antagonize the falling phase of the voltage - activated current , as illustrated in fig . 5 d. therefore , the differential effects of 4-ap do not rule out the possibility that ik and il may be comprised by the same set of ion channels . ( b ) time course of the peak amplitude of the photocurrent evoked by a flash of fixed intensity in asw and during administration of 100 m 4-ap by local superfusion via a puffer pipette ( indicated by the horizontal bar ) ; holding potential 30 mv . ( inset ) raw traces of the light - evoked currents . at this concentration , the reduction in the peak of ik is negligible , and only a modest depression of the plateau level is observed . ( d ) voltage steps administered to the same cell during steady illumination in the presence of the antagonist . a differential effect of a channel antagonist would be immediately informative if the blockade affected only the transient voltage - dependent k current , irrespective of mechanism , without compromising the photocurrent , because in such case the notion that il stems from removal of ik inactivation would become untenable . we screened a wide variety of toxins known to target different types of voltage - gated k channels ; the high specificity of action , at least in some cases , and blocking affinities that can reach into the nanomolar range made this class of antagonists particularly appealing . the list included agitoxin 2 , dendrotoxin - k , and margatoxin , especially promising because they affect some transient voltage - gated k channels ( of the kv 1 family ) ; in addition , we examined the effects of apamin , bds - i , bds - ii , charybdotoxin , e4031 , and tertiapin . all proved inert on both voltage- and light - evoked currents , except for apamin , a toxin known for its powerful effects on small - conductance ca - activated k channels . the changes in the peak amplitude of the light response before , during , and after application of apamin are plotted as a function of time in fig . 6 b. by contrast , the toxin was completely inert on the voltage - activated currents . c shows families of traces in asw and during application of the same concentration of apamin . the i v relations for the peak and the plateau amplitude of the depolarization - activated current in the two conditions are plotted in fig . it is interesting to note that an apamin - sensitive , cgmp - dependent k channel has been previously described ( mule et al . the peak amplitude of the photocurrent is plotted as a function of time , as the cell was stimulated with one flash every minute . having failed to identify suitable toxin blockers of the voltage - gated transient k current , we turned to inorganic blockers , even though their affinity is typically orders of magnitude lower and their action is seldom as selective . barium is known to occlude the permeation pore in a variety of voltage - dependent potassium channels , including delayed rectifiers ( eaton and brodwick , 1980 ) , calcium - activated maxi - k channels ( miller et al . 7 a shows the outward currents evoked by repetitive depolarizing 100-ms voltage steps to 0 mv , administered every 30 s. upon switching to ba - asw ( replacing ca on an equimolar basis ) the transient outward current was virtually abolished ; the sustained component of the voltage - activated outward current appears to be less susceptible , and was depressed by 40% . by contrast , no detectable effect of replacing calcium with barium was observed on the amplitude of the response elicited by a repetitive flash of fixed intensity , as shown by fig . these results unambiguously indicate that a completely normal photocurrent can be obtained under conditions that suppress the transient outward current , and thus strongly argue against the possibility that the light - dependent current may arise from the removal of steady - state inactivation of the voltage - dependent conductance , and against the notion that the two currents share a common permeation pathway . finally , we also evaluated the effect of illumination in the time course of the depolarization - evoked outward currents in the presence of barium . 8 shows two families of depolarization - activated currents measured in the same cell superfused with 10 mm ba , in the dark and during steady light . clearly , light was still capable of modulating the kinetics of the currents , but under these conditions , the time course changed from nearly rectangular in the dark ( owing to the ba - induced suppression of the transient phase ) , to a sluggish outward relaxation reaching a larger plateau when the photoreceptor was illuminated ( n = 3 ) . in conclusion , there appears to be little doubt that the light - induced loss of decay kinetics in depolarization - activated current is not a reflection of removal of inactivation , but rather the development of an outward current that compensates for the inactivation of ik . ( a ) a repetitive depolarizing step to 0 mv was administered every 30 s. after two control stimuli in asw , the solution was switched to 10 mm ba ( replacing ca on an equimolar basis ) ; the transient outward current was gradually eliminated , and the sustained component suffered a significant reduction . a photoreceptor was tested with depolarizing voltage steps while exposed to 10 mm ba ; under these conditions , the outward current virtually lacked the transient component . cyclic nucleotide - activated channels are genetically related to the family of voltage - gated k channels ( kaupp , 1991 ; guy et al . it is therefore plausible that in the course of the evolutionary modifications that may have given rise to the family of cngcs , some structural elements implicated in regulating the flow of ions in their ancestral voltage - dependent cousins may have been retained as the chemically operated gate . in the case of ciliary visual receptors , an explicit proposal was that the photoresponse reflects light - induced removal of the steady - state inactivation in the transient depolarization - activated k conductance ( shimatani and katagiri , 1995 ) . our observations confirmed and extended the basic phenomenology , demonstrating that direct chemical manipulations of the light - transduction cascade at the level of both the g protein and the messenger , cgmp , mimic the effect of light on the decay of ik . generation of the photocurrent and modulation of voltage - triggered currents can thus be ascribed to a common internal messenger system . to test the hypothesis that underlying conductances are the same unfortunately , even if the permeation pathway was indeed the same , the predictions would be inextricably linked to the assumptions one makes about the gating scheme : if light removed steady - state inactivation , as in the original proposal , the population of channels available for activation by photostimulation would be the same that had been opened by voltage and had undergone inactivation ; therefore the larger the transient voltage - activated current , the larger the photocurrent that could be subsequently evoked . by contrast , if light and voltage acted on the activation gate , then opening a certain fraction of the channels by either stimulus would decrease ( by the same amount ) the fraction of channels susceptible to be opened using the other stimulus , i.e. support for the notion of a shared ionic pathway can be garnered by demonstrating similar inhibitory effects of pharmacological agents on the light - dependent and the voltage - dependent currents . micromolar concentrations of 4-ap , a range far closer to the previously reported ic50 for the light response ( gomez and nasi , 1994b ) , nearly abolished the light response without any detectable antagonism on the transient voltage - dependent current . it is thus conceivable that 4-ap at low concentration could impair the proposed mechanism of removal of steady - state inactivation , without hampering ionic conduction . on the other hand , a differential effect in the opposite direction ( namely , antagonism of the transient voltage - gated k current without interference with the photocurrent ) would be far less ambiguous . our observations with barium are of that nature , and strongly argue against the idea that the mechanism of the light response consists in the control by light of an inactivation gate in a voltage - sensitive k conductance . the proposed mechanism predicts a strong outward rectification of the photocurrent , because at negative potentials , the probability of opening the voltage - dependent channels is low and hence removal of inactivation would not be relevant . the question remains of the nature of the striking differences in the voltage step elicited currents measured in the dark or in the presence of light . clearly the phenomenon parallels the activation of the transduction cascade , and represents the gradual increase of a conductance separate from ik . we have previously reported that when the membrane potential is stepped abruptly during illumination , the current through light - dependent channels shows a distinct relaxation : with depolarizing steps , the conductance shows a time - dependent increase , while it decreases with hyperpolarizing steps . these values are quite similar to those of the inactivation kinetics of the depolarization - activated k current and have a similar voltage dependency ; the amplitudes were also in the same range . in the presence of steady illumination , therefore , the falling phase of the voltage - gated current would be compensated by the growing current through the photoconductance . our results therefore refute the intriguing hypothesis on the gating mechanism of the light - sensitive conductance in ciliary photoreceptors ; nonetheless , future investigations might still reveal that these light - controlled channels , while being distinct from the voltage - dependent k conductances expressed in the same cells , employ some common elements as mechanisms to regulate the ionic flux .

all human cd8 t cells express nkg2d , whereas nkg2d expression on cd4 t cells has primarily been reported in some pathological conditions [ 36 ] . thus , nkg2d is expressed on effector cells of both innate and adaptive immune responses and is implicated in the surveillance of viral infections and cancers as well as in some autoimmune processes and transplantation reactions [ 715 ] . nkg2d is a c - type lectin - like type receptor and belongs to the nk group 2 ( nkg2 ) of receptors as member d. it has also been classified as killer cell lectin - like receptor of the subfamily k , member 1 ( klrk1 ) . nkg2d ( klrk1 ) is encoded by the nkg2d ( klrk1 ) gene that is located within the nk gene complex ( nkc ) situated on chromosome 6 in mice and on chromosome 12 in humans [ 17 , 18 ] . although nkg2d belongs to the nkg2 family , it does not share most of their properties . in contrast to other members of the family ( nkg2a / b , nkg2c , and nkg2e ) , which form heterodimers with cd94 and recognize mhc class ib molecules ( hla - e in humans and qa1 in mouse ) , nkg2d is a homodimer and recognizes a number of stress induced mhc class i - like ligands [ 2 , 19 ] . there is no inhibitory counterpart known for nkg2d and nkg2d is capable of overriding signals provided by inhibitory receptors on nk cells engaging mhc class ia and ib molecules . thus , nkg2d plays a role as a molecular sensor detecting induced self on cells in danger , which is mostly triggered by viral infections and by factors causing dna damage and tumor transformation [ 19 , 20 ] . the nkg2d receptor consists of two disulfide - linked type ii transmembrane proteins with positively charged amino acid residues in their transmembrane domains and very short intracellular tails that do not have any signaling properties . signal transduction operates through two adaptor proteins , dap10 and dap12 , which associate with the receptor as homodimers [ 22 , 23 ] . the nkg2d signaling complex appears as a hexameric structure since each nkg2d homodimer binds two adaptor homodimers . in mice , there are two nkg2d isoforms that differ in length of their intracellular sequence by 13 amino acid residues and have different association properties for the two adaptor proteins . the short isoform of nkg2d ( nkg2d - s ) associates with both adaptor proteins , while the long one ( nkg2d - l ) only binds to dap10 [ 22 , 23 , 25 ] . dap10 has a yinm motif in its cytoplasmatic tail which , upon phosphorylation , recruits the p85 subunit of phoshpoinositide-3-kinase ( pi3k ) and grb2vav1 . this signal stimulates survival and cytotoxicity of nk cells and provides co - stimulation to activated t cells [ 2628 ] . dap12 possesses an immunoreceptor tyrosine - based activation motif ( itam ) which , after phosphorylation , recruits src family kinases zap70 and syk , responsible for cytokine release and enhancement of cytotoxicity in nk cells . even though there are some reports that dap12 can also be expressed by t cells , deficiency of dap10 results in complete loss of nkg2d signaling in t cells and therefore appears to be the most important adaptor for nkg2d signaling in these cells [ 13 , 22 , 23 ] . nkg2d is able to bind a number of mhc class i - like ligands because of its unique protein structure [ 3133 ] . in mice , it binds to the family of retinoic acid early inducible proteins ( rae1 ) [ 34 , 35 ] . there are five members of these gpi - anchored proteins ( rae1- , - , - , - , and - ) and their expression differs between mouse strains . nkg2d also binds to the closely related group of histocompatibility antigen 60 ( h60 ) glycoproteins , which consists of three members ( h60a c ) . h60a and b are transmembrane proteins , while h60c is gpi - anchored [ 3638 ] . whereas h60a is widely expressed , h60b and c proteins display highly tissue- ( but not mouse strain)-specific expression patterns . murine ul-16-binding protein - like transcript 1 ( mult1 ) , which binds nkg2d with the highest affinity of all ligands , is so far the only transmembrane glycoprotein that belongs to the third group of murine nkg2d - binding proteins [ 39 , 40 ] . in humans , nkg2d binds to the mhc class - i related proteins mica and micb ( mhc class i chain - related protein a and b ) . nkg2d also recognizes surface glycoproteins that bind human cytomegalovirus ( hcmv ) ul-16 protein ( ulbps ) [ 12 , 4143 ] . there are six members of the ulbp family of proteins , which are closely related to the rae1 molecules in mice . ulbp1 , -2 , and -3 and -6 are gpi - anchored , while raet1e and -g ( also known as ulbp4 and ulbp5 ) are transmembrane proteins . nkg2d ligands are mostly induced by cellular stress ( induced self ) , although some of them are expressed at low levels in different tissues [ 3739 , 44 ] . however , expression of the ligands is very tightly controlled at transcriptional or / and posttranscriptional levels , since unbalanced expression may trigger activation of the immune system and autoimmune responses . viruses , as intracellular pathogens , are usually efficiently controlled by nk and cd8 t cells . nk cells limit viral replication and viral load in different tissues early upon infection , while cd8 t cells are responsible for the final viral clearance and , in some cases , for the establishment of viral latency . nkg2d is expressed on both of these cell types and plays an important role in the control of viral infections . different viruses are able to induce expression of nkg2d ligands on infected cells [ 4649 ] . they cause strong activation of nk cells as well as enhancement of antiviral effector functions of activated cd8 t cells , which can be prevented by nkg2d - specific mabs [ 1 , 13 , 46 , 48 , 50 ] . thus , nkg2d - mediated control represents a powerful and efficient mechanism to cope with viral infections . however , the nkg2d - mediated immunosurveillance may exert considerable selective pressure on viruses in the course of the co - evolution with their hosts . therefore , it is not surprising that some of them have developed mechanisms to evade nkg2d - mediated activation of the immune system . cytomegaloviruses ( cmvs ) have particularly well - developed mechanisms of interference with nkg2d - mediated immune cell activation . the hcmv - encoded membrane glycoprotein ul16 binds the nkg2d ligands ulbp1 , ulbp2 , and micb [ 12 , 51 , 52 ] , thus preventing their expression on the cell surface . hcmv gene product ul142 retains ulbp-3 in the cis - golgi complex and prevents its transport to the membrane . ul142 also retains newly synthesized full - length mica ( allele 001 ) in the cis - golgi [ 54 , 55 ] . interestingly , the mica allele 008 , which lacks the cytoplasmatic domain , shows resistance to ul142 binding . the fact that the truncated form of mica is the most common allele in several human populations suggests a profound advantage of the hosts ability to control virus infection through nkg2d - dependent activation of nk cells . although mica and micb genes are transcribed in all cells , their translation in healthy cells is repressed by endogenous cellular mirnas . for example , the hcmv - derived mirna , mir - ul112 , protects infected cells from nkg2d - mediated lysis by acting as a sponge for micb mrna . mouse cytomegalovirus ( mcmv ) has also been shown to extensively downregulate nkg2d ligands . mcmv protein m145 downmodulates cell surface expression of mult-1 , m152 interferes with the expression of rae1 and h60 proteins [ 61 , 62 ] , while m155 is involved in downmodulation of h60 . in addition , m138/fcr-1 plays an important role in downmodulation of mult-1 , rae1 and h60 [ 64 , 65 ] . kaposi s sarcoma - associated herpesvirus ( kshv ) immune evasion gene k5 reduces cell surface expression of mica and micb as well as of the nkp80 ligand activation - induced c - type lectin ( aicl ) via ubiquitination of lysine residues in cytoplasmic tails of the ligands . epstein ebv - transformed b cell lines express a high level of mhc class i molecules rendering them nk cell - resistant . however , reactivating latent ebv in transformed b cells increases susceptibility to nkg2d- and dnam1-dependent nk cell lysis . ectromelia virus ( ectv ) belongs to the orthopoxvirus genus , which includes variola virus ( varv ) , the causative agent of smallpox , and vaccinia virus ( vv ) . depletion of nk cells in mousepox - resistant mouse strains ( c57bl/6 ) results in massive viremia and death . zoonotic orthopoxviruses ( monkeypox and cowpox viruses ) encode for a soluble antagonist of nkg2d : orthopoxvirus mhc class i - like protein ( omcp ) . omcp , which is conserved among cowpox and monkeypox viruses , is secreted by infected cells and blocks recognition of nkg2d ligands . binding of omcp causes internalization of nkg2d , thus lowering the amount of available nkg2d receptor and inhibiting nkg2d - dependent killing by nk cells . human immunodeficiency virus ( hiv)-encoded protein nef ( negative factor ) has been shown to downregulate expression of the nkg2d ligands mica , ulbp1 , and ulbp2 on jurkat and primary cd4 t cells in vitro , in addition to hla - a and -b . in contrast to nef , another hiv - encoded protein vpr ( viral protein r ) has recently been shown to upregulate expression of nkg2d ligands ulbp1 , -2 , -3 , but not mica or micb , both in vitro and in vivo . vpr causes upregulation of the ligands through activation of dna damage / stress - sensing atr kinase . it seems that vpr not only contributes to hiv-1-induced cd4 t - lymphocyte depletion but also takes part in hiv-1-induced nk cell dysfunction . in conclusion , nkg2d - mediated control appears to be an important and powerful mechanism in the immunosurveillance of viral infections . a number of viral evasion mechanisms targeting the nkg2d pathway just emphasize its importance and this fact should be seriously considered in the development of future vaccines . the potential role of nkg2d in cytotoxic anti - tumor responses was quickly recognized and discussed as a revival of the tumor surveillance model . early experiments showed that overexpression of nkg2d ligands in cancer cells caused tumor rejection after transplantation in mice [ 74 , 75 ] . in humans , it was found that expression of nkg2d ligands highly correlated with the amount of t cell infiltrates in solid tumors . paradoxically , some tumors were shown to actively upregulate nkg2d ligands , making them prone to recognition by nk cells and cytotoxic t cells . as described in the previous section , viral infection leads to upregulation of nkg2d ligands . some virally induced tumors therefore show enhanced expression of these proteins in vivo however , also tumors without viral origin can have high levels of nkg2d ligands , suggesting a different cause . one hypothesis is that induction of nkg2d ligands is the result of the oncogenic process itself . nkg2d ligands are stress - response genes and are upregulated by stimuli such as dna damage [ 20 , 78 ] , heat - shock [ 44 , 79 ] , and shifts in hormone levels . oncogenesis is usually associated with genomic instability , heat - shock proteins are often hijacked by tumor cells to promote their survival and many tumors , such as breast , endometrial and ovarian cancers use estrogens to promote their growth . upregulation of these molecules in cancer cells might therefore be a bystander effect of the oncogenic process . preventing lysis by nkg2d - expressing nk and cd8 t cells must therefore be a feature that a cancer cell acquires in order to sustain its growth . the first and most obvious strategy is downmodulation of nkg2d ligands . despite significant amounts of dna damage or expression of heat - shock proteins , many tumors have been shown to actively inhibit nkg2d ligand expression [ 9 , 8587 ] . an important mechanism for nkg2d ligand downregulation appears to be the production of immunomodulatory cytokines such as tgf , which can be directly excreted by tumor cells themselves , or by regulatory immune cells that are expanded during tumor progression [ 8891 ] . in addition to this system , tumors decrease surface nkg2d ligand expression via shedding of the extracellular domain by metalloproteases or with the assistance of the disulphide - isomerase erp5 [ 92 , 93 ] . thirdly , expression of nkg2d ligands is actively regulated by micrornas , and tumor cells also have been shown to manipulate this system via mirna overexpression . experimental evidence for the in vivo relevance of nkg2d ligand downmodulation in the formation of tumors was recently shown in mice deficient for nkg2d . in animal models for spontaneous tumor formation , absence of nkg2d in addition , these tumors were shown to have increased expression of rae1 molecules on their surface . some tumors exploit an opposite strategy to manipulate nkg2d mediated signaling and instead induce high levels of its ligands . this concept remained relatively ill - understood until it was mimicked in mice overexpressing rae1 and mica molecules [ 15 , 95 , 96 ] . in these animals , surface expression of nkg2d was greatly reduced , both on nk cells and on cd8 t cells , resulting in impaired anti - tumor responses by these immune cells [ 15 , 95 ] . interestingly , the impact of nkg2d ligand overexpression on cd8 t cell responses against pathogens appears to depend highly on the model used . infection of transgenic mice with l. monocytogenes resulted in reduced anti - bacterial cd8 t cell numbers . antiviral responses upon mcmv infection , on the other hand , did not functionally impair cd8 t cell responses . one explanation for these observations may be the differential co - stimulation of cd8 t cells upon infection with different pathogens . unlike cd28 triggering , nkg2d co - stimulation is not required for t cell function . it will therefore depend on the pathogen or kind of tumor encountered to what extent nkg2d signaling is required for t cell - mediated cytotoxicity . for nk cells , nkg2d is a directly activating receptor and nk cell function was impaired in mica- and rae1-transgenic animals . not surprisingly , both in vivo and in vitro killing of tumor cells expressing nkg2d ligands by nk cells of these mice was reduced . oppenheim and coworkers also suggested that constitutive engagement of nkg2d impaired nk cell function beyond its downmodulation and subsequent inability to engage its ligands on tumor cells . however , other studies that directly addressed this issue indicate that this is not the case [ 15 , 96 ] . interestingly , nkg2d downmodulation via hyperstimulation appears to have different effects than inhibition of nkg2d signaling by omitting the molecules involved in transducing nkg2d - signaling . downmodulation of nkg2d via antibody treatment or in rae1 transgenic animals resulted in increased tumor cell growth in a model of chemically induced cancer formation [ 95 , 98 ] . however , the same model showed no differences when nkg2d - deficient animals were compared with wild type controls . the explanation for these differences is currently lacking , but it appears likely that nkg2d hyperligation induces compensatory and/or regulatory mechanisms which are absent in nkg2d - deficient animals . in support of this notion is the observation that nkg2d stimulation promotes the specific outgrowth of regulatory t cell subsets in humans . nkg2d ligation enhances proliferation of a characteristic regulatory nkg2dcd4 t cell pool that is rare under normal conditions [ 6 , 99 ] . this cell subset produces il-10 and tgf , thus inhibiting immune responses in a paracrine fashion . in addition , these cells express high levels of fas ligand ( fasl ) , which induces apoptosis in neighboring activated t cells , whereas these regulatory cells themselves appear refractory to this fasl . apart from directly presenting nkg2d ligands in cis , human tumor cells can manipulate the immune system by releasing a soluble form of nkg2d ligands . with the help of metalloproteases or of the cell surface - bound protein erp5 , the nkg2d ligands mica , micb , and ulbp2 are cleaved of the membrane and are capable of affecting nkg2d expressing cells in trans [ 100102 ] . apart from producing soluble proteins , tumor cells produce exosomes with high levels of nkg2d ligands [ 104 , 105 ] . both soluble ligands and ligands expressed on exosomes have been shown to downmodulate nkg2d on cytotoxic cells and thus impair their anti - tumor activity . in summary , it appears that tumor cells in general adapt methods to inhibit nkg2d signaling . they do this either via downmodulation of nkg2d ligands or via hyperexpression of nkg2d ligands on their surface or in soluble form . in addition , however , there are also tumors that do not seem to modify the nkg2d signaling pathway at all , yet still escape from destruction the molecular mechanism behind this phenomenon has yet to be revealed . due to its prominent role in tumor cell biology , the nkg2d signaling pathway has been under extensive investigation in the cancer field , both as a diagnostic tool and as a therapeutic target . mica has been shown to be one of the most polymorphic genes within the group of mhc class i - related molecules and several alleles , supposedly of reduced nkg2d affinity , have been associated with cancer [ 107109 ] . also , expression of nkg2d ligands , both the soluble and the membrane - bound form , has been shown to be a reliable marker for disease progression in a variety of malignancies [ 85 , 94 , 110 ] , illustrating its value as a clinical marker . induction of nkg2d ligand expression on tumors appears to be a promising therapeutic strategy in cancer . expression of the nkg2d ligand mult1 is low under homeostatic conditions , due to its continuous ubiquitination and subsequent targeting to the proteasome . in stressed cells , this ubiquitination process is inhibited , leading to rapid upregulation of surface - bound mult1 [ 45 , 111 ] . in tumor cells , nkg2d ligand expression can therefore be specifically induced with proteasome inhibitors , making tumors sensitive to nkg2d - mediated killing . in addition , hdac inhibitors appear to stimulate nkg2d ligand expression on tumor cells and their immunogenicity for allogeneic nk cells . finally , the nkg2d receptor system has been used as a target for anti - cancer therapy . therapeutic mica - specific antibodies effectively opsonized cancer cells and induced dc - mediated cross - presentation of tumor antigens . bifunctional proteins consisting of a tumor - antigen directed antibody fused to nkg2d ligands effectively coated tumor cells with activating ligands and increased their killing [ 115 , 116 ] . in summary , the two sides of nkg2d in tumor biology make this protein a key molecule of interest for future oncological research . on one hand , the nkg2d receptor system may be exploited in anti - tumor strategies , and on the other hand , over - stimulation of nkg2d should be prevented in order to potentiate tumor surveillance . as previously mentioned , inappropriate or deregulated expression of nkg2d ligands on healthy cells can break the delicate balance between immune activation and tolerance , and trigger autoimmune response . several autoimmune diseases have therefore been associated with nkg2d signaling . insulin - dependent diabetes mellitus / type 1 diabetes ( t1d ) is a chronic autoimmune disorder in which insulin - producing langerhans islets are destroyed by autoreactive immune cells . genetic linkage studies have shown that some mica alleles are positively associated with human t1d , but the functional relevance of this polymorphism is far from clear [ 118 , 119 ] . the nonobese diabetic ( nod ) mouse is widely studied as a model of human t1d . the development of disease can be completely prevented by treatment with nkg2d - blocking mabs , which reduce expansion and function of autoreactive cd8 t cells . rheumatoid arthritis ( ra ) is a chronic systemic inflammatory disorder in which immune cells , especially t cells , cause inflammation and destruction of the joints . ra patients have high levels of il-15 and tnf- in the sera and inflamed joints , which induce expression of nkg2d on cd4cd28 subset of t cells . since mica and micb molecules are also dramatically upregulated in ra synoviocytes , they activate the t cells in an nkg2d - dependent manner . celiac disease is an autoimmune disorder of the small intestine that occurs in genetically susceptible individuals as a reaction to wheat gliadin protein . one of the diagnostic hallmarks of celiac disease is a massive infiltration of intraepithelial nkg2d cd8 t lymphocytes ( iels ) in the gut . mic proteins , normally found intracellularly in enterocytes , become strongly expressed on the surface of epithelial cells in patients with active disease . it has been shown that the number of intestinal epithelial cd4 t cells expressing nkg2d highly correlates with the amount of intestinal inflammation and therefore are thought to play an important role in disease progression . nkg2d is expressed very early in the development of nk cells , already at the stage of nk precursors ( nkps ) , the earliest nk committed cells . about 10% of cd122 nk1.1 nkps ( stage i ) exhibit this receptor on the cell surface , whereas all immature nk cells ( stage ii and iii ) express nkg2d . during these stages of nk cell development , nkg2d expression rapidly increases and remains high through all later stages of maturation [ 127 , 128 ] . recently , the intracellular signaling components of il-15r and nkg2d have been shown to be coupled . il-15 is an essential cytokine for the development and survival of nk cells [ 130 , 131 ] . mice deficient for il-15 , il-15r , or any of the components of the il-15 signaling pathway ( e.g. , jak3 , stat5 ) have severe defects in nk cell development [ 132136 ] . have created a transgenic mouse strain expressing a dap10 protein fused with the monoubiquitin ( dap10-ub ) , which directly targets this molecule for proteasomal degradation . these mice display completely abolished nkg2d expression on t cells and severely reduced expression of this receptor on nk cells , which also express the nkg2d adaptor molecule dap12 . they observed severe defects in nk cell development because of their failure to respond to il-15 stimulation . jak3 has been shown to be the essential kinase downstream of the il-15 receptor that is responsible for dap10 phosphorylation important for the activation of stat5 , a prime target of jak3 . in addition , it was found that dap10 can associate with the il-15r and ( fig . 1 ) . interestingly , in contrast to dap10-ub mice , defects in nk cell development were not reported in dap10 mice . dap12 complexes on the surface of nk cells could compensate the lack of dap10 to a major extent , despite a reduction in nkg2d expression . activation of syk by dap12 can regulate survival and cytotoxicity via activation of pi3k [ 137 , 138].fig . the figure roughly summarizes interactions between signaling components of the il-15r and nkg2d , which was proposed by horng et al . . jak3 , as a part of canonical il-15r signaling pathway , is essential for the phosphorylation of dap10 . activated dap10 recruits pi3k and grb2 , which control proliferation , survival , and cytotoxicity of nk cells . dap10 also associates with il-15r and - chains coupling of the il-15r and nkg2d signaling components in nk cells . the figure roughly summarizes interactions between signaling components of the il-15r and nkg2d , which was proposed by horng et al . . jak3 , as a part of canonical il-15r signaling pathway , is essential for the phosphorylation of dap10 . activated dap10 recruits pi3k and grb2 , which control proliferation , survival , and cytotoxicity of nk cells . dap10 also associates with il-15r and - chains considering the above - mentioned findings , the question is whether and how nkg2d is involved in the development of nk cells . in transgenic mouse models with sustained expression of nkg2d ligands ( mica , rae1 , rae1 ) and consequent downmodulation of nkg2d , impairments in the nk cell development this may be the result of low - level expression of the receptor complexes on the cell surface and persistent nk cell stimulation . to resolve this issue , recently two models of nkg2d - deficiency were reported [ 9 , 139 ] . in the klrk1 mice generated by guerra et al . , the authors did not find any major developmental defects , although they showed mild changes in some subpopulations of nk cells ( i.e. ly49a cells ) . in klrk1 mice generated in our lab , nk cell development was moderately affected . in the absence of nkg2d we observed perturbations in the size of some developmental subsets of nk cells , increased proliferation of immature nk cells ( mostly in stage ii ) , their faster maturation and increased sensitivity to apoptosis . in addition , nk cell - mediated control of mcmv infection in these mice was better than in littermate controls , which we ascribed to the dysregulation and faster maturation of nk cells in the absence of nkg2d . our targeting inserted the egfp sequence in the third exon of the klrk1 locus , which could , as a foreign genetic element , cause non - specific effects . therefore , we generated klrk1 mice in which the egfp cassette is not present ( fig . these mice were obtained from the breeding of klrk1 mice ( our unpublished data ) and cre deleter mice , which resulted in the elimination of the floxed second and third exon of the klrk1 gene in vivo , a mutation that was further propagated as klrk1 . after we confirmed that nk cells from klrk1 mice do not express nkg2d ( fig . 2b ) , we analyzed the various stages of nk cell development in klrk1 versus klrk1 mice in comparison to wt animals ( fig . klrk1 mice displayed alterations in cell surface marker expression and maturation stages on nk cell subsets that were very similar to those seen in klrk1 animals . in addition , both mouse strains controlled early mcmv replication in a highly comparable manner ( fig . 3b ) . thus , these data strongly argue that the previously observed phenotype of nk cells in our klrk1 mice is the consequence of the desired klrk1 mutation rather than of a genetic deregulation caused by the egfp sequence.fig . 2comparison of klrk1 and klrk1 mice . a different mutations of the klrk1 locus ( upper row ) in klrk1 ( middle row ) and klrk1 ( lower row ) mice are shown . b nkg2d expression on cd3cd19nk1.1 cells isolated from the spleen of klrk1 ( left panel ) and klrk1 ( right panel ) mice is shownfig . 3comparative analysis of nk cell phenotype and function between wild type , klrk1 and klrk1 mice . a nk cells isolated from the spleens of 9 weeks old klrk1 and klrk1 mice were analyzed by flow cytometry . b indicated groups of klrk1 , klrk1 and their littermate controls were infected intravenously with 5 10 pfu of m157 mcmv . the groups of mice were either nk cell depleted ( pk136 ) or treated with pbs . each symbol represents individual virus titers in indicated organs comparison of klrk1 and klrk1 mice . a different mutations of the klrk1 locus ( upper row ) in klrk1 ( middle row ) and klrk1 ( lower row ) mice are shown . b nkg2d expression on cd3cd19nk1.1 cells isolated from the spleen of klrk1 ( left panel ) and klrk1 ( right panel ) mice is shown comparative analysis of nk cell phenotype and function between wild type , klrk1 and klrk1 mice . a nk cells isolated from the spleens of 9 weeks old klrk1 and klrk1 mice b indicated groups of klrk1 , klrk1 and their littermate controls were infected intravenously with 5 10 pfu of m157 mcmv . the groups of mice were either nk cell depleted ( pk136 ) or treated with pbs . each symbol represents individual virus titers in indicated organs in conclusion , nkg2d appears to play an important role in nk cell development . it seems that its role in the early development of nk cells is different than during the mature stages where nkg2d is responsible for activation of effector functions . unleashed proliferation of immature nk cells and their increased sensitivity to apoptosis in the absence of nkg2d suggest that this receptor plays rather a regulatory role at this stage , most probably in concert with il-15r and perhaps some other receptors ( fig . 1 ) , controlling proliferation and survival of nk cells . future research must reveal whether this function is limited to nk cells or whether other cellular subsets that constitutively express nkg2d ( most notably nkt and

nkg2d is one of the most intensively studied immune receptors of the past decade . its unique binding and signaling properties , expression pattern , and functions have been attracting much interest within the field due to its potent antiviral and anti - tumor properties . as an activating receptor , nkg2d is expressed on cells of the innate and adaptive immune system . it recognizes stress - induced mhc class i - like ligands and acts as a molecular sensor for cells jeopardized by viral infections or dna damage . although the activating functions of nkg2d have been well documented , recent analysis of nkg2d - deficient mice suggests that this receptor may have a regulatory role during nk cell development . in this review , we will revisit known aspects of nkg2d functions and present new insights in the proposed influence of this molecule on hematopoietic differentiation .

The NKG2D receptor and its ligands NKG2D in the control of viral infections NKG2D in the control of cancer NKG2D in autoimmunity NKG2D in NK cell development

thus , nkg2d is expressed on effector cells of both innate and adaptive immune responses and is implicated in the surveillance of viral infections and cancers as well as in some autoimmune processes and transplantation reactions [ 715 ] . nkg2d is a c - type lectin - like type receptor and belongs to the nk group 2 ( nkg2 ) of receptors as member d. it has also been classified as killer cell lectin - like receptor of the subfamily k , member 1 ( klrk1 ) . in contrast to other members of the family ( nkg2a / b , nkg2c , and nkg2e ) , which form heterodimers with cd94 and recognize mhc class ib molecules ( hla - e in humans and qa1 in mouse ) , nkg2d is a homodimer and recognizes a number of stress induced mhc class i - like ligands [ 2 , 19 ] . there is no inhibitory counterpart known for nkg2d and nkg2d is capable of overriding signals provided by inhibitory receptors on nk cells engaging mhc class ia and ib molecules . thus , nkg2d plays a role as a molecular sensor detecting induced self on cells in danger , which is mostly triggered by viral infections and by factors causing dna damage and tumor transformation [ 19 , 20 ] . the nkg2d receptor consists of two disulfide - linked type ii transmembrane proteins with positively charged amino acid residues in their transmembrane domains and very short intracellular tails that do not have any signaling properties . the nkg2d signaling complex appears as a hexameric structure since each nkg2d homodimer binds two adaptor homodimers . the short isoform of nkg2d ( nkg2d - s ) associates with both adaptor proteins , while the long one ( nkg2d - l ) only binds to dap10 [ 22 , 23 , 25 ] . even though there are some reports that dap12 can also be expressed by t cells , deficiency of dap10 results in complete loss of nkg2d signaling in t cells and therefore appears to be the most important adaptor for nkg2d signaling in these cells [ 13 , 22 , 23 ] . nkg2d is able to bind a number of mhc class i - like ligands because of its unique protein structure [ 3133 ] . murine ul-16-binding protein - like transcript 1 ( mult1 ) , which binds nkg2d with the highest affinity of all ligands , is so far the only transmembrane glycoprotein that belongs to the third group of murine nkg2d - binding proteins [ 39 , 40 ] . in humans , nkg2d binds to the mhc class - i related proteins mica and micb ( mhc class i chain - related protein a and b ) . however , expression of the ligands is very tightly controlled at transcriptional or / and posttranscriptional levels , since unbalanced expression may trigger activation of the immune system and autoimmune responses . nkg2d is expressed on both of these cell types and plays an important role in the control of viral infections . they cause strong activation of nk cells as well as enhancement of antiviral effector functions of activated cd8 t cells , which can be prevented by nkg2d - specific mabs [ 1 , 13 , 46 , 48 , 50 ] . thus , nkg2d - mediated control represents a powerful and efficient mechanism to cope with viral infections . however , the nkg2d - mediated immunosurveillance may exert considerable selective pressure on viruses in the course of the co - evolution with their hosts . therefore , it is not surprising that some of them have developed mechanisms to evade nkg2d - mediated activation of the immune system . the hcmv - encoded membrane glycoprotein ul16 binds the nkg2d ligands ulbp1 , ulbp2 , and micb [ 12 , 51 , 52 ] , thus preventing their expression on the cell surface . hcmv gene product ul142 retains ulbp-3 in the cis - golgi complex and prevents its transport to the membrane . ul142 also retains newly synthesized full - length mica ( allele 001 ) in the cis - golgi [ 54 , 55 ] . the fact that the truncated form of mica is the most common allele in several human populations suggests a profound advantage of the hosts ability to control virus infection through nkg2d - dependent activation of nk cells . for example , the hcmv - derived mirna , mir - ul112 , protects infected cells from nkg2d - mediated lysis by acting as a sponge for micb mrna . kaposi s sarcoma - associated herpesvirus ( kshv ) immune evasion gene k5 reduces cell surface expression of mica and micb as well as of the nkp80 ligand activation - induced c - type lectin ( aicl ) via ubiquitination of lysine residues in cytoplasmic tails of the ligands . epstein ebv - transformed b cell lines express a high level of mhc class i molecules rendering them nk cell - resistant . however , reactivating latent ebv in transformed b cells increases susceptibility to nkg2d- and dnam1-dependent nk cell lysis . ectromelia virus ( ectv ) belongs to the orthopoxvirus genus , which includes variola virus ( varv ) , the causative agent of smallpox , and vaccinia virus ( vv ) . zoonotic orthopoxviruses ( monkeypox and cowpox viruses ) encode for a soluble antagonist of nkg2d : orthopoxvirus mhc class i - like protein ( omcp ) . omcp , which is conserved among cowpox and monkeypox viruses , is secreted by infected cells and blocks recognition of nkg2d ligands . binding of omcp causes internalization of nkg2d , thus lowering the amount of available nkg2d receptor and inhibiting nkg2d - dependent killing by nk cells . human immunodeficiency virus ( hiv)-encoded protein nef ( negative factor ) has been shown to downregulate expression of the nkg2d ligands mica , ulbp1 , and ulbp2 on jurkat and primary cd4 t cells in vitro , in addition to hla - a and -b . in contrast to nef , another hiv - encoded protein vpr ( viral protein r ) has recently been shown to upregulate expression of nkg2d ligands ulbp1 , -2 , -3 , but not mica or micb , both in vitro and in vivo . vpr causes upregulation of the ligands through activation of dna damage / stress - sensing atr kinase . in conclusion , nkg2d - mediated control appears to be an important and powerful mechanism in the immunosurveillance of viral infections . a number of viral evasion mechanisms targeting the nkg2d pathway just emphasize its importance and this fact should be seriously considered in the development of future vaccines . the potential role of nkg2d in cytotoxic anti - tumor responses was quickly recognized and discussed as a revival of the tumor surveillance model . early experiments showed that overexpression of nkg2d ligands in cancer cells caused tumor rejection after transplantation in mice [ 74 , 75 ] . as described in the previous section , viral infection leads to upregulation of nkg2d ligands . one hypothesis is that induction of nkg2d ligands is the result of the oncogenic process itself . nkg2d ligands are stress - response genes and are upregulated by stimuli such as dna damage [ 20 , 78 ] , heat - shock [ 44 , 79 ] , and shifts in hormone levels . upregulation of these molecules in cancer cells might therefore be a bystander effect of the oncogenic process . the first and most obvious strategy is downmodulation of nkg2d ligands . despite significant amounts of dna damage or expression of heat - shock proteins , many tumors have been shown to actively inhibit nkg2d ligand expression [ 9 , 8587 ] . thirdly , expression of nkg2d ligands is actively regulated by micrornas , and tumor cells also have been shown to manipulate this system via mirna overexpression . experimental evidence for the in vivo relevance of nkg2d ligand downmodulation in the formation of tumors was recently shown in mice deficient for nkg2d . in animal models for spontaneous tumor formation , absence of nkg2d in addition , these tumors were shown to have increased expression of rae1 molecules on their surface . in these animals , surface expression of nkg2d was greatly reduced , both on nk cells and on cd8 t cells , resulting in impaired anti - tumor responses by these immune cells [ 15 , 95 ] . interestingly , the impact of nkg2d ligand overexpression on cd8 t cell responses against pathogens appears to depend highly on the model used . for nk cells , nkg2d is a directly activating receptor and nk cell function was impaired in mica- and rae1-transgenic animals . oppenheim and coworkers also suggested that constitutive engagement of nkg2d impaired nk cell function beyond its downmodulation and subsequent inability to engage its ligands on tumor cells . interestingly , nkg2d downmodulation via hyperstimulation appears to have different effects than inhibition of nkg2d signaling by omitting the molecules involved in transducing nkg2d - signaling . however , the same model showed no differences when nkg2d - deficient animals were compared with wild type controls . the explanation for these differences is currently lacking , but it appears likely that nkg2d hyperligation induces compensatory and/or regulatory mechanisms which are absent in nkg2d - deficient animals . in support of this notion is the observation that nkg2d stimulation promotes the specific outgrowth of regulatory t cell subsets in humans . apart from directly presenting nkg2d ligands in cis , human tumor cells can manipulate the immune system by releasing a soluble form of nkg2d ligands . with the help of metalloproteases or of the cell surface - bound protein erp5 , the nkg2d ligands mica , micb , and ulbp2 are cleaved of the membrane and are capable of affecting nkg2d expressing cells in trans [ 100102 ] . apart from producing soluble proteins , tumor cells produce exosomes with high levels of nkg2d ligands [ 104 , 105 ] . both soluble ligands and ligands expressed on exosomes have been shown to downmodulate nkg2d on cytotoxic cells and thus impair their anti - tumor activity . they do this either via downmodulation of nkg2d ligands or via hyperexpression of nkg2d ligands on their surface or in soluble form . due to its prominent role in tumor cell biology , the nkg2d signaling pathway has been under extensive investigation in the cancer field , both as a diagnostic tool and as a therapeutic target . mica has been shown to be one of the most polymorphic genes within the group of mhc class i - related molecules and several alleles , supposedly of reduced nkg2d affinity , have been associated with cancer [ 107109 ] . also , expression of nkg2d ligands , both the soluble and the membrane - bound form , has been shown to be a reliable marker for disease progression in a variety of malignancies [ 85 , 94 , 110 ] , illustrating its value as a clinical marker . expression of the nkg2d ligand mult1 is low under homeostatic conditions , due to its continuous ubiquitination and subsequent targeting to the proteasome . in tumor cells , nkg2d ligand expression can therefore be specifically induced with proteasome inhibitors , making tumors sensitive to nkg2d - mediated killing . finally , the nkg2d receptor system has been used as a target for anti - cancer therapy . in summary , the two sides of nkg2d in tumor biology make this protein a key molecule of interest for future oncological research . on one hand , the nkg2d receptor system may be exploited in anti - tumor strategies , and on the other hand , over - stimulation of nkg2d should be prevented in order to potentiate tumor surveillance . as previously mentioned , inappropriate or deregulated expression of nkg2d ligands on healthy cells can break the delicate balance between immune activation and tolerance , and trigger autoimmune response . genetic linkage studies have shown that some mica alleles are positively associated with human t1d , but the functional relevance of this polymorphism is far from clear [ 118 , 119 ] . the nonobese diabetic ( nod ) mouse is widely studied as a model of human t1d . rheumatoid arthritis ( ra ) is a chronic systemic inflammatory disorder in which immune cells , especially t cells , cause inflammation and destruction of the joints . ra patients have high levels of il-15 and tnf- in the sera and inflamed joints , which induce expression of nkg2d on cd4cd28 subset of t cells . celiac disease is an autoimmune disorder of the small intestine that occurs in genetically susceptible individuals as a reaction to wheat gliadin protein . one of the diagnostic hallmarks of celiac disease is a massive infiltration of intraepithelial nkg2d cd8 t lymphocytes ( iels ) in the gut . mic proteins , normally found intracellularly in enterocytes , become strongly expressed on the surface of epithelial cells in patients with active disease . nkg2d is expressed very early in the development of nk cells , already at the stage of nk precursors ( nkps ) , the earliest nk committed cells . about 10% of cd122 nk1.1 nkps ( stage i ) exhibit this receptor on the cell surface , whereas all immature nk cells ( stage ii and iii ) express nkg2d . during these stages of nk cell development , nkg2d expression rapidly increases and remains high through all later stages of maturation [ 127 , 128 ] . recently , the intracellular signaling components of il-15r and nkg2d have been shown to be coupled . mice deficient for il-15 , il-15r , or any of the components of the il-15 signaling pathway ( e.g. , jak3 , stat5 ) have severe defects in nk cell development [ 132136 ] . have created a transgenic mouse strain expressing a dap10 protein fused with the monoubiquitin ( dap10-ub ) , which directly targets this molecule for proteasomal degradation . these mice display completely abolished nkg2d expression on t cells and severely reduced expression of this receptor on nk cells , which also express the nkg2d adaptor molecule dap12 . they observed severe defects in nk cell development because of their failure to respond to il-15 stimulation . jak3 has been shown to be the essential kinase downstream of the il-15 receptor that is responsible for dap10 phosphorylation important for the activation of stat5 , a prime target of jak3 . interestingly , in contrast to dap10-ub mice , defects in nk cell development were not reported in dap10 mice . jak3 , as a part of canonical il-15r signaling pathway , is essential for the phosphorylation of dap10 . activated dap10 recruits pi3k and grb2 , which control proliferation , survival , and cytotoxicity of nk cells . the figure roughly summarizes interactions between signaling components of the il-15r and nkg2d , which was proposed by horng et al . jak3 , as a part of canonical il-15r signaling pathway , is essential for the phosphorylation of dap10 . activated dap10 recruits pi3k and grb2 , which control proliferation , survival , and cytotoxicity of nk cells . dap10 also associates with il-15r and - chains considering the above - mentioned findings , the question is whether and how nkg2d is involved in the development of nk cells . in transgenic mouse models with sustained expression of nkg2d ligands ( mica , rae1 , rae1 ) and consequent downmodulation of nkg2d , impairments in the nk cell development this may be the result of low - level expression of the receptor complexes on the cell surface and persistent nk cell stimulation . to resolve this issue , recently two models of nkg2d - deficiency were reported [ 9 , 139 ] . in klrk1 mice generated in our lab , nk cell development was moderately affected . in the absence of nkg2d we observed perturbations in the size of some developmental subsets of nk cells , increased proliferation of immature nk cells ( mostly in stage ii ) , their faster maturation and increased sensitivity to apoptosis . in addition , nk cell - mediated control of mcmv infection in these mice was better than in littermate controls , which we ascribed to the dysregulation and faster maturation of nk cells in the absence of nkg2d . our targeting inserted the egfp sequence in the third exon of the klrk1 locus , which could , as a foreign genetic element , cause non - specific effects . therefore , we generated klrk1 mice in which the egfp cassette is not present ( fig . these mice were obtained from the breeding of klrk1 mice ( our unpublished data ) and cre deleter mice , which resulted in the elimination of the floxed second and third exon of the klrk1 gene in vivo , a mutation that was further propagated as klrk1 . 2b ) , we analyzed the various stages of nk cell development in klrk1 versus klrk1 mice in comparison to wt animals ( fig . thus , these data strongly argue that the previously observed phenotype of nk cells in our klrk1 mice is the consequence of the desired klrk1 mutation rather than of a genetic deregulation caused by the egfp sequence.fig . 3comparative analysis of nk cell phenotype and function between wild type , klrk1 and klrk1 mice . b nkg2d expression on cd3cd19nk1.1 cells isolated from the spleen of klrk1 ( left panel ) and klrk1 ( right panel ) mice is shown comparative analysis of nk cell phenotype and function between wild type , klrk1 and klrk1 mice . the groups of mice were either nk cell depleted ( pk136 ) or treated with pbs . each symbol represents individual virus titers in indicated organs in conclusion , nkg2d appears to play an important role in nk cell development . it seems that its role in the early development of nk cells is different than during the mature stages where nkg2d is responsible for activation of effector functions . unleashed proliferation of immature nk cells and their increased sensitivity to apoptosis in the absence of nkg2d suggest that this receptor plays rather a regulatory role at this stage , most probably in concert with il-15r and perhaps some other receptors ( fig .

the wnt signaling network is a major regulator of mammalian development through control of cellular functions such as proliferation and differentiation . binding of wnt ligands , a family of secreted glycosylated proteins , to the frizzled and lrp families of cell surface receptors initiates a series of signaling events via the cytoplasmic protein dishevelled ( dsh ) . upon activation , dsh recruits axin and destabilizes the destruction complex that normally acts to degrade the transcriptional cofactor -catenin . in the absence of degradation , -catenin accumulates and translocates to the nucleus , where it recruits coactivators and associates with the lef / tcf family of dna - binding proteins , thereby altering expression of a variety of genes , including cyclin d1 and c - myc . in cells not subject to stimulation by wnt ligands , -catenin degradation by the destruction complex limits -catenin - mediated gene transcription . key components of the -catenin destruction complex include axin , apc ( the protein product of the adenomatous polyposis coli tumor suppressor gene ) , and glycogen synthase kinase-3- ( gsk3- ) , which phosphorylates -catenin and thereby renders -catenin a substrate for ubiquitination and degradation by the 26s proteasome . overexpression of wnt - regulated genes can cause transformation of mammalian epithelial cells . clinically , 80% of colon cancers have defects in the apc gene , leading to high levels of -catenin . a subset of colon cancers and melanomas harbor -catenin mutations that prevent its phosphorylation and subsequent degradation . many other cancers also show evidence of inappropriate wnt pathway activation , including raised levels of -catenin . in addition , defective wnt signaling plays a key role in the generation and maintenance of cancer stem cells . validation of a pivotal role for wnt signaling in cancer is therefore very strong , and compounds that dampen wnt pathway activity could provide useful molecularly targeted therapeutics for the treatment of cancer ; indeed , the fully humanized monoclonal antibody omp-18r5 that targets frizzled receptors is efficacious in patient - derived mouse tumor models and is currently in a phase i clinical trial . despite the importance of the wnt pathway , it is only recently that small - molecule inhibitors have been identified and progressed toward clinical trial . compound 1 ( icg001 ) blocks the interaction between -catenin and the transcriptional coactivating protein creb binding protein ( cbp ) , leading to a reduction of colon adenoma formation in mouse models . the -catenin cbp interaction was shown to promote stem / progenitor marker expression , while the related -catenin p300 interaction ( not inhibited by 1 ) promoted expression of genes involved in proliferative responses such as c - myc . compounds 2 ( xav939 ) and 3 ( iwr-1 ) have been disclosed as inhibitors of wnt signaling via inhibition of the tankyrase activity required for degradation of axin ; 2 was discovered by target deconvolution from a cell - based pathway screen.4 ( iwp-2 ) , a small - molecule inhibitor of porcupine , the acyltransferase essential for secretion of functional wnt ligands , was also discovered by cell - based pathway screening ( figure 1 ) . subsequent to the identification of tankyrase and porcupine as validated molecular targets , many small - molecule inhibitors have been reported . examplar tankyrase inhibitors include 5 ( nvp - tnks656),6 ( g007-lk ) , and compound 7(27 ) from amgen ; the porcupine inhibitor 8 ( lgk974 ) is currently in a phase i clinical trial . small - molecule inhibitors of the wnt pathway : 1,2,3,4 , 5,6,7 , and 8 . many wnt pathway mutations occur at , or upstream of , -catenin . therefore , if the pathway is blocked at or below this point , an inhibitor should be active against multiple tumors driven by a wnt - activating mutation . with this in mind , we set out to discover small - molecule inhibitors of wnt signaling and employed a cell - based pathway screening strategy to identify compounds that block wnt signaling at , or downstream of , -catenin . while our work was in progress a number of reports appeared describing successful cell - based pathway screens against the wnt pathway and , for example , bmp signaling revealing hitherto undiscovered regulatory mechanisms , increasing our confidence in such an approach . we have previously reported our screening strategy and describe here the medicinal chemistry optimization of a 3,4,5-trisubstituted pyridine hit ( 9 ) to give potent and orally bioavailable small - molecule inhibitors of wnt signaling . we demonstrate a 350-fold potency enhancement in a cell - based assay of wnt pathway activity through hypothesis - driven medicinal chemistry design respectful of physicochemical properties likely to elicit good cell permeability . our synthetic strategy to most desired 3,4,5-trisubstituted pyridines involved snar displacement of the 4-chloro substituent in either 3,4,5-trichloropyridine ( 10 ) or 3-bromo-4,5-dichloropyridine ( 11 ) using the appropriate n - boc - protected pyrrolidine 12 , piperidine 13 , or nh - piperidine 14 to give the 4-substituted analogues 9 , 1522 , 39 , 41 , 43 , 45 , 47 , 49 , 51 , 53 , 57 , and 84 according to our previously published methodology . subsequent palladium - mediated cross - coupling of appropriate aryl boronic acids or esters gave the corresponding 3,4,5-trisubstituted pyridines 26 , 2838 , 40 , 42 , 44 , 46 , 48 , 50 , 52 , 54 , and 6581 ( scheme 1 ) . compounds 59 , 60 , and 62 described in table 5 were prepared by sequential cross - coupling at the 3- and 5-positions of 8-(3-bromo-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one ( 57 ) . compounds 63 and 64 were prepared directly from 8-(3-chloro-5-(4-morpholin-4-ylphenyl)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one ( 44 ) , while compound 56 was prepared by snar displacement of the 4-chloro substituent in 3-bromo-4-chloro-5-methylpyridine , followed by palladium - mediated cross - coupling at the 3-position . preparations of noncommercial aryl boronic acids or esters are described in the experimental section and the supporting information . to discover inhibitors of wnt signaling , we used an inducible luciferase reporter assay in human embryonic kidney cells ( hek293 ) that contained both estrogen receptor - dsh and tcf - luciferase - ires - gfp constructs . gfp fluorescence was used to sort cells by flow cytometry that were highly induced by estradiol . clone 7df3 was selected for use in hts format and gave a 40-fold induction of luciferase activity when dsh protein expression was induced using 10 m estradiol . we screened 63 040 compounds at an assay concentration of 20 m and selected compounds that reproducibly gave > 50% inhibition in the 7df3 luciferase reporter assay ( n = 2 ) and did not inhibit luciferase enzymatic activity nor lead to > 50% cell death at 20 m . a control cell line incorporating a thymidine kinase promoter - driven renilla luciferase reporter construct was also utilized to deselect compounds that demonstrated potent and efficacious inhibition of non - wnt - mediated transcription . hit compound 9 ( ic50 = 1.76 m ) was the only exemplar of a 3,4,5-trisubstituted pyridine in our screening library at that time ; however , despite being a singleton , it was among the most attractive chemicals , with a low molecular weight and acceptable physicochemical properties ( table 1 and figure 2 ) . compound 9 blocked activation of the pathway by the gsk-3 inhibitor bromoindirubin-3-oxime ( bio ) ( ic50 = 2.60 m ) , indicative of action at or below gsk-3-mediated phosphorylation of -catenin . we then further characterized the locus of interaction for compound 9 using a cell - based assay cascade in which wnt signaling is activated at distinct levels by inducible expression of cdna - encoded activating components of the canonical wnt pathway ; a more robust development of our previously reported cell - based assay cascade . this cell - based assay cascade included activation of wnt signaling through expression of either n - lrp6 ( a constitutively activating component of the wnt receptor ) , dvl-2 ( a disheveled homologue and activator of wnt signaling ) , axin - gid ( axin - gsk3 interaction domain , a dominant negative form of axin that binds to and inhibits gsk-3 ) , n--catenin ( a stabilized form of -catenin resistant to degradation ) , or vp16-tcf ( a tcf transcription factor that is constitutively active in the absence of -catenin ) . compound 9 demonstrated potency and efficacy in all these cell - based assays , similar to the potency observed in the 7df3 primary assay and consistent with intracellular inhibition of wnt signaling at or below the tcf locale ( ic50 s = 0.97.6 m , table 2 ) . activating components of the canonical wnt pathway : n - lrp6 ( a constitutively activating component of the wnt receptor ) , dvl-2 ( a disheveled homologue and activator of wnt signaling ) , axin - gid ( axin - gsk3 interaction domain , a dominant negative form of axin that binds to and inhibits gsk-3 ) , n--catenin ( a stabilized form of -catenin resistant to degradation ) , or vp16-tcf ( a tcf transcription factor that is constitutively active in the absence of -catenin ) . in addition to promising potency in the 7df3 luciferase cell - based reporter assay ( ic50 = 1.76 m ) , compound 9 demonstrated moderate in vitro metabolic stability in human and mouse liver microsomes ( figure 2 ) . however , we were concerned that 9 may be a nonspecific pharmacophore for ion channels by virtue of the 4-aminopyridine motif . a measured pka of the pyridine nitrogen ( pka = 3.5 ) indicated that 9 is essentially nonbasic , in contrast to 4-aminopyridine ( pka = 9.2 ) and 4-(piperidin-1-yl)pyridine ( measured pka = 9.42 ) . the inductive electron - withdrawing effect of two m - chloro substituents is likely to significantly reduce the pka ( pyridine pka = 5.24 ; 3-chloropyridine pka = 2.84 ) . we hypothesized that a twisted conformation about the pyridine piperidine bond , driven by the steric bulk of the flanking chlorine atoms , may also reduce the basicity of compound 9 by abrogating electron donation from the 4-amino substituent . indeed , a twisted conformation was evident in the small molecule x - ray crystal structure of 9 with a torsion angle of 54.4 observed between the plane of the piperidine and pyridine rings [ figure 3 and supplementary figure s4 , supporting information ( si ) ] . thus , the 3,5-dichloro substitution pattern in compound 9 reduces the pka of the pyridine nitrogen ( inductive effect ) and induces a twisted conformation about the piperidine pyridine bond ( steric effect ) , which may further reduce the pka . the measured pka of compound 9 is consistent with an observed lack of in vitro ion channel pharmacology ( herg ic50 > 10 m ) and absence of cyp450 enzyme inhibition apart from weak inhibition of cyp3a4 ( ic50 = 7 m , supplementary table s1 , si ) ; these observations are in marked contrast to classical 4-aminopyridine - based ion channel modulators . small - molecule x - ray crystal structure of 9 ( panel a ) depicting a twisted conformation about the pyridine piperidine bond with the plane of piperidine ring tilted 54.4 against the plane of the aromatic ring ( panel b and supplementary figure s4 , si ) . data has been deposited with the cambridge crystallographic data centre ( deposition number ccdc 1020858 ) . in the absence of information about the precise biochemical target of compound 9 we hypothesized , first , that the h - bond donor ( hbd ) and acceptor ( hba ) properties of the primary amide moiety would be essential for activity ; second , that the pyridine nitrogen would be an essential component of activity ; and , third , that it would be important to maintain the observed twisted conformation about the pyridine piperidine bond . we initially set out to test these three hypotheses , cognisant in our compound design of the cell - based primary assay whereby true sar versus an intracellular biochemical target may be masked by poor cell penetration . hit compound 9 ( hbd count = 2 ; hba count = 4 ) has excellent passive cell permeability ( caco-2 : papp = 36 10 cm s , no efflux ) and compounds with equivalent or lower hbd / hba count were prioritized to avoid masking of sar trends against an intracellular target by poor cell permeability . methylation of the primary amide in compound 9 to give the secondary amide 15 or tertiary amide 16 led to 7- and 15-fold loss in activity , respectively , suggesting that at least one of the h - bond donors is required for activity . the greater loss of activity with more sterically demanding amides 1719 suggested a lack of space adjacent to the amide moiety . removal of the carboxamide completely ( compound 20 ) or removal of the carbonyl moiety ( compound 21 ) led to a reduction in potency compared with compound 9 , consistent with the hypothesis that both h - bond donor and acceptor functions of the carboxamide are important components of the pharmacophore ( table 1 ) . to explore the extent to which the pyridine nitrogen is essential for activity , we prepared compound 22 , where the pyridine nitrogen was replaced with a ch ( table 1 , entry 9 ) . a significant potency drop was observed , suggesting that the hydrogen bond acceptor capability of this nonbasic nitrogen is indeed crucial for activity . we next turned our attention to the requirement for a twisted conformation about the pyridine piperidine bond while the piperidine 4-carboxamide was kept constant . replacement of chlorine atoms with protons at both the 3- and 5-positions on the pyridine ring led to loss of activity , while replacement of one chloride led to a 13-fold loss in potency ( table 3 , entries 1 and 2 ) . by contrast , replacement of both chlorine atoms with bulkier bromines led to a 5-fold potency increase ( table 3 , entry 3 ) . pleasingly , replacement of one chlorine atom with a phenyl ring in compound 26 ( table 3 , entry 4 ) resulted in a 20-fold increase in potency over compound 9 ; however , the combination of a 3-phenyl , 5-h ( table 3 , entry 5 ) abrogated activity by 37-fold in comparison with 26 ( table 3 , entry 4 ) , consistent with a requirement for substitution at both the 3- and 5-positions of the pyridine ring . the increased potency of compound 26 was of particular interest , although in vitro microsomal stability was suboptimal . we set out to explore alternatives to the phenyl ring with greater stability to oxidative metabolism . replacement with 4-substituted pyrazoles led to a loss of activity ( table 3 , entries 6 and 7 ) , although some activity loss exhibited by compound 28 ( table 3 , entry 6 ) may result from the presence of an additional h - bond donor and consequent impaired cell penetration with respect to its matched pair ( table 3 , entry 7 ) . the thiophene analogue 30 , while among the most potent replacements , exhibited unsurprising metabolic instability , and pyridine replacements had decreased potency ( table 3 , entries 9 and 10 ) . substitution at the para - position of the phenyl ring proved more fruitful , with both electron - donating and -withdrawing substituents tolerated ( entries 1113 ) ; however , the strongly electron withdrawing sulfone was not tolerated ( entry 14 ) . increased potency of the p - methoxy analogue 33 in the 7df3 cell - based luciferase reporter assay ( ic50 = 0.037 m ) compared to 9 ( ic50 = 1.76 m ) was mirrored by increased potency and overall profile in cell - based assays incorporating different activating components of wnt signaling , suggesting that the locus of action on the wnt pathway had been maintained ( table 2 ) . mlm and hlm values indicate percent turnover after 30 min of incubation . although compound 33 was among the most potent , its metabolic stability was poor ; however , the p - morpholine derivative 38 ( table 3 , entry 16 ) proved equipotent , with acceptable metabolic stability , while the corresponding meta - derivative 37 was 8-fold less potent ( table 3 , entry 15 ) . despite acceptable metabolic stability , compound 38 demonstrated in vivo clearance greater than liver blood flow upon iv dosing in mouse pharmacokinetic studies ; we suspected amidase - mediated cleavage of the primary amide , although this proved difficult to definitively confirm . with compound 38 in hand , we were keen to further explore the piperidine carboxamide in order to understand the optimal geometry for the h - bond acceptor and donor motifs and to reduce the potential for putative amidase - mediated cleavage of the primary carboxamide . replacement of the 4-carboxamide by nitrile , a highly directional h - bond acceptor , maintained activity in the reporter assay and reinforced the notion that an appropriately positioned h - bond acceptor motif was the dominant feature ( table 4 , entry 1 ) . introduction of a geminal methyl group at the 4-position of the piperidine ring also maintained potency ( table 4 , entry 2 ) and suggested that spirocyclic carboxamides might be tolerated . with this result in hand , a range of 6,5-spirocyclic ring scaffolds was explored and , pleasingly , resulted in compounds with good potency and metabolic stability . the 2,8-diazaspiro[4.5]decan-1-one 44 exhibited 25-fold improved potency over the isomeric spirocycle 46 , consistent with a specific interaction from the amide motif ( table 4 , entries 3 and 4 ) . methylation of the amide ( entry 5 ) reduced potency 24-fold , indicating that , in this spirocyclic scaffold , both h - bond donor and acceptor motifs are important for activity . an isomeric spirocyclic ring system at the 3-position of the piperidine ring lost significant potency ( entry 6 ) , although potency could be restored with the corresponding 5,5-spirocyclic ring system incorporated as a racemate ( entry 7 ) . improving the h - bond donor ability of the spirocyclic nh by incorporation of an additional carbonyl functionality to give the acidic imide 54 resulted in a very potent and metabolically stable cell - based inhibitor of wnt pathway activity ; however , poor solubility ( 0.003 mg / ml in phosphate buffer at ph 7.4 ) was an unfavorable attribute and below our criteria for compound progression . considering all the data together , compound 44 presented the most promising profile , and we were keen to explore the extent to which the chloro substituent could be varied ( table 5 ) . replacement with small alkyl , cycloalkyl , aryl , or heteroaryl groups ( table 5 , entries 27 ) , all of which were designed to maintain cell penetration , resulted in maintenance of potency for the prop-1-en-2-yl substituent in 59 ( table 5 , entry 3 ) but a loss of potency for other exemplars , which we ascribed to a lack of toleration of steric bulk in this region of the putative binding site . in order to avoid potential metabolic liabilities with the presence of an alkene in compound 59 , we henceforth adopted chloride as the preferred substituent . mlm and hlm values indicate percentage parent remaining after 30 min of incubation . to benchmark the chemical series , we conducted mouse in vivo pharmacokinetic studies on compound 44 , which revealed improved in vivo clearance compared to compound 38 ; however , clearance remained unacceptably high with corresponding low oral bioavailability ( table 6 ) . despite an unfavorable pharmacokinetic profile , other properties of compound 44 were attractive : it was inactive when tested at 10 m across a panel of 55 receptors , ion channels , and enzymes and demonstrated no significant activity at 1 m across a broad panel of 209 nonmutated kinases ( see supplementary tables s2 and s3 , si ) ; in addition , 44 did not strongly inhibit the known intracellular wnt pathway target tankyrase ( tnks1 ic50 = 19.0 0 m , tnks2 ic50 = 16.0 1.41 m ) . permeability , as assessed by caco-2 flux , was high ( papp = 37 10 cm s ) with no efflux , while kinetic and thermodynamic aqueous solubilities were acceptable ( 164 m and 0.017 mg / ml in phosphate buffer at ph 7.4 , respectively ) and no plasma instability was observed . we concluded that the poor oral bioavailability was largely the result of high first pass clearance and adopted a more accurate measure of intrinsic metabolic clearance ( clint ) across three species to better inform our selections for in vivo pharmacokinetic studies . in these in vitro metabolism studies , compound 44 showed high intrinsic clearance in the mouse consistent with our in vivo observations ( table 7 ) . compound 44 also exhibited increased inhibition of cyp enzymes ( cyp2c9 , ic50 = 2.9 m ; cyp3a4 , ic50 = 2.1 m ) in comparison with the original hit 9 and generated a glutathione adduct in an in vitro reactive metabolite assay ( data not shown ) ; we were therefore keen to replace the morpholine moiety that has been implicated in reactive metabolite formation . a diverse set of compounds was prepared bearing saturated rings or aromatic heterocycles as morpholine replacements ( table 8 , entries 14 and 510 , respectively ) as well as 6,6- and 6,5-fused ring systems as isosteres for the 4-morpholinophenyl ( table 8 , entries 1113 ) . removal of either morpholine heteroatom reduced potency versus the parent compound 44 ( table 8 , entries 1 and 2 ) . incorporation of an unsubstituted piperazine proved detrimental , whereas the n - methylpiperazine 68 restored potency with some improvement in metabolic stability ; however , an unfavorable caco-2 permeability profile ( efflux ratio > 500 ) precluded further progression of this compound . pleasingly , 5- or 6-membered heterocyclic replacements for the morpholine provided compounds with good potency and equivalent or improved metabolic stability . the electron - withdrawing oxadiazole 70 proved the least potent modification in the set , consistent with the poor activity of the electron - withdrawing sulfone 36 ( table 3 , entry 14 ) . the n - methylpyrazole 74 proved more potent than its desmethyl matched pair 73 , possibly due to poorer cell penetration of the latter compound , while the regioisomeric pyrazole 71 also displayed lower potency . replacement of the 4-morpholinophenyl moiety with fused 6,6- or 5,6-heterocycles ( table 8 , entries 1113 ) retained potency but at the expense of higher metabolic clearance compared to compound 74 , which provided the optimal balance of potency and metabolic stability in this subseries . we were keen to explore the combination of the preferred 1-methyl-4-phenyl-1h - pyrazole with selected spirocyclic carboxamides from table 4 . in comparison with compound 74 , we noted that the corresponding 5,5-spirocyclic carboxamide 78 was significantly less metabolically stable as a racemate ( table 9 ) . the most potent enantiomer 80 ( absolute stereochemistry not assigned ) displayed the highest metabolic stability of the pair ; however , this was still inferior to the parent molecule 74 and halted further investigation of this compound . combining a spirocyclic imide with our favored 1-methyl-4-phenyl-1h - pyrazole substituent gave the potent and stable inhibitor 81 ; however , in common with other imides prepared in this series , aqueous solubility proved to be very poor ( < 0.001 mg / ml in phosphate buffer at ph 7.4 ) , which precluded further progression of this compound . 8-(3-chloro-5-(4-(1-methyl-1h - pyrazol-4-yl)phenyl)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one ( cct251545 , 74 ) was therefore selected for progression to in vivo studies . pharmacokinetic studies on compound 74 revealed moderate clearance in both mouse and rat with moderate to high oral bioavailability ; the volume of distribution in both species was low , consistent with the pharmacokinetic profile of a neutral compound ( table 10 ) . compound 74 displayed high flux ( papp = 24 10 cm s ) , acceptable efflux ( er = 2.5 ) , and minimal herg inhibition ( ic50 > 10 m ) ; aqueous kinetic and thermodynamic solubilities were also sufficient for further progression ( 94 m and 0.006 mg / ml , respectively , in ph 7.4 phosphate buffer ) . compound 74 demonstrated minimal activity when tested across a panel of 55 receptors , ion channels , and enzymes at 1 m ( table s4 , si ) ; weak inhibition of cyps 2c8 , 2c9 , and 3a4 was observed ( see supplementary table s1 , si ) ; however , this was not considered sufficient to preclude further investigation . testing versus a panel of 291 kinases at 1 m revealed weak inhibition of gsk3 and - as the only hits ( ic50 = 0.462 and 0.690 m , respectively ; see supplementary table s5 , si ) , insufficiently potent to account for the observed effects on cell - based wnt pathway activity ; moreover , inhibition of gsk3 compound 74 demonstrated weak inhibition of tankyrase enzymes ( tnks1 ic50 > 10 m , tnks2 ic50 = 15.0 0 m ) . to confirm activity on the wnt pathway , rt - pcr of 7df3 cells treated with 74 demonstrated a 12-fold inhibition of endogenous cmyc transcription ( ic50 = 2.3 nm , supplementary figure s1 , si ) , a commonly studied wnt target gene . compound 74 was also tested in human colorectal cancer cell lines ls174 t ( -catenin mutant ) and sw480 ( apc mutant ) that have constitutively activated wnt signaling or pa-1 human teratocarcinoma cells that are wnt ligand dependent ; 74 potently inhibited reporter - based readouts of basal wnt pathway activity in ls174 t and sw480 cells in the absence of wnt ligand or other stimulants ( ic50 = 0.023 0.011 m and 0.190 0.030 m , respectively ) and demonstrated potent inhibition of wnt3a ligand - dependent reporter readout in pa-1 cells ( ic50 = 0.007 m ) . no activity was observed in rko - f1522 cells bearing a firefly luciferase reporter downstream of the ef1- promoter , a commonly used , ubiquitously active , house - keeping promoter that has high activity in most mammalian cells , irrespective of their wnt dependence ( ic50 > 10 m ) . we developed an apc mutant human colorectal cancer cell line ( colo205-f1756 clone 4 ) engineered to express a modified luciferase - based wnt pathway reporter . compound 74 demonstrated potent inhibition of wnt pathway activity in this clone ( ic50 = 0.035 0.003 m , n = 8) ; in addition , we were able to demonstrate that compound 74 did not affect endogenous levels of tcf1 or tcf4 at concentrations that reduce luciferase expression in this cell line ( supplementary figure s2 , si ) . taken together , these data confirm that suppression of the wnt pathway activity by 74 is not unique to a specific cell line , is independent of exogenous wnt pathway stimulation , does not result from destabilization of tcf1 or tcf4 , and is not due to interference with the luciferase - based reporter readout . extension of wnt pathway inhibition to the whole animal setting was achieved using the apc mutant human colorectal cancer cell line colo205-f1756 clone 4 . mice bearing established colo205-f1756 clone 4 tumor xenografts were treated with compound 74 ( 70 mg / kg po bid for 9 days ) , at which point wnt reporter activity was measured by whole body bioluminescent imaging and therapeutic response by excised tumor weights . significant inhibition of wnt signaling was observed , and treated tumors were smaller than controls : flux normalized to tumor weight was reduced by 80% ( p = 0.0003 ) and tumor weights by 37.5% ( p = 0.0379 , mann whitney u test ; figure 4a , b ) . plasma and tumor levels of compound 74 were determined 2 and 6 h after the last dose , and at these time points , total plasma and tumor levels significantly exceeded those required for inhibition of luciferase reporter activity in a cell - based assay . taken together , these data demonstrate inhibition of wnt pathway activity in a human colorectal cancer xenograft model and some indication of tumor growth inhibition following oral dosing at exposures consistent with inhibition of wnt pathway activity . ( b ) reduced tumor growth with compound 74 in colo205 human colon cancer xenografts after oral dosing ( 70 mg / kg bid ) . ( c ) plasma and tumor total concentrations of 74 at 2 and 6 h after the last dose . we have described the optimization of a small - molecule inhibitor of the wnt pathway identified as a singleton from an hts of a diverse 63 040 compound library versus a cell - based luciferase reporter assay . application of classical hypothesis - driven , ligand - based sar enabled the discovery of highly potent cell - based wnt pathway inhibitors , with compound 74 demonstrating good oral pharmacokinetics in mouse and rat . with knowledge from our cell - based assay cascade , we hypothesized that this series of compounds acts at an intracellular target . therefore , our medicinal chemistry optimization necessitated design criteria likely to maintain cell permeability to avoid masking true wnt - pathway sar , and with this in mind , attention to h - bond donor and acceptor count was critical . the sar is consistent with the requirement for a distinct twisted conformation about the pyridine piperidine bond , as observed in the small - molecule x - ray crystal structure of the original hit compound 9 , and also is consistent with the lower pka of the pyridine nitrogen and the lack of significant ion channel pharmacology across exemplars of the chemical series . enhanced potency was achieved through maintenance of this twisted conformation and replacement of one of the chlorine atoms with a para - substituted aryl ring . methylpyrazole was found to be the optimal para - substituent in terms of potency , metabolic stability , and membrane permeability . combination of the favored methylpyrazole substituent with a 6,5-spirocyclic carboxamide , in place of the original piperidine carboxamide in hit compound 9 , led to 74 , a potent small - molecule inhibitor of the wnt pathway with good oral pharmacokinetics and in vivo activity against the wnt pathway . compound 74 also demonstrated a lack of transporter - mediated efflux and a lack of activity across broad in vitro panels of enzyme , receptor , and ion channel targets . further details of the in vivo pharmacological profile of this chemical tool , including studies to determine its molecular target , will be reported in due course . the canonical wnt pathway is of significant interest in oncology , and the paucity of druggable targets on the pathway has hampered drug discovery efforts . pathway - based screening approaches are becoming increasingly widespread as greater appreciation of the benefits of this strategy become apparent . in particular , hits from such screens , by necessity , have good cell permeability , and components of protein complexes are more likely to be in a relevant endogenous conformation , which may reveal new druggable binding sites or alternative targetable conformations of known sites ; moreover , key signal - determining pathway nodes are more likely to be discovered as a result of cell - based readouts at the transcriptional level . here we describe a successful example of hypothesis - driven medicinal chemistry optimization from a singleton hit against a cell - based assay readout for the wnt pathway in the absence of knowledge of the biochemical target . commercially available starting materials , reagents , and dry solvents were used as supplied . flash column chromatography was performed using merck silica gel 60 ( 0.0250.04 mm ) . column chromatography was also performed on a biotage sp1 purification system using thomson or biotage flash silica cartridges . ion exchange chromatography was performed using acidic isolute flash scx - ii columns or basic isolute flash nh2 columns . preparative hplc was conducted using a phenomenex luna column ( 5 m , 250 21.2 mm , c18 , phenomenex , torrance , ca ) using a gilson gx-281 liquid handler system combined with a gilson 322 hplc pump ( gilson , middleton , wi ) , over a 15 min gradient elution from 10:90 to 100:0 meoh : water ( both modified with 0.1% formic acid ) at a flow rate of 20 ml / min or over a 15 min gradient elution from 40:60 to 100:0 meoh : water ( both modified with 0.1% formic acid ) at a flow rate of 20 ml / min . vis spectra were acquired at 254 nm on a gilson 156 uv vis detector ( gilson , middleton , wi ) . collection was triggered by uv signal and collected using a gilson gx-281 liquid handler system ( gilson , middleton , wi ) . samples were prepared as solutions in a deuterated solvent and referenced to the appropriate internal nondeuterated solvent peak . the following internal references were used : cdcl3 ( c 77.0 ) , cd3od ( c 49.0 ) , and dmso - d6 ( c 39.5 ) . unobserved resonances for quaternary carbon atoms are denoted by cq not observed . lc ms and hrms analyses were performed on an agilent 1200 series hplc and diode array detector coupled to a 6210 time - of - flight mass spectrometer with dual multimode atmospheric pressure ci / esi source or coupled to an agilent 6520 quadrupole - time - of - flight mass spectrometer with dual multimode atmospheric pressure ci / esi source . lc ms analysis was also performed on a waters alliance 2795 separations module and waters 2487 dual wavelength absorbance detector coupled to a waters / micromass lct time - of - flight mass spectrometer with esi source . for method a , analytical separation was carried out at 40 c on a merck purospher star column ( rp-18e , 30 4 mm ) using a flow rate of 3 ml / min in a 2 min gradient elution with detection at 254 nm . the mobile phase was a mixture of methanol ( solvent a ) and water containing formic acid at 0.1% ( solvent b ) . gradient elution was as follows : 1:9 ( a / b ) to 9:1 ( a / b ) over 1.25 min , 9:1 ( a / b ) for 0.5 min , and then reversion back to 1:9 ( a / b ) over 0.15 min , and finally 1:9 ( a / b ) for 0.1 min . for method b , analytical separation was carried out at 30 c on a merck chromolith speedrod column ( rp-18e , 50 4.6 mm ) using a flow rate of 2 ml / min in a 4 min gradient elution with detection at 254 nm . the mobile phase was a mixture of methanol ( solvent a ) and water containing formic acid at 0.1% ( solvent b ) . gradient elution was as follows : 1:9 ( a / b ) to 9:1 ( a / b ) over 2.5 min , 9:1 ( a / b ) for 1 min , and then reversion back to 1:9 ( a / b ) over 0.3 min , and finally 1:9 ( a / b ) for 0.2 min . for method c , analytical separation was carried out at 30 c on a merck purospher star column ( rp-18e , 30 4 mm ) using a flow rate of 1.5 ml / min in a 4 min gradient elution with detection at 254 nm . the following reference masses were used for hrms analysis : caffeine [ m + h ] 195.087652 ; hexakis(1h,1h,3h - tetrafluoropentoxy)phosphazene [ m + h ] 922.009798 and hexakis(2,2-difluoroethoxy)phosphazene [ m + h ] 622.02896 or reserpine [ m + h ] 609.280657 . all compounds submitted for biological testing were determined to be > 95% pure by method a , b , or c. to a stirred suspension of 3,4,5-trichloropyridine ( 10 ) ( 500 mg , 2.74 mmol ) and piperidine-4-carboxamide ( 350 mg , 2.73 mmol ) in nmp ( 15 ml ) at ambient temperature was added triethylamine ( 770 l , 5.48 mmol ) in one portion . the separated aqueous layer was extracted with etoac ( 2 100 ml ) , and the combined organic extracts were washed with water ( 50 ml ) and brine ( 50 ml ) , dried over magnesium sulfate , filtered , and concentrated in vacuo to give a crude oily solid . purification was accomplished by flash chromatography on silica gel eluting with ch2cl2/1 m methanolic ammonia ( 95:5 to 92:8 ) to yield impure title compound ( 963 mg ) as a white solid . the solid was triturated in hot et2o ( 25 ml ) to give the title compound 9 as a white solid ( 519 mg , 69% yield ) : h nmr ( 500 mhz , cdcl3 ) 1.891.97 ( m , 4h ) , 2.332.40 ( m , 1h ) , 3.273.33 ( m , 2h ) , 3.383.44 ( m , 2h ) , 5.62 ( br s , 1h ) , 5.79 ( br s , 1h ) , 8.32 ( s , 2h ) ; c nmr ( 126 mhz , cdcl3 ) 29.4 , 42.4 , 50.1 , 128.6 , 149.1 , 151.7 , 176.9 ; lc ms ( method a , esi , m / z ) tr = 1.16 min , 274/276/278 ( m + h ) ; esi - hrms calcd for c11h14cl2n3o ( m + h ) 274.0508 , found 274.0509 . to a stirred suspension of 1-(3,5-dichloropyridin-4-yl)piperidine-4-carboxamide ( 9 ) ( 100 mg , 0.365 mmol ) , phenylboronic acid ( 49 mg , 0.401 mmol ) , and tetrakis(triphenylphosphine)palladium(0 ) ( 21 mg , 0.018 mmol ) in mecn ( 4 ml ) was added 0.5 m aqueous sodium carbonate solution ( 1.0 ml , 0.50 mmol ) . the reaction was heated to 150 c under microwave irradiation for 30 min . once cooled , the reaction was concentrated in vacuo and azeotroped with toluene ( 2 50 ml ) to give a gray , oily solid . purification was accomplished by flash chromatography on silica gel eluting with ch2cl2/meoh ( 97/3 ) and by semipreparitive hplc ( gemini column 250 10 , 15 min gradient , mecn / h2o 10:90 to 90:10 ) to give the title compound 26 as a white solid ( 41 mg , 36% yield ) : h nmr ( 500 mhz , cdcl3 ) 1.731.80 ( m , 4h ) , 2.122.20 ( m , 1h ) , 2.62 ( dt , j = 13.4 , 7.1 hz , 2h ) , 3.153.21 ( m , 2h ) , 5.40 ( s , 2h ) , 7.247.30 ( m , 2h ) , 7.387.51 ( m , 3h ) , 8.21 ( s , 1h ) , 8.44 ( s , 1h ) ; c nmr ( 126 mhz , cdcl3 ) 29.1 , 42.4 , 50.9 , 127.8 , 128.3 , 128.6 , 129.3 , 134.1 , 137.6 , 149.6 , 150.7 , 152.8 , 176.8 ; lc ms ( method a , esi , m / z ) tr = 0.99 min , 316/318 ( m + h ) ; esi - hrms calcd for c17h19cln3o ( m + h ) 316.1211 , found 316.1206 . to a stirred suspension of 1-(3,5-dichloropyridin-4-yl)piperidine-4-carboxamide ( 9 ) ( 70 mg , 0.26 mmol ) , 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)morpholine ( 92 mg , 0.32 mmol ) , and tetrakis(triphenylphosphine)palladium(0 ) ( 15 mg , 5 mol % ) in mecn ( 3 ml ) , at ambient temperature , was added 0.5 m aqueous sodium carbonate solution ( 720 l , 0.360 mmol ) . the reaction was heated to 150 c for 45 min under microwave irradiation . once cooled , the reaction was concentrated in vacuo and then dry - loaded onto silica . purification was accomplished by flash chromatography on silica gel ( ch2cl2/etoh 98:2 to 93:7 ) to yield the title compound 38 ( 53 mg , 52% yield ) as a pale orange / pink solid : h nmr ( 500 mhz , dmso - d6 ) 1.531.60 ( m , 4h ) , 2.042.11 ( m , 1h ) , 2.492.57 ( m , 2h ) , 3.003.06 ( m , 2h ) , 3.153.20 ( m , 4h ) , 3.733.78 ( m , 4h ) , 6.73 ( br s , 1h ) , 7.03 ( d , j = 8.7 hz , 2h ) , 7.18 ( d , j = 8.7 hz , 3h ) , 8.13 ( br s , 1h ) , 8.40 ( br s , 1h ) ; c nmr ( 126 mhz , cdcl3 ) 29.2 , 42.4 , 48.9 , 51.0 , 67.0 , 115.4 , 128.3 130.1 , 134.0 , 147.9 , 149.7 , 150.9 , 153.6 , 176.9 , ( 1 cq not observed ) ; lc ms ( method b , esi , m / z ) tr = 1.96 min , 401/403 ( m + h ) ; esi - hrms calcd for c21h26cln4o2 ( m + h ) 401.1739 , found 401.1746 . to a stirred suspension of 3,4,5-trichloropyridine ( 10 ) ( 509 mg , 2.79 mmol ) and 2,8-diazaspirodecan-1-one hydrochloride ( 410 mg , 2.66 mmol ) in nmp ( 14.6 ml ) , at ambient temperature , was added triethylamine ( 1.10 ml , 7.89 mmol ) . the reaction mixture was heated to 220 c under microwave irradiation for 60 min . nahco3 , the separated aqueous layer was extracted with etoac ( 2 100 ml ) , and the combined organic extracts were washed with brine ( 50 ml ) , dried over magnesium sulfate , filtered , and concentrated in vacuo to give a crude pale yellow oil . purification was accomplished by flash chromatography on silica gel eluting with ch2cl2/etoh ( 97.5:2.5 to 97:3 ) to give 8-(3,5-dichloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one ( 43 ) ( 422 mg , 53% yield ) as a white solid : h nmr ( 500 mhz , cdcl3 ) 1.53 ( br d , j = 13.1 hz , 2h ) , 2.072.18 ( m , 4h ) , 3.33 ( dt , j = 11.9 , 2.6 hz , 2h ) , 3.373.45 ( m , 4h ) , 6.66 ( br s , 1h ) , 8.33 ( br s , 2h ) ; lc ms ( method b , esi , m / z ) tr = 2.66 min , 300/302/304 ( m + h ) . to a stirred suspension of 8-(3,5-dichloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one ( 43 ) ( 70 mg , 0.23 mmol ) , 4-morpholinophenylboronic acid ( 60 mg , 0.29 mmol ) , and tetrakis(triphenylphosphine)palladium(0 ) ( 14 mg , 0.012 mmol ) in mecn ( 3 ml ) , at ambient temperature , was added 0.5 m aqueous sodium carbonate solution ( 650 l , 0.326 mmol ) . the reaction mixture was heated to 150 c under argon in the microwave for 60 min . once cooled , the reaction mixture was concentrated in vacuo and then dry - loaded onto silica . purification was accomplished by flash chromatography on silica gel eluting with ch2cl2/meoh ( 98:2 to 96:5 ) to give the title compound 44 ( 19 mg , 19% yield ) as a white solid : h nmr ( 500 mhz , cdcl3 ) 1.341.39 ( m , 2h ) , 1.922.01 ( m , 4h ) , 2.742.81 ( m , 2h ) , 3.17 ( dt , j = 13.0 , 4.0 hz , 2h ) , 3.213.25 ( m , 4h ) , 3.29 ( t , j = 6.9 hz , 2h ) , 3.873.90 ( m , 4h ) , 5.93 ( br s , 1h ) , 6.98 ( d , j = 8.7 hz , 2h ) , 7.20 ( d , j = 8.7 hz 2h ) , 8.17 ( br s , 1h ) , 8.39 ( br s , 1h ) ; c nmr ( 126 mhz , cdcl3 ) 31.8 , 32.5 , 38.7 , 41.8 , 47.7 , 49.1 , 67.0 , 115.4 , 128.3 , 128.9 , 130.2 , 134.0 , 149.2 , 150.8 , 151.0 , 152.9 , 181.7 ; lc ms ( method b , esi , m / z ) tr = 2.20 min , 427/429 ( m + h ) ; esi - hrms calcd for c23h28cln4o2 ( m + h ) 427.1895 , found 427.1903 . to a stirred suspension of 3,4,5-trichloropyridine ( 10 ) ( 215 mg , 1.18 mmol ) and 2,8-diazaspirodecan-1,3-dione ( 180 mg , 1.07 mmol ) in nmp ( 4.5 ml ) , at ambient temperature , was added triethylamine ( 450 l , 3.20 mmol ) . the reaction mixture was heated to 220 c under microwave irradiation for 60 min . once cooled , the mixture was diluted with meoh ( 20 ml ) and placed on an scx column ( 10 g ) . recrystallization from etoac gave 8-(3,5-dichloropyridin-4-yl)-2,8-diazaspiro[4.5]decane-1,3-dione ( 53 ) ( 206 mg , 62% yield ) as a cream solid : h nmr ( 500 mhz , cdcl3 ) 1.69 ( d , j = 13.4 hz , 2h ) , 2.28 ( ddd , j = 13.4 , 10.7 , 4.1 hz , 2h ) , 2.72 ( s , 2h ) , 3.32 ( ddd , j = 13.2 , 10.7 , 2.6 hz , 2h ) , 3.48 ( dt , j = 13.2 , 4.1 hz , 2h ) , 8.26 ( br s , 1h ) , 8.37 ( br s , 2h ) ; lc ms ( method b , esi , m / z ) tr = 2.43 min , 314/316/318 ( m + h ) . ten microwave vials were prepared as follows : to a stirred suspension of 8-(3,5-dichloropyridin-4-yl)-2,8-diazaspiro[4.5]decane-1,3-dione ( 53 ) ( 300 mg , 0.764 mmol ) , 4-morpholinophenylboronic acid ( 222 mg , 0.963 mmol ) , and tetrakis(triphenylphosphine)palladium ( 0 ) ( 100 mg , 0.0850 mmol ) in mecn ( 12 ml ) at ambient temperature was added 0.5 m aqueous sodium carbonate solution ( 3 ml ) . once cooled , the reaction mixtures from the ten vials were combined , concentrated in vacuo , and dry - loaded onto silica . purification was accomplished by flash chromatography ( hexane / etoac 30:70 ) to obtain the title compound 54 ( 950 mg , 25% yield ) as a white solid : h nmr ( 500 mhz , cdcl3 ) 1.481.54 ( m , 2h ) , 2.072.14 ( m , 2h ) , 2.55 ( s , 2h ) , 2.75 ( t , j = 11.7 hz , 2h ) , 3.183.23 ( m , 2h ) , 3.233.27 ( m , 4h ) , 3.883.92 ( m , 4h ) , 6.977.05 ( m , 2h ) , 7.177.24 ( m , 2h ) , 8.10 ( s , 1h ) , 8.25 ( s , 1h ) , 8.47 ( s , 1h ) ; c nmr ( 126 mhz , cdcl3 ) 33.3 , 40.6 , 44.2 , 47.2 , 48.7 , 66.8 , 115.2 , 127.8 , 130.0 , 148.1 , 150.0 , 150.9 , 153.0 , 174.9 , 181.4 , ( 2 cq not observed ) ; lc ms ( method b , esi , m / z ) tr = 2.20 min , 441/443 ( m + h ) ; esi - hrms calcd for c23h26cln4o3 ( m + h ) 441.1688 , found 441.1693 . a solution of 3-bromo-4-chloro-5-methylpyridine ( 300 mg , 1.45 mmol ) , triethylamine ( 610 l , 4.36 mmol ) , and 2,8-diazaspiro[4.5]decan-1-one hydrochloride ( 291 mg , 1.53 mmol ) in nmp ( 4.4 ml ) was heated to 220 c under microwave irradiation for 5 h at 220 c . nahco3 ( 100 ml each ) , the separated organic layer was extracted with etoac ( 2 75 ml ) , and the combined organic extracts were washed with brine ( 75 ml ) , dried over mgso4 , filtered , and concentrated in vacuo to give a crude yellow solid . the solid was recrystallized in hot etoac , filtered , washed with ether , and dried in vacuo to yield 8-(3-bromo-5-methylpyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one ( 55 ) ( 44 mg , 9% yield ) as an off white solid : h nmr ( 500 mhz , cdcl3 ) 1.511.58 ( m , 2h ) , 2.062.15 ( m , 2h ) , 2.16 ( t , j = 6.8 hz , 2h ) , 2.31 ( s , 3h ) , 3.18 ( br s , 2h ) , 3.33 ( br s , 2h ) , 3.39 ( t , j = 6.8 hz , 2h ) , 6.18 ( br s , 1h ) , 8.21 ( br s , 1h ) , 8.45 ( br s , 1h ) ; lc ms ( method c , esi , m / z ) tr = 1.42 min , 324/326 ( m + h ) . to a stirred solution of 8-(3-bromo-5-methylpyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one ( 55 ) ( 36 mg , 0.11 mmol ) , pd(dppf)cl2ch2cl2 ( 4.5 mg , 5.6 mol ) , and 4-morpholinophenylboronic acid ( 25 mg , 0.12 mmol ) in mecn ( 830 l ) , at ambient temperature , was added 0.5 m aqueous sodium carbonate solution ( 310 l , 0.150 mmol ) . the reaction mixture was sealed in a microwave vial under argon and heated to 120 c for 60 min under microwave irradiation . the reaction was concentrated in vacuo and then diluted with meoh / dcm and dry - loaded onto silica . purification was accomplished by flash chromatography on silica gel eluting with dcm / etoh ( 99:1 to 90:10 ) to yield the title compound 56 ( 20 mg , 44% yield ) as a clear colorless oil , which solidified on standing : h nmr ( 500 mhz , cdcl3 ) 1.281.34 ( m , 2h ) , 1.881.96 ( m , 4h ) , 2.31 ( s , 3h ) , 2.63 ( br t , j = 12.3 hz , 2h ) , 2.912.97 ( m , 2h ) , 3.203.24 ( m , 4h ) , 3.28 ( t , j = 6.9 hz , 2h ) , 3.873.91 ( m , 4h ) , 6.25 ( br s , 1h ) , 6.97 ( d , j = 8.7 hz , 2h ) , 7.18 ( d , j = 8.7 hz , 2h ) , 8.17 ( br s , 1h ) , 8.27 ( br s , 1h ) ; c nmr ( 126 mhz , cdcl3 ) 16.4 , 31.0 , 32.5 , 38.7 , 42.1 , 47.6 , 49.1 , 66.9 , 115.1 , 130.3 , 130.4 , 150.4 , 150.6 , 150.7 , 155.3 , 181.8 ; lc ms ( method c , esi , m / z ) tr = 1.70 min , 407 ( m + h ) ; esi - hrms calcd for c24h31n4o2 ( m + h ) 407.2442 , found 407.2447 . n - butyllithium ( 1.6 m in hexane , 7.15 ml , 11.4 mmol ) was added to a solution of diisopropylamine ( 1.70 ml , 11.9 mmol ) in thf ( 30 ml ) at 78 c . the reaction was stirred for 30 min and then 3-bromo-5-chloropyridine ( 2.00 g , 10.4 mmol ) in thf ( 10 ml ) was added dropwise over 7 min . the reaction was stirred for 45 min , resulting in a yellow / brown suspension . hexachloroethane ( 4.92 g , 20.8 mmol ) in thf ( 7 ml ) was added at 78 c and the dark brown reaction mixture was stirred at 78 c for 75 min . the cooling bath was removed and the brown suspension was allowed to warm to rt ( about 30 min ) . aq nh4cl ( 100 ml ) and the aqueous layer was extracted with ether ( 3 70 ml ) . the combined organic layers were washed with water ( 2 100 ml ) and brine ( 70 ml ) , dried over mgso4 , and filtered , and the solvent was evaporated in vacuo . the crude product was purified by flash chromatography ( ch2cl2/cyclohexane , 1:4 to 1:3 ) to give the 3-bromo-4,5-dichloropyridine ( 11 ) ( 2.05 g , 87% yield ) as a white solid : h nmr ( 500 mhz , cdcl3 ) 8.54 ( s , 1h ) , 8.64 ( s , 1h ) ; lc ms ( method b , esi , m / z ) tr = 3.01 min , 225/227/229 ( m + h ) . 3-bromo-4,5-dichloropyridine ( 11 ) ( 1.00 g , 4.41 mmol ) and boc-2,8-diazaspiro[4.5]decan-1-one ( 1.35 g , 5.29 mmol ) were introduced into a microwave vial with 1-methoxy-2-propanol ( 11 ml ) and triethylamine ( 1.80 ml , 13.2 mmol ) . the reaction mixture was heated under microwave irradiation for 2.5 h at 220 c . the solvent was evaporated and the crude material was purified via biotage column chromatography ( ch2cl2/etoh , 99.8:0.2 to 97:3 ) to give 8-(3-bromo-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one ( 57 ) ( 1.14 g , 75% yield ) as a white solid : h nmr ( 500 mhz , cdcl3 ) 1.521.58 ( m , 2h ) , 2.112.20 ( m , 4h ) , 3.323.41 ( m , 6h ) , 6.00 ( br s , 1h ) , 8.35 ( s , 1h ) , 8.49 ( s , 1h ) ; lc ms ( method b , esi , m / z ) tr = 2.68 min , 344/346/348 ( m + h ) . 8-(3-bromo-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one ( 57 ) ( 200 mg , 0.580 mmol ) , isopropenylboronic acid ( 120 l , 0.638 mmol ) , and pd(dppf)cl2ch2cl2 ( 24 mg , 0.029 mmol ) were loaded in a microwave vial under argon , and then acetonitrile ( 10.4 ml ) and 0.5 m aqueous sodium carbonate solution ( 1.60 ml , 0.812 mmol ) were added . the reaction mixture was heated at 120 c for 60 min under microwave irradiation , concentrated , and purified via biotage column chromatography ( dcm / etoh 99:1 to 95:5 ) to give 8-(3-chloro-5-(prop-1-en-2-yl)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one ( 58 ) as a colorless oil ( 122 mg , 69% yield ) : h nmr ( 500 mhz , cdcl3 ) 1.501.56 ( m , 2h ) , 2.072.14 ( m , 5h ) , 2.16 ( t , j = 6.9 hz , 2h ) , 3.153.20 ( m , 2h ) , 3.363.42 ( m , 4h ) , 4.974.98 ( m , 1h ) , 5.265.28 ( m , 1h ) , 5.81 ( s , 1h ) , 8.15 ( s , 1h ) , 8.39 ( s , 1h ) ; lc ms ( method c , esi , m / z ) tr = 1.78 min , 306/308 ( m + h ) . 8-(3-chloro-5-(prop-1-en-2-yl)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one ( 58 ) ( 40 mg , 0.13 mmol ) , 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)morpholine ( 57 mg , 0.20 mmol ) , sodium carbonate ( 35 mg , 0.33 mmol ) , and dichlorobis(tricyclohexylphosphine)palladium(ii ) ( 9.7 mg , 0.013 mmol ) were loaded in a microwave vial under argon , and then acetonitrile ( 1.0 ml ) and water ( 260 l ) were added . the reaction mixture was heated at 150 c for 35 min under microwave irradiation . the solvent was evaporated and the crude material was purified via biotage column chromatography ( dcm / etoh 98:2 to 90:10 ) to give the title compound 59 as a colorless oil ( 24.5 mg , 44% yield ) : h nmr ( 500 mhz , cdcl3 ) 1.211.25 ( m , 2h ) , 1.801.88 ( m , 2h ) , 1.96 ( t , j = 6.9 hz , 2h ) , 2.13 ( s , 3h ) , 2.722.79 ( m , 2h ) , 3.113.16 ( m , 2h ) , 3.213.24 ( m , 4h ) , 3.27 ( t , j = 6.9 hz , 2h ) , 3.873.91 ( m , 4h ) , 5.005.02 ( m , 1h ) , 5.245.26 ( m , 1h ) , 5.52 ( s , 1h ) , 7.00 ( d , j = 8.8 hz , 2h ) , 7.30 ( d , j = 8.8 hz , 2h ) , 8.17 ( s , 1h ) , 8.18 ( s , 1h ) ; lc ms ( method c , esi , m / z ) tr = 1.87 min , 433 ( m + h ) ; esi - hrms calcd for c26h33n4o2 ( m + h ) 433.2598 , found 433.2595 . 8-(3-(4-morpholinophenyl)-5-(prop-1-en-2-yl)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one ( 59 ) ( 5 mg , 0.01 mmol ) was solubilized in ethanol ( 0.5 ml ) and a few drops of dcm . then 1 m hcl ( 35 l , 0.035 mmol ) and palladium 10% on carbon ( 3 mg , 0.01 mmol ) were added . the reaction was hydrogenated under a balloon of hydrogen at rt for 2 days and filtered over celite . the crude material was filtered on a flash nh2 column and concentrated to give the title compound 60 as a colorless oil ( 4 mg , 80% yield ) : h nmr ( 500 mhz , cdcl3 ) 1.281.33 ( m , 8h ) , 1.882.00 ( m , 4h ) , 2.582.68 ( m , 2h ) , 2.852.92 ( m , 2h ) , 3.213.25 ( m , 4h ) , 3.253.29 ( m , 2h ) , 3.373.45 ( m , 1h ) , 3.883.92 ( m , 4h ) , 5.64 ( s , 1h ) , 6.966.99 ( m , 2h ) , 7.20 ( d , j = 8.3 hz , 2h ) , 8.15 ( s , 1h ) , 8.42 ( s , 1h ) ; c nmr ( 126 mhz , cdcl3 ) 24.0 , 26.2 , 30.5 , 32.4 , 38.6 , 42.0 , 48.5 , 49.2 , 66.9 , 115.1 , 130.4 , 130.6 , 134.3 , 140.9 , 147.9 , 150.3 , 150.5 , 154.5 , 181.6 ; lc ms ( method c , esi , m / z ) tr = 1.85 min , 435 ( m + h ) ; esi - hrms calcd for c26h35n4o2 ( m + h ) 435.2755 , found 435.2747 . 8-(3-bromo-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one ( 57 ) ( 35 mg , 0.10 mmol ) , 1-methyl-4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)piperazine ( 34 mg , 0.11 mmol ) , and pd(dppf)cl2ch2cl2 ( 3.7 mg , 5.1 mol ) were loaded in a microwave vial under argon , and then degassed mecn ( 1.8 ml ) and 0.5 m aqueous sodium carbonate solution ( 284 l , 0.142 mmol ) were added . the reaction mixture was heated under microwave irradiation at 120 c for 60 min . the solvent was evaporated and the crude material was dry - loaded and purified via biotage column chromatography ( ch2cl2/meoh 97/3 to 85/15 ) to give the title compound 68 ( 38 mg , 85% yield ) as a cream solid : h nmr ( 500 mhz , cdcl3 ) 1.321.38 ( m , 2h ) , 1.942.02 ( m , 4h ) , 2.39 ( s , 3h ) , 2.602.65 ( m , 4h ) , 2.702.78 ( m , 2h ) , 3.113.17 ( m , 2h ) , 3.273.31 ( m , 6h ) , 5.54 ( br s , 1h ) , 7.00 ( d , j = 8.7 hz , 2h ) , 7.19 ( d , j = 8.7 hz , 2h ) , 8.18 ( s , 1h ) , 8.39 ( s , 1h ) ; c nmr ( 126 mhz , cdcl3 ) 31.4 , 32.4 , 38.8 , 42.0 , 46.1 , 47.7 , 48.6 , 55.0 , 115.7 , 128.3 , 128.7 , 130.1 , 133.9 , 149.1 , 150.6 , 151.0 , 152.9 , 181.8 ; lc ms ( method c , esi , m / z ) tr = 1.43 min , 440/442 ( m + h ) ; esi - hrms calcd for c24h31cln5o ( m + h ) 440.2212 , found 440.2205 . 8-(3-bromo-5-chloro - pyridin-4-yl)-2,8-diaza - spiro[4.5]decan-1-one ( 57 ) ( 250 mg , 0.725 mmol ) , 1-methyl-4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-1h - pyrazole ( 82 ) ( 268 mg , 0.943 mmol ) , and pd(dppf)cl2ch2cl2 ( 30 mg , 0.036 mmol ) were introduced into a microwave vial , and then mecn ( 13 ml ) and aqueous sodium carbonate solution ( 0.5 m , 2.00 ml , 1.02 mmol ) were added . the reaction mixture was heated under microwave irradiation at 120 c for 1 h and concentrated . the crude material was purified via biotage column chromatography ( ch2cl2/etoh , 96:4 to 91:9 ) , and trituration with hot etoac gave the title compound 74 ( 700 mg , 76% yield ) as a white solid : h nmr ( 500 mhz , cdcl3 ) 1.331.39 ( m , 2h ) , 1.942.01 ( m , 4h ) , 2.712.79 ( m , 2h ) , 3.143.19 ( m , 2h ) , 3.28 ( t , j = 6.8 hz , 2h ) , 3.97 ( s , 3h ) , 5.58 ( br s , 1h ) , 7.29 ( d , j = 8.3 hz , 2h ) , 7.56 ( d , j = 8.3 hz , 2h ) , 7.68 ( s , 1h ) , 7.82 ( s , 1h ) , 8.22 ( s , 1h ) , 8.44 ( s , 1h ) ; c nmr ( 126 mhz , cdcl3 ) 31.6 , 32.4 , 38.7 , 39.3 , 41.8 , 47.8 , 122.7 , 125.6 , 127.2 , 128.3 , 129.8 , 132.3 , 133.7 , 135.7 , 136.9 , 149.6 , 150.9 , 152.9 , 181.6 ; lc ms ( method c , esi , m / z ) tr = 2.37 min , 422/424 ( m + h ) ; esi - hrms calcd for c23h25cln5o ( m + h ) 422.1742 , found 422.1730 . two microwave vials were prepared as follows : 1-methyl-4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-1h - pyrazole ( 82 ) ( 52 mg , 0.18 mmol ) , 8-(3-bromo-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decane-1,3-dione ( 84 ) ( 52 mg , 0.18 mmol ) , and pd(dppf)cl2ch2cl2 ( 5.7 mg , 7.0 mol ) were loaded into a microwave vial , and then 0.5 m aqueous sodium carbonate solution ( 390 l , 0.195 mmol ) and mecn ( 2.5 ml ) were added . the reaction mixture was heated at 120 c for 60 min under microwave irradiation . the reaction mixtures from the two vials were combined and the volatiles were evaporated . the crude material was purified via biotage column chromatography ( ch2cl2/etoh , 97:3 to 92:8 ) to give the title compound 81 ( 70 mg , 58% yield ) as a white solid : h nmr ( 500 mhz , dmso - d6 ) 1.461.52 ( m , 2h ) , 1.771.84 ( m , 2h ) , 2.47 ( s , 2h ) , 2.622.69 ( m , 2h ) , 3.023.08 ( m , 2h ) , 3.89 ( s , 3h ) , 7.34 ( d , j = 8.2 hz , 2h ) , 7.69 ( d , j = 8.2 hz , 2h ) , 7.95 ( d , j = 0.8 hz , 1h ) , 8.22 ( s , 1h ) , 8.23 ( s , 1h ) , 8.48 ( s , 1h ) , 11.12 ( br s , 1h ) ; c nmr ( 126 mhz , dmso - d6 ) 32.9 , 39.2 , 39.7 , 44.0 , 47.7 , 121.8 , 125.4 , 127.9 , 128.5 , 130.1 , 132.6 , 134.1 , 134.9 , 136.6 , 149.2 , 151.1 , 152.6 , 177.4 , 183.7 ; lc ms ( method c , esi , m / z ) tr = 2.30 min , 436/438 ( m + h ) ; esi - hrms calcd for c23h23cln5o2 ( m + h ) 436.1535 , found 436.1524 . 1-chloro-4-iodobenzene ( 6.39 g , 26.8 mmol ) , 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1h - pyrazole ( 5.58 g , 26.8 mmol ) , sodium carbonate ( 6.25 g , 59.0 mmol ) , and pd(dppf)cl2ch2cl2 ( 2.20 g , 2.68 mmol ) were loaded into a flask , and then a mixture of thf / h2o 3/1 ( 117 ml ) was added . the solvents were evaporated , and the residue was purified by column chromatography ( cyclohexane / etoac 99:1 to 70:30 ) to afford 4-(4-chlorophenyl)-1-methyl-1h - pyrazole as a white solid ( 3.80 g , 74% yield ) : h nmr ( 500 mhz , cdcl3 ) 3.93 ( s , 3h ) , 7.31 ( d , j = 8.7 hz , 2h ) , 7.38 ( d , j = 8.7 hz , 2h ) , 7.57 ( s , 1h ) , 7.72 ( s , 1h ) ; lc ms ( method b , esi , m / z ) tr = 2.88 min , 193/195 ( m + h ) . 4-(4-chlorophenyl)-1-methyl-1h - pyrazole ( 3.30 g , 17.1 mmol ) , bis(pinacolato)diboron ( 5.20 g , 20.6 mmol ) , potassium acetate ( 5.00 g , 51.4 mmol ) , xphos ( 650 mg , 1.37 mmol ) , and pd2dba3 ( 310 mg , 0.343 mmol ) were loaded in a flask , and dioxane ( 34 ml ) was added . the solvent was evaporated and the crude product purified by column chromatography ( cyclohexane / etoac , 90:10 to 70:30 ) to afford 1-methyl-4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-1h - pyrazole ( 82 ) as a white solid ( 3.90 g contaminated by 10% of 1-methyl-4-phenyl-1h - pyrazole , corrected yield 75% ) : h nmr ( 500 mhz , cdcl3 ) 1.35 ( s , 12h ) , 3.93 ( s , 3h ) , 7.47 ( d , j = 8.3 hz , 2h ) , 7.64 ( s , 1h ) , 7.79 ( d , j = 8.3 hz , 2h ) , 7.79 ( s , 1h ) ; lc ms ( method b , esi , m / z ) tr = 3.06 min , 284/285 ( m + h ) . to a suspension of 8-benzyl-2,8-diazaspiro[4.5]decane-1,3-dione ( 1.20 g , 4.65 mmol ) and concentrated acetic acid ( 270 l , 4.65 mmol ) in ethanol ( 20 ml ) was added palladium hydroxide ( 20 wt % on carbon , wet , 360 mg , 4.65 mmol ) and the mixture stirred under a h2 atmosphere for 24 h at rt . the mixture was filtered over celite and washed with etoh and 1 m nh3 in meoh . the filtrate was concentrated and purified on an scx2 cartridge ( loading with dcm , byproduct elution with dcm / meoh 9/1 , elution of product with dcm / meoh / nh3 9/1/0.01 ) to give 2,8-diazaspiro[4.5]decane-1,3-dione ( 83 ) ( 754 mg , 97% ) as a white solid : h nmr ( 500 mhz , dmso - d6 ) 1.41 ( dd , j = 12.5 , 1.4 hz , 2h ) , 1.65 ( td , j = 12.5 , 3.9 hz , 2h ) , 2.442.53 ( m , 2h ) , 2.56 ( s , 2h ) , 2.86 ( dt , j = 12.5 hz , 3.9 , 2h ) ; lc ms ( method b , esi , m / z ) tr = 0.17 min , 169 ( m + h ) . 3-bromo-4,5-dichloropyridine ( 11 ) ( 1.00 g , 4.41 mmol ) and 2,8-diazaspiro[4.5]decane-1,3-dione ( 83 ) ( 1.10 g , 6.61 mmol ) were introduced in a microwave vial , and then nmp ( 11 ml ) and triethylamine ( 1.90 ml , 13.2 mmol ) were added . the reaction mixture was heated under microwave irradiation for 1 h at 220 c . the reaction mixture was poured into water ( 110 ml ) and the precipitate was filtered to give 8-(3-bromo-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decane-1,3-dione ( 84 ) ( 1.08 g , 68% yield ) as a white solid : h nmr ( 500 mhz , dmso - d6 ) 1.641.70 ( m , 2h ) , 1.942.01 ( m , 2h ) , 2.69 ( s , 2h ) , 3.243.30 ( m , 4h ) , 8.47 ( s , 1h ) , 8.57 ( s , 1h ) , 11.18 ( br s , 1h ) ; lc ms ( method b , esi , m / z ) tr = 2.50 min , 358/360/362 ( m + h ) . compounds ( 10 m ) were incubated with male cd1 mouse liver microsomes ( 1 mg / ml ) protein in the presence of 1 mm nadph , 2.5 mm udp - glucuronic acid ( udpga ) , and 3 mm mgcl2 in 10 mm pbs at 37 c . control incubations were generated by the omission of nadph and udpga from the incubation reaction . compounds ( 10 m ) were incubated with mixed - gender , pooled human liver microsomes ( 1 mg / ml ) protein in the presence of 1 mm nadph , 2.5 mm udpga , and 3 mm mgcl2 in 10 mm pbs at 37 c . control incubations were generated by the omission of nadph and udpga from the incubation reaction . microsomes ( final concentration 0.5 mg / ml ) , 50 mm phosphate buffer ph 7.4 , and compound ( final concentration 1 m ) were added to the assay plate and allowed to preincubate for 5 min at 37 c . the reaction was initiated by the addition of nadph ( final concentration 1.5 mm ) and the plate was shaken at 800 rpm at 37 c . after 0 , 5 , 10 , 20 , and 30 min , aliquots were taken , and the reaction was stopped using cold acetonitrile . the samples were centrifuged at 4000 rpm for 30 min at 4 c and analyzed by lc caco-2 cells ( tc7 clone ) were maintained in dmem in an atmosphere of 8.5% co2 . for transport experiments , 0.125 10 cells / well were seeded on polycarbonate filter inserts and allowed to grow and differentiate for 1014 days before the cell monolayers were used for experiments . drug transport experiments were carried out using a cocktail approach in a 4-dimensional setting . apparent permeability coefficients were determined for a > b and b > a directions with and without the presence of cyclosporine a as a transporter inhibitor . up to five test items and reference compounds were dissolved in hank s balanced salt solution at ph 7.4 to yield a final concentration of 1 m . the assays were performed in hbss containing 25 mm hepes ( ph 7.4 ) in an atmosphere of 5% co2 at 37 c . prior to the study , the monolayers were washed in prewarmed hbss . at the start of the experiments , prewarmed hbss containing the test items was added to the donor side of the monolayer and hbss without test items was added to the receiver side . after 2 h , the transwell insert containing the monolayer was carefully removed and placed in a new plate , and aliquots of both the receiver and donor sides were taken and diluted with an equal volume of acn containing the internal standard . the apparent permeability coefficients ( papp ) were calculated using the formula papp = ( vrec / a c0donor ) dcrec / dt 10 with dcrev / dt being the change in concentration in the receiver compartment with time , vrec the volume of the sample in the receiver compartment , c0donor the concentration in the donor compartment at time 0 , and a the area of the compartment with the cells . protein binding of test compounds was determined by ultrafiltration using serum from mouse , rat , and human . the test items ( final concentration 5 m ) were incubated in triplicate with three different serum dilutions ( 1:2 , 1:5 , and 1:10 ) for 30 min at 37 c using slight agitation . after the incubation , the 96-well filter plates were centrifuged for 45 min at 3500 rpm and 37 c ; 25 l portions of filtrate samples were treated with 50 l of ethanol and 50 l of internal standard solution and analyzed by lc ms / ms . the fraction unbound was calculated from the drug concentrations in the filtrate samples . female nmri mice ( n = 5 ) received either a single intravenous ( bolus ) injection or a single oral administration ( by gavage ) of the compound in a cocktail preparation . doses of 0.2 mg / kg ( per compound , intravenous ) and 0.5 mg / kg ( per compound , orally ) were given as solutions in dmso / peg 200/water . consecutive blood samples were taken retroorbitally under isofluorane inhalation from n = 3 animals per route of administration after 0.1 ( iv only ) , 0.25 ( po only ) , 0.5 , 1 , 2 , 4 , and 6 h and were further processed to obtain plasma . in addition , feces of two mice per route of administration were collected over the time interval from 0 to 24 ( pooled for analysis , no blood sampling ) . male wistar rats ( n = 6 ) received either a single intravenous ( bolus ) injection or a single oral administration ( by gavage ) of the compound in a cocktail preparation . doses of 0.2 mg / kg ( per compound , intravenous ) and 0.5 mg / kg ( per compound , orally ) were given as solutions in dmso / peg 200/water . consecutive blood samples were taken sublingually under isofluorane inhalation from n = 3 animals per route of administration after 0.1 ( iv only ) , 0.25 ( po only ) , 0.5 , 1 , 2 , 4 , and 6 h and were further processed to obtain plasma . in addition , feces and urine of three rats per route of administration were collected over the time interval from 0 to 24 ( pooled for analysis , no blood sampling ) . the concentrations of compound in plasma and feces were quantified using an uplc method with tandem mass spectrometric detection ( lc ms / ms ) . ms system consisted of a waters acquity uplc coupled to an ab sciex mass spectrometer api 5500 q - trap . the uplc separation was carried out on a reversed - phase column ( acquity uplc bec c8 , 1.7 m , 2.1 50 mm ) using a mobile phase gradient with 0.1% formic acid and acetonitrile as eluents . the detection of the drug was performed using multiple reaction monitoring in the positive ionization mode . plasma samples were spiked with internal standard , and the analyte was extracted from the matrix using tert - butyl methyl ether ( tbme ) . feces samples were homogenized with 4 times their volume of ethanol / water ( 4:1 ) . aliquots of the aqueous ethanolic extracts were further diluted with acetonitrile/0.1% formic acid , spiked with internal standard , and directly injected into the system . the autoparsylation assay was run in two steps : first , the enzymatic reaction in which gst - tagged tnks1 or tnks2 transferred biotinylated adp - ribose to itself from biotinylated nad as the cosubstrate and , second , the detection reaction where a time - resolved fret between cryptate - labeled anti - gst bound to the gst tag of the enzyme and xlent - labeled streptavidin bound the biotin - parsylation residue was analyzed . the autoparsylation activity was detectable directly via the increase in htrf signal . the autoparsylation assay was performed in 384-well htrf ( cisbio , codolet , france ) assay format in greiner low - volume 384-well microtiter plates ; 250 nm gst - tagged tnks1 ( 10231327 aa ) or 250 nm gst - tagged tnks2 ( 8731166 aa ) and 5 m bio - nad ( biolog , life science inst . , bremen , germany ) as cosubstrate were incubated in a total volume of 5 l ( 50 mm hepes , 4 mm mgcl2 , 0.05% pluronic f-68 , 1.4 mm dtt , 0.5% dmso , ph 7.7 ) in the absence or presence of the test compound ( 10 dilution concentrations ) for 90 min at 30 c . the reaction was stopped by the addition of 1 l of 50 mm edta solution , and 2 l of the detection solution [ 1.6 m sa - xlent ( cisbio , codolet , france ) , 7.4 nm anti - gst - k ( eu - labeled anti - gst , cisbio , codolet , france ) in 50 mm hepes , 800 mm kf , 0.1% bsa , 20 mm edta , 0.1% chaps , ph 7.0 ) ] was added . after 1 h of incubation at rt the htrf was measured with an envision multimode reader ( perkinelmer las germany gmbh ) at an excitation wavelength of 340 nm ( laser mode ) and emission wavelengths of 615 and 665 nm . the pharmacological zero value used was xav-939 ( tocris ) in a final concentration of 5 m . the inhibitory values ( ic50 ) were determined using either the program symyx assay explorer or condosseo from genedata . 7df3 cells were seeded at 20 000 cells / well into 96-well cell culture plates . after overnight incubation the cells were treated with 74 at concentrations ranging from 9.1 m to 0.068 nm . after 2 or 6 h of incubation , cells were washed with pbs and stored at 80 c . cells were lysed in cells - to - cdna ii cell lysis buffer ( life technologies ltd . , paisley , uk ) and transferred to pcr plates . lysates were dnase treated ( 1 l / well life technologies ) at 37 c for 15 min and heat inactivated at 75 c for 5 min . cdna synthesis was performed using the high - capacity cdna reverse transcription kit ( life technologies ) . taqman reactions were performed in 384-well plates using 1.5 l cdna and 1 fam - labeled primer probe for the measurement of target gene and 1 vic - labeled primer probe for lrp0 and were multiplexed in each well as the internal reference control with taqman universal master mix ( life technologies ) . plates were run for 40 cycles using an abi via7 sequence detection system and software . ct values for target genes were normalized relative to the lrp0 control before being expressed relative to the vehicle treated control sample . 7df3 cells were seeded at 5000 per well in 50 l of dulbecco s modified eagle s medium ( dmem ) with 4 mm supplemental l - glutamine in 384-well white tissue culture plates ( corning 3570 ) . after incubating overnight at 37 c/5% co2 , compounds ranging in final concentration from 90 m to 0.3 nm were added in duplicate to the wells . after 2 h of further incubation as above , -oestradiol was added to a final concentration of 10 m . the cells were incubated for 24 h at 37 c/5% co2 and then 25 l of luciferase reagent ( steadyglo , promega ) was added , and the cells were mixed . after leaving the plate for 60 min at room temperature , luminescence was read on a plate luminescence reader ( topcount , perkinelmer ) . the percentage inhibition for each compound concentration was calculated using total counts ( no compound ) and low counts ( no -oestradiol ) as highs and lows , respectively . ic50 was subsequently determined using a curve - fitting software package in excel ( excelfit , idbs ) . t - rex hek293 tet - o cells ( life technologies ) were transfected with the tcf - firefly luciferase - ires - gfp ( tlig ) reporter , and a highly responsive reporter clone was derived using an analogous process to that previously used to derive the hek293-based 7df3 cell line . expression constructs coding for wnt inducers that act at different levels of the pathway were introduced downstream of the tet - o promoter by flp - frt - site - directed recombination . this generated a set of cell lines that contain an identical tcf - luciferase reporter in combination with tet - inducible cdnas encoding n - lrp6 , dvl2 , axin - gid , n--catenin , and vp16-tcf4 . cells were seeded from frozen at 40 000/well into 96-well plates in 80 l of medium . on day 3 , eight compound concentrations ranging from 30 m to 14 nm were prepared at 333 final concentration in dmso and then diluted to 5 final concentration in optimem ( life technologies ) supplemented with 0.05 g / ml doxycycline hyclate ( sigma - aldrich ) . these solutions were then pipetted at 20 l / well into each well of the plate . on day 4 , medium was removed , and cells were lysed in 30 l / well glolysis buffer ( promega ) for 30 min . brightglo firefly luciferase substrate ( promega ) was pipetted at 30 l / well . firefly luciferase activity was measured on a fluostar plate reader ( bmg ) in luminescence mode . sw480 human colorectal cancer cells were cotransfected with topflash and cmv - lacz reporter plasmids as described previously . rko ( atcc crl-2577 ) cells were transfected with plasmid f1522 encoding pest - destabilized firefly luciferase ( luc2p , designated fluc2p ; promega ) and ires - driven pest - destabilized renilla ( hrlucp ; promega ) downstream of the ef-1- promoter . ls174 t ( atcc cl-188 ) and colo205 ( atcc , # ccl-222 ) human colorectal cancer cells were transduced with a tcf / lef lentiviral firefly luciferase reporter construct . this construct was generated by synthesizing and cloning 16 repeats of the tcf / lef responsive element ( 5-agatcaaagg-3 ) upstream of the minimal pta promoter driving pest - destabilized firefly luciferase fluc2p ( t1/2 < 1 h ) from plasmid pgl4.24 ( promega ) . the tcf / lef - pta - fluc2p cassette was subcloned into lentiviral vector pcdf1-mcs2-ef1-puro ( systems biosciences ) to generate the final reporter construct f1756 , which contained a puromycin resistance gene for selection purposes . f1756 was cotransfected with ppackf1 packaging mix ( systems biosciences ) into the hek293 cell line . f1756 reporter lentivirus was recovered in cell media after 48 h , purified , and used to transduce cell lines . after 72 h , successfully transduced cells were selected using 5 g / ml puromycin and stable colonies left to grow for 2 weeks . colonies showing high - level basal luciferase expression on addition of luciferin to the flask and imaging on an ivis 200 ( perkinelmer inc . ) subsequent studies identified clone 4 as a suitable candidate for further studies . reporter activity ( and its response to inhibition by compounds ) was measured in vitro in 384-well plates using steady - glo luciferase reagent ( promega ) for firefly luciferase . pa-1 human teratocarcinoma cells were stably transfected with a topflash tcf reporter plasmid ( merck millipore ) that was modified by introduction of a neomycin resistance marker . single clones were selected and tested for wnt - driven reporter pathway activation by addition of wnt-3a supernatant produced by l wnt-3a cells ( atcc , crl-2647 ) . reporter activity was measured in 96-well plates using steady - glo luciferase reagent ( promega ) . cells were treated with compound for 6 h and subsequently stimulated with wnt-3a supernatant for 24 h before reporter assays were conducted . female athymic crtac : ncr - foxn1 mice from taconic ( 68 weeks of age ) were injected subcutaneously in the flank with 3 million colo205-f1756 clone 4 cells , and when tumor xenografts reached a mean diameter of 56 mm ( equating to a mean volume of about 100 mm ) , mice ( n = 8 per group ) were dosed orally by gavage with compound 74 ( 70 mg / kg bid for 8 days ) or vehicle ( 10% dimethyl sulfoxide ; 5% tween 20 in sterile saline ) . luminescent flux as a measure of wnt pathway activity was quantified by whole body imaging in cohorts of four animals at 2 and 6 h after the final dose . this was achieved by sc injection of 100 l of 30 mg / ml d - luciferin firefly substrate ( xr-1001 , perkinelmer ) into anaesthetized mice and imaging with a xenogen ivis 200 ( xr-1001 , perkinelmer ) , utilizing the software living image ( version 4 ) . at these time points , heparinized blood samples were collected by cardiac puncture under terminal anesthesia , and plasma was separated by centrifugation . tumors were excised and weighed , and both plasma and tumor samples were snap frozen for pk analyses . the concentrations of compound in plasma and tumor were quantified using an uplc method with tandem mass spectrometric detection ( lc ms / ms ) . ms system consisted of a 1290 binary pump from agilent coupled to an agilent 6420 triple quadrupole mass spectrometer . the uplc separation was carried out on a reversed - phase column ( kinetic column c18 , 2.6 m , 2.1 50 mm purchased from phenomenex ) using a mobile phase gradient with 0.1% formic acid and methanol as eluent . detection of the drug was performed using multiple reaction monitoring in the positive ionization mode . plasma samples were spiked with internal standard , and the analyte was extracted from the matrix ( plasma and tumor homogenized with 3 v / w pbs ) with 34 volumes of methanol and directly injected into the system .

wnt signaling is frequently deregulated in malignancy , particularly in colon cancer , and plays a key role in the generation and maintenance of cancer stem cells . we report the discovery and optimization of a 3,4,5-trisubstituted pyridine 9 using a high - throughput cell - based reporter assay of wnt pathway activity . we demonstrate a twisted conformation about the pyridine piperidine bond of 9 by small - molecule x - ray crystallography . medicinal chemistry optimization to maintain this twisted conformation , cognisant of physicochemical properties likely to maintain good cell permeability , led to 74 ( cct251545 ) , a potent small - molecule inhibitor of wnt signaling with good oral pharmacokinetics . we demonstrate inhibition of wnt pathway activity in a solid human tumor xenograft model with evidence for tumor growth inhibition following oral dosing . this work provides a successful example of hypothesis - driven medicinal chemistry optimization from a singleton hit against a cell - based pathway assay without knowledge of the biochemical target .

Introduction Chemistry Results and Discussion Conclusions Experimental Section

in addition , defective wnt signaling plays a key role in the generation and maintenance of cancer stem cells . validation of a pivotal role for wnt signaling in cancer is therefore very strong , and compounds that dampen wnt pathway activity could provide useful molecularly targeted therapeutics for the treatment of cancer ; indeed , the fully humanized monoclonal antibody omp-18r5 that targets frizzled receptors is efficacious in patient - derived mouse tumor models and is currently in a phase i clinical trial . despite the importance of the wnt pathway , it is only recently that small - molecule inhibitors have been identified and progressed toward clinical trial . compounds 2 ( xav939 ) and 3 ( iwr-1 ) have been disclosed as inhibitors of wnt signaling via inhibition of the tankyrase activity required for degradation of axin ; 2 was discovered by target deconvolution from a cell - based pathway screen.4 ( iwp-2 ) , a small - molecule inhibitor of porcupine , the acyltransferase essential for secretion of functional wnt ligands , was also discovered by cell - based pathway screening ( figure 1 ) . small - molecule inhibitors of the wnt pathway : 1,2,3,4 , 5,6,7 , and 8 . with this in mind , we set out to discover small - molecule inhibitors of wnt signaling and employed a cell - based pathway screening strategy to identify compounds that block wnt signaling at , or downstream of , -catenin . while our work was in progress a number of reports appeared describing successful cell - based pathway screens against the wnt pathway and , for example , bmp signaling revealing hitherto undiscovered regulatory mechanisms , increasing our confidence in such an approach . we have previously reported our screening strategy and describe here the medicinal chemistry optimization of a 3,4,5-trisubstituted pyridine hit ( 9 ) to give potent and orally bioavailable small - molecule inhibitors of wnt signaling . we demonstrate a 350-fold potency enhancement in a cell - based assay of wnt pathway activity through hypothesis - driven medicinal chemistry design respectful of physicochemical properties likely to elicit good cell permeability . hit compound 9 ( ic50 = 1.76 m ) was the only exemplar of a 3,4,5-trisubstituted pyridine in our screening library at that time ; however , despite being a singleton , it was among the most attractive chemicals , with a low molecular weight and acceptable physicochemical properties ( table 1 and figure 2 ) . we then further characterized the locus of interaction for compound 9 using a cell - based assay cascade in which wnt signaling is activated at distinct levels by inducible expression of cdna - encoded activating components of the canonical wnt pathway ; a more robust development of our previously reported cell - based assay cascade . this cell - based assay cascade included activation of wnt signaling through expression of either n - lrp6 ( a constitutively activating component of the wnt receptor ) , dvl-2 ( a disheveled homologue and activator of wnt signaling ) , axin - gid ( axin - gsk3 interaction domain , a dominant negative form of axin that binds to and inhibits gsk-3 ) , n--catenin ( a stabilized form of -catenin resistant to degradation ) , or vp16-tcf ( a tcf transcription factor that is constitutively active in the absence of -catenin ) . compound 9 demonstrated potency and efficacy in all these cell - based assays , similar to the potency observed in the 7df3 primary assay and consistent with intracellular inhibition of wnt signaling at or below the tcf locale ( ic50 s = 0.97.6 m , table 2 ) . activating components of the canonical wnt pathway : n - lrp6 ( a constitutively activating component of the wnt receptor ) , dvl-2 ( a disheveled homologue and activator of wnt signaling ) , axin - gid ( axin - gsk3 interaction domain , a dominant negative form of axin that binds to and inhibits gsk-3 ) , n--catenin ( a stabilized form of -catenin resistant to degradation ) , or vp16-tcf ( a tcf transcription factor that is constitutively active in the absence of -catenin ) . in addition to promising potency in the 7df3 luciferase cell - based reporter assay ( ic50 = 1.76 m ) , compound 9 demonstrated moderate in vitro metabolic stability in human and mouse liver microsomes ( figure 2 ) . we hypothesized that a twisted conformation about the pyridine piperidine bond , driven by the steric bulk of the flanking chlorine atoms , may also reduce the basicity of compound 9 by abrogating electron donation from the 4-amino substituent . indeed , a twisted conformation was evident in the small molecule x - ray crystal structure of 9 with a torsion angle of 54.4 observed between the plane of the piperidine and pyridine rings [ figure 3 and supplementary figure s4 , supporting information ( si ) ] . thus , the 3,5-dichloro substitution pattern in compound 9 reduces the pka of the pyridine nitrogen ( inductive effect ) and induces a twisted conformation about the piperidine pyridine bond ( steric effect ) , which may further reduce the pka . small - molecule x - ray crystal structure of 9 ( panel a ) depicting a twisted conformation about the pyridine piperidine bond with the plane of piperidine ring tilted 54.4 against the plane of the aromatic ring ( panel b and supplementary figure s4 , si ) . in the absence of information about the precise biochemical target of compound 9 we hypothesized , first , that the h - bond donor ( hbd ) and acceptor ( hba ) properties of the primary amide moiety would be essential for activity ; second , that the pyridine nitrogen would be an essential component of activity ; and , third , that it would be important to maintain the observed twisted conformation about the pyridine piperidine bond . we initially set out to test these three hypotheses , cognisant in our compound design of the cell - based primary assay whereby true sar versus an intracellular biochemical target may be masked by poor cell penetration . we next turned our attention to the requirement for a twisted conformation about the pyridine piperidine bond while the piperidine 4-carboxamide was kept constant . pleasingly , replacement of one chlorine atom with a phenyl ring in compound 26 ( table 3 , entry 4 ) resulted in a 20-fold increase in potency over compound 9 ; however , the combination of a 3-phenyl , 5-h ( table 3 , entry 5 ) abrogated activity by 37-fold in comparison with 26 ( table 3 , entry 4 ) , consistent with a requirement for substitution at both the 3- and 5-positions of the pyridine ring . increased potency of the p - methoxy analogue 33 in the 7df3 cell - based luciferase reporter assay ( ic50 = 0.037 m ) compared to 9 ( ic50 = 1.76 m ) was mirrored by increased potency and overall profile in cell - based assays incorporating different activating components of wnt signaling , suggesting that the locus of action on the wnt pathway had been maintained ( table 2 ) . replacement of the 4-carboxamide by nitrile , a highly directional h - bond acceptor , maintained activity in the reporter assay and reinforced the notion that an appropriately positioned h - bond acceptor motif was the dominant feature ( table 4 , entry 1 ) . improving the h - bond donor ability of the spirocyclic nh by incorporation of an additional carbonyl functionality to give the acidic imide 54 resulted in a very potent and metabolically stable cell - based inhibitor of wnt pathway activity ; however , poor solubility ( 0.003 mg / ml in phosphate buffer at ph 7.4 ) was an unfavorable attribute and below our criteria for compound progression . replacement with small alkyl , cycloalkyl , aryl , or heteroaryl groups ( table 5 , entries 27 ) , all of which were designed to maintain cell penetration , resulted in maintenance of potency for the prop-1-en-2-yl substituent in 59 ( table 5 , entry 3 ) but a loss of potency for other exemplars , which we ascribed to a lack of toleration of steric bulk in this region of the putative binding site . testing versus a panel of 291 kinases at 1 m revealed weak inhibition of gsk3 and - as the only hits ( ic50 = 0.462 and 0.690 m , respectively ; see supplementary table s5 , si ) , insufficiently potent to account for the observed effects on cell - based wnt pathway activity ; moreover , inhibition of gsk3 compound 74 demonstrated weak inhibition of tankyrase enzymes ( tnks1 ic50 > 10 m , tnks2 ic50 = 15.0 0 m ) . to confirm activity on the wnt pathway , rt - pcr of 7df3 cells treated with 74 demonstrated a 12-fold inhibition of endogenous cmyc transcription ( ic50 = 2.3 nm , supplementary figure s1 , si ) , a commonly studied wnt target gene . compound 74 was also tested in human colorectal cancer cell lines ls174 t ( -catenin mutant ) and sw480 ( apc mutant ) that have constitutively activated wnt signaling or pa-1 human teratocarcinoma cells that are wnt ligand dependent ; 74 potently inhibited reporter - based readouts of basal wnt pathway activity in ls174 t and sw480 cells in the absence of wnt ligand or other stimulants ( ic50 = 0.023 0.011 m and 0.190 0.030 m , respectively ) and demonstrated potent inhibition of wnt3a ligand - dependent reporter readout in pa-1 cells ( ic50 = 0.007 m ) . no activity was observed in rko - f1522 cells bearing a firefly luciferase reporter downstream of the ef1- promoter , a commonly used , ubiquitously active , house - keeping promoter that has high activity in most mammalian cells , irrespective of their wnt dependence ( ic50 > 10 m ) . compound 74 demonstrated potent inhibition of wnt pathway activity in this clone ( ic50 = 0.035 0.003 m , n = 8) ; in addition , we were able to demonstrate that compound 74 did not affect endogenous levels of tcf1 or tcf4 at concentrations that reduce luciferase expression in this cell line ( supplementary figure s2 , si ) . taken together , these data confirm that suppression of the wnt pathway activity by 74 is not unique to a specific cell line , is independent of exogenous wnt pathway stimulation , does not result from destabilization of tcf1 or tcf4 , and is not due to interference with the luciferase - based reporter readout . significant inhibition of wnt signaling was observed , and treated tumors were smaller than controls : flux normalized to tumor weight was reduced by 80% ( p = 0.0003 ) and tumor weights by 37.5% ( p = 0.0379 , mann whitney u test ; figure 4a , b ) . plasma and tumor levels of compound 74 were determined 2 and 6 h after the last dose , and at these time points , total plasma and tumor levels significantly exceeded those required for inhibition of luciferase reporter activity in a cell - based assay . taken together , these data demonstrate inhibition of wnt pathway activity in a human colorectal cancer xenograft model and some indication of tumor growth inhibition following oral dosing at exposures consistent with inhibition of wnt pathway activity . ( b ) reduced tumor growth with compound 74 in colo205 human colon cancer xenografts after oral dosing ( 70 mg / kg bid ) . we have described the optimization of a small - molecule inhibitor of the wnt pathway identified as a singleton from an hts of a diverse 63 040 compound library versus a cell - based luciferase reporter assay . application of classical hypothesis - driven , ligand - based sar enabled the discovery of highly potent cell - based wnt pathway inhibitors , with compound 74 demonstrating good oral pharmacokinetics in mouse and rat . therefore , our medicinal chemistry optimization necessitated design criteria likely to maintain cell permeability to avoid masking true wnt - pathway sar , and with this in mind , attention to h - bond donor and acceptor count was critical . the sar is consistent with the requirement for a distinct twisted conformation about the pyridine piperidine bond , as observed in the small - molecule x - ray crystal structure of the original hit compound 9 , and also is consistent with the lower pka of the pyridine nitrogen and the lack of significant ion channel pharmacology across exemplars of the chemical series . enhanced potency was achieved through maintenance of this twisted conformation and replacement of one of the chlorine atoms with a para - substituted aryl ring . combination of the favored methylpyrazole substituent with a 6,5-spirocyclic carboxamide , in place of the original piperidine carboxamide in hit compound 9 , led to 74 , a potent small - molecule inhibitor of the wnt pathway with good oral pharmacokinetics and in vivo activity against the wnt pathway . in particular , hits from such screens , by necessity , have good cell permeability , and components of protein complexes are more likely to be in a relevant endogenous conformation , which may reveal new druggable binding sites or alternative targetable conformations of known sites ; moreover , key signal - determining pathway nodes are more likely to be discovered as a result of cell - based readouts at the transcriptional level . here we describe a successful example of hypothesis - driven medicinal chemistry optimization from a singleton hit against a cell - based assay readout for the wnt pathway in the absence of knowledge of the biochemical target . 8-(3-bromo-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one ( 57 ) ( 35 mg , 0.10 mmol ) , 1-methyl-4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)piperazine ( 34 mg , 0.11 mmol ) , and pd(dppf)cl2ch2cl2 ( 3.7 mg , 5.1 mol ) were loaded in a microwave vial under argon , and then degassed mecn ( 1.8 ml ) and 0.5 m aqueous sodium carbonate solution ( 284 l , 0.142 mmol ) were added . 4-(4-chlorophenyl)-1-methyl-1h - pyrazole ( 3.30 g , 17.1 mmol ) , bis(pinacolato)diboron ( 5.20 g , 20.6 mmol ) , potassium acetate ( 5.00 g , 51.4 mmol ) , xphos ( 650 mg , 1.37 mmol ) , and pd2dba3 ( 310 mg , 0.343 mmol ) were loaded in a flask , and dioxane ( 34 ml ) was added . compounds ( 10 m ) were incubated with male cd1 mouse liver microsomes ( 1 mg / ml ) protein in the presence of 1 mm nadph , 2.5 mm udp - glucuronic acid ( udpga ) , and 3 mm mgcl2 in 10 mm pbs at 37 c . , bremen , germany ) as cosubstrate were incubated in a total volume of 5 l ( 50 mm hepes , 4 mm mgcl2 , 0.05% pluronic f-68 , 1.4 mm dtt , 0.5% dmso , ph 7.7 ) in the absence or presence of the test compound ( 10 dilution concentrations ) for 90 min at 30 c . the reaction was stopped by the addition of 1 l of 50 mm edta solution , and 2 l of the detection solution [ 1.6 m sa - xlent ( cisbio , codolet , france ) , 7.4 nm anti - gst - k ( eu - labeled anti - gst , cisbio , codolet , france ) in 50 mm hepes , 800 mm kf , 0.1% bsa , 20 mm edta , 0.1% chaps , ph 7.0 ) ] was added . female athymic crtac : ncr - foxn1 mice from taconic ( 68 weeks of age ) were injected subcutaneously in the flank with 3 million colo205-f1756 clone 4 cells , and when tumor xenografts reached a mean diameter of 56 mm ( equating to a mean volume of about 100 mm ) , mice ( n = 8 per group ) were dosed orally by gavage with compound 74 ( 70 mg / kg bid for 8 days ) or vehicle ( 10% dimethyl sulfoxide ; 5% tween 20 in sterile saline ) . luminescent flux as a measure of wnt pathway activity was quantified by whole body imaging in cohorts of four animals at 2 and 6 h after the final dose .

amyloids are large aggregates of misfolded proteins with a highly stable cross -structure , which are associated with a variety of degenerative illnesses such as alzheimer s , parkinson s , and huntington s diseases . proteins with different functionalities and native structures ranging from -helical and -sheet rich to intrinsically unordered are able to form amyloid fibrils in vitro with a characteristic cross- core structure . this observation leads to the conclusion that protein fibrillation is a generic property of a polypeptide chain . there are numerous research reports demonstrating that a general fibrillation mechanism involves a partially unfolded protein as the first intermediate state . steps to follow include the formation of small aggregates and a -sheet rich nucleus , which generates further protein aggregation and the formation of mature fibrils . a reduced amount of hydrogen sulfide ( h2s ) in the brain tissue of patients with alzheimer s disease has been recently reported . for centuries , people have been interested in h2s for its role as a poisonous chemical . at high concentrations , more recently , it has been demonstrated that h2s has gasotransmitter functions , similar to co and no . for example , a suspended animation - like state in mice has been achieved by administering ppm levels of h2s at low temperatures . the metabolic rate and body core temperature decrease and fully recover after such exposure a promising medical benefit that reduces physiological damage after trauma . in the past two decades , significant attention has been paid to understanding the physiological role of h2s and its endogenous production . h2s is biosynthesized in mammalian tissue by nonenzymatic reactions and by the enzymatic degradation of cysteine by cystathione synthetase ( cbs ) , cystathione lyase ( cse ) , cysteine aminotransferase ( cat ) , and cysteine lyase ( cl ) . moreover , aged garlic extract has been shown to cause a reduction of in vivo a fibrils and soluble amyloid as well as a decrease in tau conformational changes . this indirect evidence concerning the role of h2s in neurodegenerative diseases has motivated us to investigate the effects of h2s on the formation of amyloid fibrils . there is extensive literature on the inhibitory activity of various small molecules on protein fibrillation . recently , arioso and coauthors have reviewed the development of amyloid inhibitors , such as antibodies and chaperones , small molecules ( e.g. , congo red and polyphenols ) , colloidal inhibitors , and organic / inorganic nanoparticles , as possible participants in the various states of protein aggregation . these states include the inhibition of primary nucleation ( monomer - to - oligomer transition ) , secondary nucleation ( oligomer elongation ) , and postelongation . however , we have not found any published reports on the role of h2s in protein aggregation . it is well documented that h2s reacts with disulfide bonds , leading one to hypothesize that this reaction could have a significant effect on the mechanism of protein fibrillation . kumar and co - workers have reported that protecting disulfide bridges with iodoacetamide in an alkaline solution limits the lysozyme fibril growth to 50% . this group has concluded that changing the dynamics of disulfide to aberrant disulfide bonds would redirect the process toward the formation of native - like lysozyme aggregates . it has been reported that treating antibodies with h2s has resulted in ss bond modifications , including the formation of trisulfide bonds ( sss ) assessed by liquid chromatography and mass spectrometry ( lc - ms ) . surprisingly , no changes in antibody stability and function have been observed . h2s can be incorporated as a sulfane sulfur , a divalent sulfur with six valence electrons , and an oxidation number of zero ( s ) that only binds to other sulfur atoms to form polysulfides . several research groups have also reported that the sulfur atom of h2s can be endogenously incorporated into a large amount of proteins by sulfuration , also known as sulfhydration of cysteines . this leads to the formation of protein persulfides ( ssh ) , which could play an intermediary role in protein sss formation . in the current study , we have investigated the effect of h2s on the aggregation of lysozyme , a glycoside hydroxylase responsible for antimicrobial protection in most mammalian species . hewl is a single chain protein stabilized by four ss bonds in positions cys6-cys127 , cys30-cys115 , cys64-cys80 , and cys76-cys94 . the effect of h2s has been investigated under typical fibrillation conditions such as high temperature and acidic ph using duvrr and nonresonance raman spectroscopy , fluorescence , lc - ms , and atomic force microscopy ( afm ) . we have shown that in the presence of h2s hewl forms spherical aggregates of unordered protein under fibrillation conditions . cytotoxicity tests reveal that these spherical aggregates have no cell toxicity by contrast with typical hewl fibrils . our spectroscopic results , buttressed by data that have been published , indicate that h2s reacts with protein disulfide bonds to form trisulfide bridges . this reaction results in significant lysozyme denaturation and the formation of spherical aggregates of unordered proteins , which prevent protein fibrillation . this discovery indicates possible new roles for h2s and trisulfide bridges in protein biochemistry in vivo . louis , mo ) : 99.7% acetic acid ( 695092 ) , sodium chloride ( nacl ) ( s771 - 3 ) , hewl ( l6876 ) , sodium sulfidenonahydrate salt ( 208043 ) , dipropyl disulfide ( 149225 ) , and trisulfide ( 6028 - 61 - 1 ) . lysozyme was dissolved ( 25 mg / ml ) in 20% acetic acid and 100 mm nacl at ph 2.0 and incubated at 60 c to form fibrils under initial ( control ) conditions . to study the effect of h2s , sodium sulfide nonahydrate salt ( 12 mm ) was added to the control solution in a molar ratio of 1:5 ( hewl : h2s ) , prior to the temperature elevation . powder samples of native and aggregated hewl were prepared by drying the solutions under nitrogen at room temperature , which removed the acetic acid . raman spectra ( 785 nm excitation ) of hewl powder samples and pure dipropyl di- and trisulfide liquids were recorded using a renishaw invia confocal raman spectrometer equipped with a research grade leica microscope and 50 objective ( numerical aperture , 0.55 ) . five accumulations of 30 s each were collected for each sample in the range of 4001800 cm . a laser power of approximately 11.5 mw was used to avoid sample photodegradation . a reaction with tris(2-carboxyethyl)phosphine ( tcep ) reducing agent hen egg white lysozyme ( hewl ) in native and aggregated form was incubated at ph 2.0 and room temperature for 90 min in the presence of tcep . lc - ms experiments were performed on thermo ltq orbitrap xl mass spectrometer ( thermo , bremen , germany ) . samples were injected using an agilent 1200 nano - hplc system ( agilent , palo alto , ca ) and agilent 1200 autosampler . agilent zorbax c18 column ( 150 4.6 mm ) was used for separation of mixtures . the injection volume was up to 8 l , and a flow rate of the mobile phase was 250 l / min . mobile phase consisted of 0.2% of formic acid ( solvent a ) and 0.2% formic acid in acetonitrile ( solvent b ) with the gradient from 5% to 40% of solvent b in 40 min followed by washing the column by 90% of solvent b and equilibration . the mass spectrometer operated in esi mode in the mass range m / z 100500 with detection of positively and negatively charged ions . the resolution was 30 000 , and the accuracy of mass measurements was better than 2 ppm . powder samples of hewl aggregates incubated at different concentrations of tcep were prepared for nonresonance raman spectroscopic analysis by drying the corresponding solutions under a nitrogen flow at room temperature . duvrr spectra ( 199.7 nm excitation ) of 25 mg / ml hewl were collected using a home - built instrument equipped with a ccd camera ( roper scientific , inc . ) each spectrum recorded an average of 20 accumulations with 30 s acquisition time . grams / ai 7.0 software ( thermo galactic , salem , nh ) was used for data processing . fluorescence spectra were measured in a jobinyvon fluoromax-3 spectrofluorometer ( jobinyvon , edison , nj ) . intrinsic tryptophan fluorescence of 25 mg / ml hewl was measured in a 10 m path length cell without dilutions . the uv absorption was < 0.05 at an excitation wavelength of 295.5 nm . three spectral accumulations were taken , and the spectra were averaged for each sample . hewl fibrils formed after 90 min of incubation were also characterized using intrinsic tryptophan fluorescence . fibrils were washed in acetic acid solution twice in a procedure which included sonication for 10 min , centrifugation for 4 min at 13 000 rpm , the removal of supernatant liquid , and resuspension in an acetic acid solution . in the tht fluorescence assay , aliquots of 25 mg / ml hewl were diluted in a molar ratio of 1:10 ( hewl : thioflavin t ( tht ) dye ) to a final concentration of 2.5 mm tht . aliquots of hewl incubated at 60 c , ph 2.0 , 100 mm nacl , were cooled to room temperature and deposited on freshly cleaved mica . after a few minutes of exposure , the mica surface was rinsed with mq water and dried . afm images were collected using the smartspm 1000 system ( aist - nt , novato , ca ) . images were acquired in the tapping mode using silicon cantilevers with a 1025 nm tip curvature radius . human neuroblastoma sh - sy5y cells were cultured in a 1:1 ( v / v ) mixture of dmem ( sigma - aldrich , st . louis , mo ) and ham s f-12 nutrient mixture ( sigma - aldrich , st . louis , mo ) supplemented with 10% fbs ( sigma - aldrich , st . louis , mo ) and antibiotic - antimitotic solution ( sigma - aldrich , st . the cells were incubated at 37 c in an incubator with 95% humidified air and 5% co2 . cells were plated onto flat - bottom 96-well plates ( 1 10 cells per well ) and incubated overnight at 37 c . after cell attachment , the initial medium was replaced with serum - free medium using different concentrations of mature hewl fibrils or hewl aggregates ( formed with h2s ) and incubated for 24 h. cytotoxicity was assessed using the mtt reduction inhibition assay . mtt was added to the culture medium to yield a final concentration of 0.5 mg / ml . cells were incubated with the mtt medium for 4 h in a co2 incubator , followed by the addition of 200 l of an isopropanol 0.04 n hcl solution to dissolve formazan crystals . absorbance was measured at 540 nm using a synergy h1 plate reader ( biotek , vermont ) . control experiments were performed by exposing cells to solutions of an equivalent volume of the same initial buffer for the same length of time . cell viability was measured relative to control cells that were not exposed to the hewl aggregates / hewl fibrils solutions . to form fibrils , hewl was incubated at 60 c in 20% acetic acid ( ph 2.0 ) and 100 mm nacl , from here on referred to as control conditions . the morphology of lysozyme aggregates formed in the course of incubation under the fibrillogenic conditions and in the presence of h2s was characterized by afm . the presence of typical long rodlike fibrils was evident after 90 min of incubation under control conditions ( figure 1a ) . however , incubation of hewl in the presence of h2s resulted in the formation of spherical aggregates instead of fibrils , as evident from afm images ( figure 1b ) . lysozyme forms -sheet - rich fibrils under fibrillogenic control conditions and spherical aggregates of unordered protein under fibrillogenic conditions with h2s incubation . afm images of ( a ) hewl fibrils formed after incubation of the control solution for 90 min and ( b ) hewl aggregates formed after incubation of the solution in the presence of h2s for 48 h ; scale bars are 1 m . ( c ) aggregation kinetics ( tht fluorescence ) of hewl incubated under control conditions ( red ) and in the presence of h2s ( black ) . ( d ) duvrr spectra of native hewl ( blue ) , hewl fibrils ( red ) and hewl spherical aggregates ( black ) formed in the presence of h2s ; all spectra were normalized using the aromatic amino acid raman band ( approximately 1600 cm ) for comparison . the amide i vibrational mode ( am i ) is dominated by c = o stretching , with minor contributions from c amide ii ( am ii ) and amide iii ( am iii ) bands involve significant c the ch bending vibrational mode involves ch symmetric bending and c c stretching . tht fluorescence intensity increased dramatically after 70 min of incubation of lysozyme under control conditions , indicating the formation of amyloid fibrils ( figure 1c ) . however , no increase in tht fluorescence intensity was observed for the hewl solution incubated with h2s within 48 h. we investigated changes in the lysozyme secondary structure during incubation with and without h2s using deep uv resonance raman ( duvrr ) spectroscopy . duvrr has been used to study structural rearrangements of hewl at all stages of fibrillation . the duvrr spectrum of hewl excited at 199.7 nm was mainly composed of the amide chromosphere and the aromatic amino acid ( phe and tyr ) contributions . a noticeable increase in the intensity and sharpness of the am i band ( approximately 1672 cm ) indicated the appearance of -sheets due to the formation of fibrils . the duvrr spectrum of fibrillated lysozyme under control conditions confirmed the formation of -sheets . the spectrum of hewl after 30 min of incubation under control conditions ( figure 1d , red ) is similar to that reported previously for hewl fibrils . however , the duvrr spectrum of lysozyme incubated in the presence of h2s confirmed the lack of -sheet formation . in this case , the am i band ( approximately 1670 cm ) did not show a significant intensity change ( figure 1d , black ) . instead , the am i band shifted slightly to a higher frequency , signifying the formation of an unordered protein . this was further supported by the increase in ch band intensity at 1390 cm that was indicative of -helix melting . a significant change in raman bands for am iii ( approximately 1250 cm ) and am ii ( approximately 1555 cm ) was consistent with the transition of -helix to unordered protein . therefore , afm , tht fluorescence and duvrr spectroscopy indicated the formation of unordered spherical aggregates of hewl by contrast with -sheet - rich fibrils in the presence of h2s . tryptophan ( trp ) fluorescence is an efficient intrinsic marker of local environments , which is often used for monitoring tertiary structural changes in proteins . native lysozyme at neutral ph shows a maximum trp emission at 340 nm . at ph 2.0 ( 20% acetic acid ) , the trp fluorescence peak shifts to 345 nm , indicating a partial denaturation of lysozyme . a further minor shift to 347 nm due to hewl fibril formation under control conditions was observed ( figure 2b , c ) . to confirm that the intrinsic trp fluorescence is dominated by the signal from hewl fibrils , the solutions ( after incubation for 40 and 90 min ) were sonicated , centrifuged and resuspended in 20% acetic acid to remove possible monomeric and oligomeric forms of the protein ( figure 2b ) . time - dependent trp fluorescence changes of lysozyme ( a ) incubated in the presence of h2s for 0 min ( - - - ) , 10 min ( ) , 90 min ( ) , and 48 h ( ) and ( b ) incubated with control solution for 0 min ( ) , 40 min ) , and 90 min ( ) . ( c ) trp maximum emission wavelength of hewl incubated with h2s ( a , ) and fibrillation under control conditions ( b , ) . a significant shift of the trp emission maximum , from 345 to 357 nm , was observed after 90 min of lysozyme incubation in the presence of h2s ( figure 2a , c ) , with no further changes for at least 48 h. this significant red - shift is consistent with the previously reported maximum emission at 352 nm for fully denatured lysozyme in 6 m guanidinium - hcl at ph 7.0 . therefore , we conclude that incubation of lysozyme in the presence of h2s results in a stronger denaturation than that which occurs during control fibrillation conditions . nonresonance raman spectroscopy of proteins offers a unique opportunity for characterizing the conformation of disulfide bridges . the ss symmetric stretching vibrational mode is typically represented as a strong raman band in the range of 505550 cm . the raman spectrum of hewl was found to change significantly in the ss signature region with incubation time ( figure 3a ) . a strong 507 cm peak in the raman spectrum of native hewl represents the gauche gauche gauche ( g - g - g ) configuration of three ss bonds , and a small 523 cm peak can be attributed to the gauche gauche trans ( g - g - t ) configuration of the fourth ss bond of lysozyme . the amplitudes of these peaks decreased , and a new peak appeared at 490 cm as a result of hewl incubation in the presence of 12 mm h2s , indicating significant rearrangements of ss bonds ( figure 3a ) . evolution of lysozyme disulfide bonds in the presence of h2s probed by normal raman spectroscopy . raman spectra of hewl incubated ( a ) in the presence of h2s and ( b ) under control conditions , where 507 and 523 cm bands correspond to g - g - g and g - g - t ss configurations , respectively . synchronous kinetic change in the area of the 507 and 490 cm bands is assigned to the newly formed rsssr group . ( c ) kinetics of rsssr formation ( 490 cm ) and the decrease in the amount of rssr ( 507 cm ) during the incubation of hewl in the presence of h2s . ( d ) difference spectrum between normal raman spectra of hewl aggregated in the presence of h2s acquired at 90 and 0 min incubation ( shown in ( a ) , gray solid line ) . the latter spectrum is represented by the expected spectral change demonstrating the disulfide - to - trisulfide transition symbolized by the inverted raman spectrum of dipropyl disulfide ( - - ) and dipropyltrisulfide ( red ) . the concentration of 12 mm h2s corresponded to a 5:1 ( h2s : hewl ) molar ratio , chosen so that a sufficient number of h2s molecules could react with all four lysozyme ss bonds assuming a 1:1 stoichiometric ratio . the 1003 cm peak corresponding to phenylalanine was used to normalize raman spectra in figure 3a ( region not shown ) . figure 3c shows synchronous kinetic change in the area of the 507 and 490 cm bands with incubation time up to 90 min . no further changes were observed during 48 h of additional incubation in the presence of h2s ( data not shown ) . as discussed in detail below , the dipropyl trisulfide ( dpts ) raman spectrum contains a 485 cm band ( figure 3d ) characteristic of the trisulfide moiety that motivated us to investigate the possibility of assigning the 490 cm band in the hewl aggregate raman spectrum to the sss group . the nonresonance raman spectroscopy of hewl fibrillation under control conditions indicates that the 507 cm peak does not change significantly during fibril formation ( figure 3b ) . therefore , in the absence of h2s , the hewl ss bands remain intact and the g - g - g conformation dominates , in agreement with our previous report . to test the hypothesis about the formation of trisulfide groups , we investigated the reaction of hewl aggregates with tcep reduction agent by normal raman spectroscopy and mass spectrometry . tcep reaction with ss groups is well - known to result in oxidation of tcep and formation of tcep(o ) and r sh groups . more recently , cumnock et al . reported that tcep reacted preferentially with sss moieties in the presence of ss bridges until the majority of sss groups were consumed according to eqs 1 and 2 . figure 4 shows raman spectra of hewl aggregates after incubation with different concentrations of tcep ( 0.5 , 1 , 2.5 , and 10 mm ) . the amount of aggregated hewl molecules in these samples was kept about 3.0 mm.12 normal raman spectra of hewl aggregates in the presence of reducing agent tcep with concentration 0 mm ( ) , 0.5 mm ( ) , 1 mm ( - - - ) , 2.5 mm ( ) , and 10 mm ( - - ) . selected spectral regions with characteristic raman bands of disulfide and trisulfide moieties ( a ) as well as sulfhydryl ( sh ) group ( b ) are shown . the phenylalanine raman band at 1003 cm was used to normalize the spectra ( spectral region not shown ) . the raman spectrum of hewl aggregates was found to change significantly in the sss / ss vibrational signature region with the addition of tcep ( figure 4a ) . the sss band at 490 cm decreases after 0.5 mm tcep addition which is in a good agreement with predominant reaction of tcep with sss groups . the amplitudes of both 490 and 507 cm bands ( sss and ss , respectively ) decreased as a result of hewl incubation in the presence of higher concentration of tcep ( 110 mm ) , indicating significant reduction of ss and sss groups and formation of r sh moiety in agreement with an increase in 2575 cm band intensity ( figure 4b ) . lc - ms was utilized to monitor the formation of tcep(s ) and tcep(o ) as products of sss and ss reduction , respectively ( eqs 1 and 2 ) . the reduction of native hewl with an excess of tcep ( 10 mm ) resulted in products with retention times of 6.68 , 9.01 , and traces at 17.51 min ( figure 5a ) . accurate mass measurements confirmed that the products correspond to tcep , tcep(o ) , and tcep(s ) , respectively . hewl aggregates treated with 0.5 mm tcep showed tcep(o ) and tcep(s ) , with retention times of 9.0 and 17.53 min ( figure 5b ) . all initial tcep was completely consumed due to the reaction with sss and ss groups . combined lc - ms ( esi in negative ion mode ) chromatograms of ions at m / z 249 , m / z 265 , and m / z 281 corresponding to deprotonated tcep , tcep(o ) , and tcep(s ) , respectively , for native lysozyme incubated with 10 mm tcep ( a ) and lysozyme aggregates incubated with 0.5 mm tcep ( b ) . the solution of native hewl incubated with an excess of tcep contained significant amounts of tcep and tcep(o ) and traces of tcep(s ) . the main expected products of tcep reaction with rssr are tcep(o ) and 2rsh . we believe that the traces of tcep(s ) could form due to a prolonged exposure of native hewl to the excess of tcep prior to lc - ms analysis . this suggestion is based on the reported reaction 3 of triphenylphosphine ( ph3p ) with dialkyl disulfides ( rssr ) , which resulted in formation of ph3p(s ) . we hypothesize that the trace amount of tcep(s ) is formed in the reaction of tcep with native hewl due to a similar mechanism . however , the amount of tcep(s ) is low because the second step in reaction 3 is slow and reaction 2 dominates.3overall , the analysis of reaction products resulting from the incubation of native and aggregated lysozyme in the presence of tcep using lc - ms is consistent with the presence of sss in aggregated hewl . the variation in the amplitude of the 490 cm raman band with the concentration of tcep is in agreement with its assignment to the sss group as well . to compare the cytotoxicity of hewl fibrils and spherical aggregates formed in the presence of h2s , samples of mature fibrils and spherical aggregates were posteriorly transferred to pbs buffer at ph 7.4 in order to avoid drastic changes in the ph of the cells . to achieve this , mature fibrils were washed three times in pbs buffer , and spherical aggregates were dialyzed against pbs buffer . the latter procedure did not change fibrils and spherical aggregates according to their normal raman spectra ( data not shown ) . specifically , the 490 cm band remained intact after buffer exchange to pbs . the incubation of confluent sh - sy5y cells with mature fibrils ( 0.536 m ) for 24 h resulted in a significant reduction ( 40% ) of the cellular viability . mean cell viability and one standard deviation of two independent experiments performed in triplicate are presented . the most common methods used for studying the fibrillation process include afm , tht , and trp fluorescence . duvrr spectroscopy has been shown to be uniquely suitable for the structural characterization of proteins at all stages of the fibrillation process . we utilized these complementary methods for studying the effect of h2s on the morphology and structure of lysozyme aggregates . although fibril formation was not detected by afm and tht fluorescence assays , the intrinsic trp fluorescence marker suggested that significant tertiary structure changes had taken place minutes after h2s incubation began . the red - shift of trp fluorescence of greater than 10 nm is typical for unfolded lysozyme . changes were also evident for ss bridges at the same time scale , as discussed in the next section . the changes observed in the tryptophan local environment and in ss bonds indicate substantial changes in hewl tertiary structure . duvrr spectroscopy was utilized to investigate changes in hewl secondary structure during the incubation with and without h2s . it was found that h2s prevented the formation of -sheet and resulted in a significant transition of -helix to unordered protein . moreover , we utilized duvrr spectra of aggregated lysozyme to evaluate the protein secondary structure composition . xu et al . have reported on the quantitative analysis of lysozyme duvrr spectral changes during its denaturation . according to that work , the amount of -helix melting can be estimated from the intensity of ch bending band . -sheet and unordered structures only contribute to ch bending duvrr band , while the -helix does not make a noticeable input . it is evident from amide i raman bands in duvrr spectra presented in figure 1d that no fibril - type -sheet is formed in hewl aggregates since the am i intensity does not increase . therefore , the increase in the ch band intensity in the spectrum of hewl aggregates relative to that of native protein could be assigned to newly formed unordered structures . we normalized the duvrr spectra of hewl aggregates and native protein with the denatured - reduced hewl spectrum reported by xu et al . and estimated the amount of -helix in hewl aggregates as 11% . assuming that the amount of -sheet in hewl aggregates is approximately the same as in the native protein , we estimated the secondary structural composition of hewl aggregates as 83% unordered , 11% -helix , and 6% -sheet . to summarize the results concerning the significant tertiary structural rearrangements , -helix melting , and lack of -sheet formation , we conclude that h2s causes more significant denaturation of lysozyme than that taking place during the initial stages of protein fibrillation , which is typically reported as partial protein denaturation . we hypothesize that this significant lysozyme denaturation results in rapid protein aggregation , the formation of spherical species , and the prevention of the formation of -sheets and fibrillation . in other words , this observation is consistent with an earlier report by wang and colleagues which demonstrated that fully denatured lysozyme forms amorphous aggregates that prevent fibril formation . the protein has been fully denatured by reducing ss bonds with dttred . as a result , fully denatured lysozyme may lack the hydrophobic regions which are present in the partially unordered intermediates formed at the early stage of fibril formation . in addition , it is possible that amorphous aggregates decreased the effective concentration of hewl available for fibril formation . in agreement with wang s report , our results suggest that lysozyme denatures strongly in the presence of h2s and forms unordered aggregates that prevent -sheet formation and fibrillation . according to figure 3 , the contributions of both g - g - g ( 507 cm band ) and g - g - t ( 523 cm band ) conformations of ss bonds to the raman spectrum of hewl decreased significantly during its incubation with h2s . simultaneously , a new peak appeared at 490 cm ( figure 3a , c ) . nielsen and colleagues proposed that sss bridges can form in proteins in the presence of h2s via the thiol we investigated the possibility of assigning a new raman band at 490 cm to the sss moiety . initially , we reproduced raman spectra of two model compounds , dipropyl disulfide ( dpds ) and dipropyl trisulfide ( dpts ) , shown in figure 3d . in agreement with other published studies , these compounds exhibit strong raman bands at 509 and 485 cm , respectively , in agreement with the raman spectra of native hewl and hewl spherical aggregates formed in the presence of h2s . furthermore , we obtained the difference spectrum by subtracting hewl spectra after 0 and 90 min of incubation in the presence of h2s and compared it to the expected spectral change representing the ss to sss transition . the latter spectral change is depicted as a combination of dipropyl disulfide and dipropyl trisulfide spectra ( figure 3d ) . this spectral comparison provides further support for the hypothetical assignment of the 490 cm raman band to the sss moiety . several studies have identified a 490 cm raman band in inorganic compounds and small organic molecules containing sss , and we report the appearance of this band in proteins for the first time . wieser and krueger have assigned the 488 cm raman peak of h sss h to a symmetric ss stretch with a contribution from the sss bend . freeman has reported the raman spectra of organic ss and sss compounds , found in natural products where a strong 485 cm stretching band has been observed in cyclic and acyclic trisulfides . janz et al . have reported raman spectra of inorganic sss from bas3 where 458 and 476 cm bands were assigned to the symmetric stretching of sss . kimbaris et al . have reported the gc / ms and raman spectra of garlic oil , which contains a variety of compounds with ss and sss groups . we noticed an intense band at 489 cm in these spectra that could potentially originate from an sss moiety , although the assignment of the band was not discussed in the article . overall , our hypothetical assignment of the 490 cm raman band to the sss moiety is in agreement with data from the literature . the mechanism of sss formation in proteins is unclear despite the significant interest that this topic has gained in recent years . there is emerging evidence indicating that sulfane sulfur ( s ) , which is generated from h2s , is responsible for sulfuration through the formation of persulfide or trisulfide in proteins . it would be interesting to investigate whether these sss form by intra- or intermolecular processes . it is noteworthy that the 490 cm raman band can not be assigned to rssh groups . these groups could form as a result of disulfide bond reduction in the presence of h2s by a process known as sulfuration or sulfhydration . however , r sh and r ssh groups have a characteristic raman band at 2575 cm which is not evident in the experimental spectra ( figures s1 and s2 ) . in addition , these groups do not have a vibrational mode at 490 cm . there is evidence in the literature that indicates the possibility of ss bond reduction in the presence of h2s and the formation of rssh groups in basic environments . the results allowed us to eliminate rssh as a possible candidate for newly formed species with a characteristic 490 cm raman band . ss bonds preserve the three - dimensional structure of proteins , and their cleavage typically results in significant disruption of the native conformations of proteins . it is well established that ss bonds play a significant role in amyloid fibrillation . dobson and colleagues have reported that the reduction of ss bridges significantly accelerated the rate of human lysozyme aggregation . it has also been demonstrated that reduction of four ss bonds to three ss bonds of apo--la accelerates its fibrillation and leads to the formation of a new fibril polymorph with a different morphology and structure compared to fibrils formed from the wild - type la . at the same time , ss bonds of insulin remain intact and preserve their conformation during the fibrillation process . similar to insulin , the conformation of the ss bonds in hewl remains intact during the fibrillation of hewl in control solution , as we have described here . it has been suggested that a partial denaturation of lysozyme precedes fibril formation because the native tertiary structure would not allow rearrangement to the cross--sheet structure due to steric constraints . it has also been reported that partial denaturation , the first step of lysozyme fibrillation , is an irreversible process . at the same time , a fully denatured lysozyme forms amorphous aggregates that prevent fibril formation . it is believed that the fully denatured protein lacks the hydrophobic side chains present in partially unordered intermediates . in addition , amorphous aggregates potentially decrease the effective concentration of hewl available for fibril formation . in agreement with these observations , our results suggest that lysozyme denatures strongly in the presence of h2s and forms unordered aggregates that prevent -sheet formation and fibrillation . amyloid fibrils are associated with many neurodegenerative diseases including alzheimer s , parkinson s , and systemic amyloidosis . it has recently been found that there is a reduced amount of hydrogen sulfide ( h2s ) in the brain tissue of patients with alzheimer s disease , leading us to investigate the effects of h2s on the formation of amyloid fibrils . our objective was to utilize several complementary techniques , including raman spectroscopy , afm , and intrinsic tryptophan and tht fluorescence for studying the kinetics of hewl fibrillation , a well - characterized model amyloidogenic protein , in the presence of h2s . lysozyme forms typical -sheet - rich fibrils after incubation in 20% acetic acid solution at 60 c for approximately 70 min . the addition of 12 mm h2s in a molar ratio of 5:1 ( h2s : hewl ) completely prevented the formation of the -sheet conformation as measured by deep uv resonance raman ( duvrr ) spectroscopy and tht fluorescence . by contrast , the melting of the -helix resulted in unordered protein in the form of spherical aggregates , which were distinctly different from amyloid fibrils . according to the intrinsic tryptophan fluorescence , hewl exhibited a more significant perturbation of its tertiary structure in the presence of h2s than that which occurred during the partial protein denaturation at the early stage of fibrillation . lysozyme has four ss bonds , which stay intact during the fibrillation process . however , based on our data from nonresonance raman spectroscopy , ss bonds exhibit significant rearrangements in the presence of h2s . the peak area of the 507 cm raman band corresponding to the ss segment in the g - g - g conformation was reduced significantly , and a new band appeared at 490 cm . we assigned this new band to the sss group based on previously reported data , the raman spectra we acquired for model compounds , and lc - ms analysis of tcep reduction products . cytotoxicity tests revealed that the spherical aggregates formed by lysozyme in the presence of h2s are nontoxic to cells by contrast with fibrils . overall , we report for the first time that h2s prevents fibrillation of hewl in vitro and results in the formation of small spherical aggregates of unordered protein . we also hypothesize that the mechanism of this h2s effect involves the formation of sss bridges . our findings that h2s inhibits fibril formation and that putative sss forms open a new and intriguing topic of biochemical and biomedical research . the fact that the spherical aggregates formed are nontoxic to cells by contrast with fibrils is worth further investigation in vivo . given that there are new drugs under development for a slow release of h2s with a variety of therapeutic targets , including s - sildenafil for urological conditions , s - diclofenac for inflammation , s - latanoprost for neurodegenerative illnesses , s - levodopa for parkinson s disease , and s - aspirin for cardiovascular conditions , our results suggest possible new roles of sss in vivo .

amyloid fibrils are large aggregates of misfolded proteins , which are often associated with various neurodegenerative diseases such as alzheimer s , parkinson s , huntington s , and vascular dementia . the amount of hydrogen sulfide ( h2s ) is known to be significantly reduced in the brain tissue of people diagnosed with alzheimer s disease relative to that of healthy individuals . these findings prompted us to investigate the effects of h2s on the formation of amyloids in vitro using a model fibrillogenic protein hen egg white lysozyme ( hewl ) . hewl forms typical -sheet rich fibrils during the course of 70 min at low ph and high temperatures . the addition of h2s completely inhibits the formation of -sheet and amyloid fibrils , as revealed by deep uv resonance raman ( duvrr ) spectroscopy and tht fluorescence . nonresonance raman spectroscopy shows that disulfide bonds undergo significant rearrangements in the presence of h2s . raman bands corresponding to disulfide ( rssr ) vibrational modes in the 550500 cm1 spectral range decrease in intensity and are accompanied by the appearance of a new 490 cm1 band assigned to the trisulfide group ( rsssr ) based on the comparison with model compounds . the formation of rsssr was proven further using a reaction with tcep reduction agent and lc - ms analysis of the products . intrinsic tryptophan fluorescence study shows a strong denaturation of hewl containing trisulfide bonds . the presented evidence indicates that h2s causes the formation of trisulfide bridges , which destabilizes hewl structure , preventing protein fibrillation . as a result , small spherical aggregates of unordered protein form , which exhibit no cytotoxicity by contrast with hewl fibrils .

Introduction Materials and Methods Results Discussion Conclusions

amyloids are large aggregates of misfolded proteins with a highly stable cross -structure , which are associated with a variety of degenerative illnesses such as alzheimer s , parkinson s , and huntington s diseases . steps to follow include the formation of small aggregates and a -sheet rich nucleus , which generates further protein aggregation and the formation of mature fibrils . a reduced amount of hydrogen sulfide ( h2s ) in the brain tissue of patients with alzheimer s disease has been recently reported . this indirect evidence concerning the role of h2s in neurodegenerative diseases has motivated us to investigate the effects of h2s on the formation of amyloid fibrils . it is well documented that h2s reacts with disulfide bonds , leading one to hypothesize that this reaction could have a significant effect on the mechanism of protein fibrillation . it has been reported that treating antibodies with h2s has resulted in ss bond modifications , including the formation of trisulfide bonds ( sss ) assessed by liquid chromatography and mass spectrometry ( lc - ms ) . this leads to the formation of protein persulfides ( ssh ) , which could play an intermediary role in protein sss formation . in the current study , we have investigated the effect of h2s on the aggregation of lysozyme , a glycoside hydroxylase responsible for antimicrobial protection in most mammalian species . the effect of h2s has been investigated under typical fibrillation conditions such as high temperature and acidic ph using duvrr and nonresonance raman spectroscopy , fluorescence , lc - ms , and atomic force microscopy ( afm ) . we have shown that in the presence of h2s hewl forms spherical aggregates of unordered protein under fibrillation conditions . cytotoxicity tests reveal that these spherical aggregates have no cell toxicity by contrast with typical hewl fibrils . this reaction results in significant lysozyme denaturation and the formation of spherical aggregates of unordered proteins , which prevent protein fibrillation . to study the effect of h2s , sodium sulfide nonahydrate salt ( 12 mm ) was added to the control solution in a molar ratio of 1:5 ( hewl : h2s ) , prior to the temperature elevation . a reaction with tris(2-carboxyethyl)phosphine ( tcep ) reducing agent hen egg white lysozyme ( hewl ) in native and aggregated form was incubated at ph 2.0 and room temperature for 90 min in the presence of tcep . cell viability was measured relative to control cells that were not exposed to the hewl aggregates / hewl fibrils solutions . the morphology of lysozyme aggregates formed in the course of incubation under the fibrillogenic conditions and in the presence of h2s was characterized by afm . however , incubation of hewl in the presence of h2s resulted in the formation of spherical aggregates instead of fibrils , as evident from afm images ( figure 1b ) . lysozyme forms -sheet - rich fibrils under fibrillogenic control conditions and spherical aggregates of unordered protein under fibrillogenic conditions with h2s incubation . afm images of ( a ) hewl fibrils formed after incubation of the control solution for 90 min and ( b ) hewl aggregates formed after incubation of the solution in the presence of h2s for 48 h ; scale bars are 1 m . ( c ) aggregation kinetics ( tht fluorescence ) of hewl incubated under control conditions ( red ) and in the presence of h2s ( black ) . ( d ) duvrr spectra of native hewl ( blue ) , hewl fibrils ( red ) and hewl spherical aggregates ( black ) formed in the presence of h2s ; all spectra were normalized using the aromatic amino acid raman band ( approximately 1600 cm ) for comparison . tht fluorescence intensity increased dramatically after 70 min of incubation of lysozyme under control conditions , indicating the formation of amyloid fibrils ( figure 1c ) . however , no increase in tht fluorescence intensity was observed for the hewl solution incubated with h2s within 48 h. we investigated changes in the lysozyme secondary structure during incubation with and without h2s using deep uv resonance raman ( duvrr ) spectroscopy . a noticeable increase in the intensity and sharpness of the am i band ( approximately 1672 cm ) indicated the appearance of -sheets due to the formation of fibrils . the spectrum of hewl after 30 min of incubation under control conditions ( figure 1d , red ) is similar to that reported previously for hewl fibrils . however , the duvrr spectrum of lysozyme incubated in the presence of h2s confirmed the lack of -sheet formation . therefore , afm , tht fluorescence and duvrr spectroscopy indicated the formation of unordered spherical aggregates of hewl by contrast with -sheet - rich fibrils in the presence of h2s . to confirm that the intrinsic trp fluorescence is dominated by the signal from hewl fibrils , the solutions ( after incubation for 40 and 90 min ) were sonicated , centrifuged and resuspended in 20% acetic acid to remove possible monomeric and oligomeric forms of the protein ( figure 2b ) . time - dependent trp fluorescence changes of lysozyme ( a ) incubated in the presence of h2s for 0 min ( - - - ) , 10 min ( ) , 90 min ( ) , and 48 h ( ) and ( b ) incubated with control solution for 0 min ( ) , 40 min ) , and 90 min ( ) . a significant shift of the trp emission maximum , from 345 to 357 nm , was observed after 90 min of lysozyme incubation in the presence of h2s ( figure 2a , c ) , with no further changes for at least 48 h. this significant red - shift is consistent with the previously reported maximum emission at 352 nm for fully denatured lysozyme in 6 m guanidinium - hcl at ph 7.0 . therefore , we conclude that incubation of lysozyme in the presence of h2s results in a stronger denaturation than that which occurs during control fibrillation conditions . a strong 507 cm peak in the raman spectrum of native hewl represents the gauche gauche gauche ( g - g - g ) configuration of three ss bonds , and a small 523 cm peak can be attributed to the gauche gauche trans ( g - g - t ) configuration of the fourth ss bond of lysozyme . the amplitudes of these peaks decreased , and a new peak appeared at 490 cm as a result of hewl incubation in the presence of 12 mm h2s , indicating significant rearrangements of ss bonds ( figure 3a ) . evolution of lysozyme disulfide bonds in the presence of h2s probed by normal raman spectroscopy . raman spectra of hewl incubated ( a ) in the presence of h2s and ( b ) under control conditions , where 507 and 523 cm bands correspond to g - g - g and g - g - t ss configurations , respectively . synchronous kinetic change in the area of the 507 and 490 cm bands is assigned to the newly formed rsssr group . ( c ) kinetics of rsssr formation ( 490 cm ) and the decrease in the amount of rssr ( 507 cm ) during the incubation of hewl in the presence of h2s . ( d ) difference spectrum between normal raman spectra of hewl aggregated in the presence of h2s acquired at 90 and 0 min incubation ( shown in ( a ) , gray solid line ) . no further changes were observed during 48 h of additional incubation in the presence of h2s ( data not shown ) . as discussed in detail below , the dipropyl trisulfide ( dpts ) raman spectrum contains a 485 cm band ( figure 3d ) characteristic of the trisulfide moiety that motivated us to investigate the possibility of assigning the 490 cm band in the hewl aggregate raman spectrum to the sss group . the nonresonance raman spectroscopy of hewl fibrillation under control conditions indicates that the 507 cm peak does not change significantly during fibril formation ( figure 3b ) . to test the hypothesis about the formation of trisulfide groups , we investigated the reaction of hewl aggregates with tcep reduction agent by normal raman spectroscopy and mass spectrometry . the amount of aggregated hewl molecules in these samples was kept about 3.0 mm.12 normal raman spectra of hewl aggregates in the presence of reducing agent tcep with concentration 0 mm ( ) , 0.5 mm ( ) , 1 mm ( - - - ) , 2.5 mm ( ) , and 10 mm ( - - ) . the raman spectrum of hewl aggregates was found to change significantly in the sss / ss vibrational signature region with the addition of tcep ( figure 4a ) . the amplitudes of both 490 and 507 cm bands ( sss and ss , respectively ) decreased as a result of hewl incubation in the presence of higher concentration of tcep ( 110 mm ) , indicating significant reduction of ss and sss groups and formation of r sh moiety in agreement with an increase in 2575 cm band intensity ( figure 4b ) . lc - ms was utilized to monitor the formation of tcep(s ) and tcep(o ) as products of sss and ss reduction , respectively ( eqs 1 and 2 ) . combined lc - ms ( esi in negative ion mode ) chromatograms of ions at m / z 249 , m / z 265 , and m / z 281 corresponding to deprotonated tcep , tcep(o ) , and tcep(s ) , respectively , for native lysozyme incubated with 10 mm tcep ( a ) and lysozyme aggregates incubated with 0.5 mm tcep ( b ) . we believe that the traces of tcep(s ) could form due to a prolonged exposure of native hewl to the excess of tcep prior to lc - ms analysis . this suggestion is based on the reported reaction 3 of triphenylphosphine ( ph3p ) with dialkyl disulfides ( rssr ) , which resulted in formation of ph3p(s ) . however , the amount of tcep(s ) is low because the second step in reaction 3 is slow and reaction 2 dominates.3overall , the analysis of reaction products resulting from the incubation of native and aggregated lysozyme in the presence of tcep using lc - ms is consistent with the presence of sss in aggregated hewl . to compare the cytotoxicity of hewl fibrils and spherical aggregates formed in the presence of h2s , samples of mature fibrils and spherical aggregates were posteriorly transferred to pbs buffer at ph 7.4 in order to avoid drastic changes in the ph of the cells . it was found that h2s prevented the formation of -sheet and resulted in a significant transition of -helix to unordered protein . according to that work , the amount of -helix melting can be estimated from the intensity of ch bending band . therefore , the increase in the ch band intensity in the spectrum of hewl aggregates relative to that of native protein could be assigned to newly formed unordered structures . assuming that the amount of -sheet in hewl aggregates is approximately the same as in the native protein , we estimated the secondary structural composition of hewl aggregates as 83% unordered , 11% -helix , and 6% -sheet . to summarize the results concerning the significant tertiary structural rearrangements , -helix melting , and lack of -sheet formation , we conclude that h2s causes more significant denaturation of lysozyme than that taking place during the initial stages of protein fibrillation , which is typically reported as partial protein denaturation . we hypothesize that this significant lysozyme denaturation results in rapid protein aggregation , the formation of spherical species , and the prevention of the formation of -sheets and fibrillation . as a result , fully denatured lysozyme may lack the hydrophobic regions which are present in the partially unordered intermediates formed at the early stage of fibril formation . in agreement with wang s report , our results suggest that lysozyme denatures strongly in the presence of h2s and forms unordered aggregates that prevent -sheet formation and fibrillation . nielsen and colleagues proposed that sss bridges can form in proteins in the presence of h2s via the thiol we investigated the possibility of assigning a new raman band at 490 cm to the sss moiety . in agreement with other published studies , these compounds exhibit strong raman bands at 509 and 485 cm , respectively , in agreement with the raman spectra of native hewl and hewl spherical aggregates formed in the presence of h2s . furthermore , we obtained the difference spectrum by subtracting hewl spectra after 0 and 90 min of incubation in the presence of h2s and compared it to the expected spectral change representing the ss to sss transition . these groups could form as a result of disulfide bond reduction in the presence of h2s by a process known as sulfuration or sulfhydration . there is evidence in the literature that indicates the possibility of ss bond reduction in the presence of h2s and the formation of rssh groups in basic environments . it has also been demonstrated that reduction of four ss bonds to three ss bonds of apo--la accelerates its fibrillation and leads to the formation of a new fibril polymorph with a different morphology and structure compared to fibrils formed from the wild - type la . similar to insulin , the conformation of the ss bonds in hewl remains intact during the fibrillation of hewl in control solution , as we have described here . in agreement with these observations , our results suggest that lysozyme denatures strongly in the presence of h2s and forms unordered aggregates that prevent -sheet formation and fibrillation . amyloid fibrils are associated with many neurodegenerative diseases including alzheimer s , parkinson s , and systemic amyloidosis . it has recently been found that there is a reduced amount of hydrogen sulfide ( h2s ) in the brain tissue of patients with alzheimer s disease , leading us to investigate the effects of h2s on the formation of amyloid fibrils . our objective was to utilize several complementary techniques , including raman spectroscopy , afm , and intrinsic tryptophan and tht fluorescence for studying the kinetics of hewl fibrillation , a well - characterized model amyloidogenic protein , in the presence of h2s . lysozyme forms typical -sheet - rich fibrils after incubation in 20% acetic acid solution at 60 c for approximately 70 min . the addition of 12 mm h2s in a molar ratio of 5:1 ( h2s : hewl ) completely prevented the formation of the -sheet conformation as measured by deep uv resonance raman ( duvrr ) spectroscopy and tht fluorescence . by contrast , the melting of the -helix resulted in unordered protein in the form of spherical aggregates , which were distinctly different from amyloid fibrils . according to the intrinsic tryptophan fluorescence , hewl exhibited a more significant perturbation of its tertiary structure in the presence of h2s than that which occurred during the partial protein denaturation at the early stage of fibrillation . however , based on our data from nonresonance raman spectroscopy , ss bonds exhibit significant rearrangements in the presence of h2s . the peak area of the 507 cm raman band corresponding to the ss segment in the g - g - g conformation was reduced significantly , and a new band appeared at 490 cm . we assigned this new band to the sss group based on previously reported data , the raman spectra we acquired for model compounds , and lc - ms analysis of tcep reduction products . cytotoxicity tests revealed that the spherical aggregates formed by lysozyme in the presence of h2s are nontoxic to cells by contrast with fibrils . overall , we report for the first time that h2s prevents fibrillation of hewl in vitro and results in the formation of small spherical aggregates of unordered protein . given that there are new drugs under development for a slow release of h2s with a variety of therapeutic targets , including s - sildenafil for urological conditions , s - diclofenac for inflammation , s - latanoprost for neurodegenerative illnesses , s - levodopa for parkinson s disease , and s - aspirin for cardiovascular conditions , our results suggest possible new roles of sss in vivo .

the documented use of brachytherapy in the treatment of choroidal melanoma dates back to the use of radon seeds in the late 1930s , and episcleral plaques embedded with the radionuclide co-60 were described by stallard in the 1960s . another plaque of that era , which remains in use to this day , employed the embedded radionuclide ru-106 [ 2 , 3 ] . in subsequent years plaques containing i-125 and ir-192 seeds were introduced . a significant advance in eye plaque technology occurred in the mid 1980s , when the collaborative ocular melanoma study ( coms ) introduced a set of standardized plaques designed for i-125 seeds . the selection of the low energy i-125 radionuclide by the coms , and more recently pd-103 by others , meant that a plaque with a thin gold shell would be sufficient to block essentially all of the primary radiation that would otherwise irradiate the patient 's brain and orbit as well as the surgeon and other attending personnel . 1 , intraocular tumors such as choroidal melanoma typically appear as small dome - shaped mounds rising inward from the scleral shell of the eye . the plaque containing the radionuclide is surgically positioned on the sclera below the base of the tumor and then sutured to the sclera to affix the plaque at that location for the 3 to 7 day duration of the implant . a 5 mm tall , 10 mm diameter posterior tumor with a 2 mm retinal margin surrounding the base of the tumor . anterior to the equator , it is modeled as an oblate spheroid which intersects the cornea at the limbus . the photosensitive portion of the retina , drawn as the thick black line , is modeled as being inset 1 mm from the exterior of the sclera and extending from the posterior pole to the ora serata the most notable vision - impairing complications subsequent to episcleral plaque therapy are cataract , radiation maculopathy ( rm ) and to a lesser extent radiation retinopathy ( rr ) , optic neuropathy and neovascular glaucoma . cataracts are now of lesser concern owing to the introduction of intraocular lens ( iol ) replacements . it was recently reported that an antibody to vascular endothelial growth factor ( vegf ) which inhibits neovascularization and reduces retinal edema appears to reduce the severity of rm . these complications are progressive and may have a delayed onset of months to years after radiation treatment . the severity is dependent on the dose rate , the total dose of radiation and the amount of retina irradiated . the obvious way to reduce vision - impairing complications is , to the extent practical , to conform the prescribed isodose surface to the target volume and reduce the dose delivered to those photosensitive portions of the retina that fall outside of the target volume , with particular attention to the macula . 1 , is typically defined by the apex of the tumor and a small retinal margin ( e.g. 2 mm ) surrounding the tumor base . this margin accounts for microscopic extension of the tumor and uncertainties associated with plaque placement . therefore , the objective of conformal plaque therapy is to enclose the tumor apex and base + margin within the prescribed isodose surface while sparing as much of the surrounding tissue as possible . fortunately , the gold shell of the plaque absorbs virtually all primary radiation emanating from the radionuclide seeds that is initially directed away from the eye , and because the dome portion of the tumor is surrounded by radiobiologically inert vitreous humor , it is not critical for the isodose surface to closely conform to the shape of the dome in order to achieve the radiobiological goals of conformal therapy . it is primarily the dose delivered to the target volume and to the retina that are of radiobiological concern , and therefore it is sufficient that the prescribed isodose surface covers , and preferably conforms to , the shape of the base ( + margin ) of the tumor . image - based treatment simulation software for ophthalmic plaques that could model the conformal treatment of intraocular tumors was initially developed and adopted for clinical use at the university of southern california ( usc ) in the early 1990s and has been in continuous development ever since [ 1016 ] . this software fuses fundus photography , ultrasound , and either computed tomography ( ct ) or magnetic resonance ( mr ) images to build interactive three - dimensional ( 3d ) virtual models of each patient 's ocular anatomy , the plaques and the radiation sources . the software was licensed for commercial distribution in 1994 under the title plaque simulator ( eckert & ziegler bebig gmbh , robert - rssle - str.10 , d-13125 berlin , germany ) . gold plaques featuring collimating slots for each radiation source , introduced in the late 1990s demonstrated that highly conformal treatment of ocular tumors was possible . a fundamental requirement of all conformal external beam radiotherapy treatments ( e.g. radiosurgery ) is precise patient and beam positioning . a conformal isodose plan with a steep dose gradient outside the target volume is of little clinical value if it can not be reliably delivered if the plaque is not positioned on the eye at the location and orientation intended by the plan , then portions of the tumor may receive less dose than intended , and the healthy retina could receive more dose than intended . historically , the information regarding tumor size , shape and location within the eye that was often provided for plaque treatment planning purposes consisted of approximations and hand - drawn sketches . occasionally , at the time of surgery , a plaque might be found to be too small , perhaps due to intervening tumor growth , requiring the plaque to be reconfigured while the patient remained under sedation or risk inadequate coverage of the tumor base . the position on the eye at which the plaque would be affixed was not determined until the time of surgery during which a bright light was used to transilluminate the eye and cast a shadow of the tumor base onto the underlying translucent sclera . this shadow was traced with a marker pen and the plaque positioned to cover the tumor base . in aviation parlance , this approach is analogous to flying by the seat of one 's pants compared to using a predetermined flight plan and instruments . under these conditions , using image - based 3d treatment simulation , the tumor location can be accurately determined and plaque position can be planned in advance of surgery . to facilitate accurate positioning at the planned position , the simulation software provides targeting support in the form of suture eyelet coordinates on the sclera in a format familiar to the ophthalmic surgeon . 1 ) for each suture eyelet of the plaque [ 12 , 16 ] . in practice , the meridian clock hours can be estimated to with a few minutes by the surgeon , and the chord distance on the meridian precisely measured using an adjustable castroviejo caliper . the 2 mm ptv margin surrounding the tumor base takes into account the uncertainty in locating the meridian . nearly two decades of experience using this coordinate system at usc has demonstrated that it is simple and reliable . providing suture coordinates also frees the plaque shell from having to physically conform to the shape of the tumor base , it is sufficient that the radiation sources mounted within the shell provide the conformal isodose coverage when the shell is positioned as planned . freeing the shape of the shell from the physical shape of the tumor enables the suture eyelets to be relocated to more convenient anterior locations where suturing is easier and more accurate . although both the hardware , software and a targeting system now exist for planning and delivering conformal treatment of intraocular tumors , until now a means of quantitatively comparing rival treatment plan options has been lacking . in conventional radiation therapy treatment planning , the dose - volume histogram ( dvh ) has proven itself a useful tool for comparing rival treatment plans . in this work an adaptation of the dvh concept , the retina dose - area histogram ( rdah ) is introduced as a quantitative tool for the comparison of rival plaque designs and treatment plan options with the ultimate goal of reducing radiation retinopathy . the plaque simulator ( ps ) software [ 1016 ] was modified to version 5.7 to support the simultaneous calculation and display of rdahs for up to 4 rival plaque configurations . in this version of the software histograms are calculated for the photosensitive region of the retina between the posterior pole and the ora serrata , the macula , the tumor base , the base plus retinal margin and for photosensitive retina outside the base and margin . in the ps software , the sclera posterior to the equator sclera anterior to the equator is modeled as a 1 mm thick oblate spheroidal shell which intersects the dome - shaped cornea . the 1 mm scleral shell thickness is an assumption carried forward from the model of the eye that was recommended by the coms , but it appears to be adequate for treatment planning purposes . 1 , the photosensitive portion of the retina is modeled as a lining covering most of the interior surface of the sclera . the ora serrata delineates the transition from the simple non - photosensitive area of the retina near the ciliary body to the complex , multi - layered photosensitive region . the location of the ora serrata in the computer model is specified by a user adjustable ora - angle . describing the photosensitive extent of the retina by a single angle enables the model to adapt to eyes of different spherical and oblate radii of curvature with minimal user intervention . the determination of the size , shape and location of the tumor base on the retina begins with a digitized fundus - photo collage of the retina which must include the macula , the optic disc , and as much of the tumor as can be photographed . the center of the optic disk and the posterior pole near the macula are readily identifiable landmarks on the collage . 2a , the 3d coordinate of every pixel in the collage can be calculated relative to those landmarks once the 3d coordinates of those landmarks are determined . returning the fundus collage to 3d space also requires that the radius of curvature of the retina be known , and , in what direction the camera was looking when photographing each of the smaller pictures that make up the collage . in fig . 2a , the approximate centers of most of the photos that make up the collage are marked to indicate where the central ray of the camera was pointing when photographing that region of the retina . the 4 image is mostly obscured by the other 3 so it has been ignored . the fundus collage can be calibrated and fused with 3d - ct imaging studies by finding the posterior pole and the center of the optic disc on the collage and in 3d - ct space . b ) the size and shape of the eye , and the center of the optic disc are derived from a transverse axial ct slice that bisects the eye through the poles and the optic nerve retinal diagram ( rd ) of a left eye , illustrating a 3.4 mm apical height , 10 6 mm elliptically shaped tumor and a 2 mm retinal margin surrounding the base of the tumor . also projected onto the diagram are generic approximations of where the six extraocular muscle insert into the underlying sclera , the shell of a coms 14 mm plaque and its suture eyelets , the surgical suture coordinates of those eyelets expressed as meridian clock hour and chord distance from the limbus , and placeholder markers for the radionuclide seeds in the silicone carrier of the plaque . all objects , including the fundus - photo collage have been circumferentially warped as a function of latitude for correct mapping to the polar rd the ps coordinates of the posterior pole , the center of the optic disc , and the radius of curvature of the retina initially default to the coms recommended standard model of the eye . if available , a transverse axial ct image which bisects the eye through the anterior pole at the cornea , the lens , the posterior pole at the macula , and which shows the optic nerve entering the eye can be used to customize the ps model to match the spherical or oblate curvature of the actual patient eye as illustrated in fig . in general , the slice spacing is too great , and the resolution of ct and mri is too low to reliably determine tumor size and shape within the eye . however , by finding a common pair of landmarks in both the fundus photos and ct images , the pixels of the fundus collage can be transformed to 3d space and fused with the ct or mri study to reveal the tumor location for a specific patient . occasionally , the taller tumors are visible in ct and mri and this has proven useful for confirming the fundus collage 3d transform . ct and mri are also very useful companions to ultrasound for estimating the size , shape and location of anterior tumors which can not be photographed . the tumor location is determined by projecting the digitized fundus collage onto a two dimensional ( 2d ) map of the retinal surface and then manually outlining the tumor base . using the earth as an analogy , the spherical surface of the earth is most commonly represented using the cylindrical mercator projection in which size and shape distortion is minimal at the equator and maximum at the poles . in the eye , however , the region of greatest visual interest surrounds the posterior pole , not the equator . the retinal diagram ( rd ) is a polar map of the retinal surface from the posterior pole to the limbus . on a polar map , size and shape distortion is minimal at the polar center of the diagram and increases with the radial distance from the pole . 3 , observe that on the diagram the distance representing a unit distance ( e.g. 1 mm ) in 3d space is radially constant , but stretches circumferentially with radial distance from the polar center of the diagram . for example , the rd circle representing the great circle of latitude at the equator of the eye is drawn with a smaller diameter , and the circumferential distance between 1 mm tic marks on the equatorial circle of the rd is smaller than for the circle on the rd which represents the circle of latitude at the ora serrata which lies in the anterior hemisphere of the eye . the circle of latitude at the ora is , of course , three dimensionally much smaller than the circle at the equator . there is an exact transformation between the retinal surface in 3d space and the rd , and most objects of interest near the retina , such as the tumor apex , the extraocular muscle insertion locations , the plaque perimeter and the radiation sources can be projected onto the rd as illustrated in fig . 3 . note that all 3d objects mapped onto the retinal diagram , including the fundus photo collage , must be circumferentially stretched according to their latitude as was illustrated for the circle of latitude at the ora . after the perimeter of the tumor base has been outlined on the rd , the points defining the perimeter polygon can be transformed back into 3d coordinates on the retina , and a ptv - like margin added by expanding the perimeter polygon on the spherical and/or oblately shaped surface of the retina as determined from the ct images . a 2 mm margin is illustrated as a thin brown line surrounding the nominally elliptical tumor base in fig . the 3d model of the tumor is completed by using ultrasound imaging to determine the shape and height of the tumor dome resulting in an interactive 3d model that includes the ocular anatomy , the tumor , the plaque , the suture coordinates and the calculated radiation dosimetry as illustrated in fig . 3d fusion of ct imaging , fundus photography , ultrasound measurements , plaque , suture eyelets , surgical coordinates and radiation dosimetry once the 3d model and its rd projection have been created , rival treatment planning options , such as the choice of radionuclide , and the number and location of radiation sources , may be quantitatively compared using a retina dose - area histogram . the dose - area histogram for the surface of the retina is calculated by first plotting a raster image analogous to the rd seen in fig . 3 , except that the image contains only the tumor and margin against a plain white background . the area on the spherical surface of the retina represented by a pixel on the rasterized rd varies with its latitude , so the area of each pixel in the rd image is calculated as a function of latitude , and then the coordinates of the center of that that pixel are transformed from rd space to 3d space and the dose delivered at that point is calculated . the areas are binned as a function of dose for the object they represent on the rd , and plotted in a manner analogous to the ubiquitous dose - volume histogram . the objects for which dose - area will be calculated are : ( 1 ) the entire photosensitive surface of the retina represented by rd pixels that fall within the ora circle , ( 2 ) pixels that fall within the tumor perimeter anywhere on the diagram , ( 3 ) pixels that fall outside of the tumor perimeter and within the ora circle , ( 4 ) pixels that fall within the margin surrounding the tumor base , ( 5 ) pixels that fall outside of the margin and within the ora circle and ( 6 ) pixels that fall within the macular region which is approximated as a 5 mm diameter disc surrounding the posterior pole . in practice , this is easily accomplished by filling the tumor base and margin perimeter polygons on the raster image with different colors which encode what objects those pixels belong to and simply sorting according to the color of each pixel when binning the object areas . the raster image method is robust in that it works for polygons of arbitrary size and shape . the spatial resolution of the histogram will increase , as will the calculation time , as a function of the number of pixels that comprise the raster image . the surface area a of a spherical dome , or equivalently , a spherical bowl is where r c is the radius of curvature of the sphere and h is the height of the dome , or equivalently , the depth of the bowl as illustrated in fig . the height h can be calculated from the chord diameter d of the dome at its base and the curvature of the sphere as h = rc ( 1 cos(asin((d/2)/rc ) ) ) diagram illustrating the terms used in the analytical solution of the spherical surface of a tumor with a circular base of chord diameter ( d ) , spherical radius of curvature ( rc ) and dome height ( h ) for a 24 mm equatorial diameter spherical eye , and assuming that the retina is inset 1 mm from the external surface of the eye , the radius of curvature of the retina r c = 11 mm . the retinal surface area covered by a tumor with a circularly shaped base of chord diameter d = 10 mm is 83.08 mm . the spacial resolution of the histogram , and hence , the visual smoothness of the plotted dose - area curves , increases with the number of pixels that comprise the rd on the raster image . however , increasing the number of pixels proportionally increases the calculation time . table 1 summarizes results obtained when varying the rd image resolution for the same eye and tumor description for which the analytical solution was calculated above . the tumor is assumed to have a circular base of chord diameter 10 mm approximated by a polygon consisting of 36 edges and a retinal spherical radius of curvature of 11 mm . because the tumor perimeter is defined by a polygon , we expect the calculated area to be slightly less than the 83.08 mm derived from the analytical solution above . the third column in table 1 is the number of colored pixels that filled the polygon on the rd image . recall that although pixels on the rasterized rd image are all identical squares , the area of spherical surface that each pixel represents varies with its radial distance , or latitude , from the pole of the rd . the rightmost column is the area on the spherical surface of the retina occupied by the tumor base . it is calculated by numerically integrating the areas of the pixels that filled the tumor polygon . the leftmost column is the diameter , measured in pixels , of the rd in the rasterized image that is used to calculate the rdah . the retina was modeled as being spherical with a radius of curvature of 11 mm . the second column is the number of polygon edges that were used to approximate the circular base of a 10 mm chord diameter tumor . the third column is the number of pixels that filled the polygon on the rd image . note that although pixels on the rd image are all identical squares , the area that each pixel represents on the spherical surface varies with its radial distance from the pole of the rd . the rightmost column is the area on the spherical surface of the retina occupied by the tumor base . it is calculated by numerically integrating the areas of the pixels that filled the tumor polygon . the analytical solution for the area of this tumor on the spherical surface is 83.08 mm in table 2 the number of polygon edges used to draw the tumor on the rd image is varied from 24 to 180 . as expected , the numerical integration approaches the analytical solution as the perimeter of the polygon approaches a circle . the dose - area histograms in the remainder of this work were calculated using a rd diameter of 1024 pixels which provides very smooth dose - area curves . calculation speed was typically a few seconds on a 3.3 ghz quad - core intel xeon processor apple macpro computer . to illustrate how the rdah might be used as a clinical tool , consider the example patient of fig . 2 and 3 . the tumor is in the left eye , it is 3.4 mm tall at the apex which was determined from ultrasound measurements , it has an elliptically shaped base of about 10 6 mm which was digitized with a 147 edge polygon . the 2 mm retinal margin surrounding the base was automatically calculated by the ps software . the description of table 2 is the same as table 1 except that here the number of polygon edges used to draw the tumor on the rd image is varied from 24 to 180 . the analytical solution for the area of this tumor on the spherical surface is 83.08 mm . as expected , the numerical integration approaches the analytical solution as the perimeter of the polygon approaches a circle the 14 mm diameter coms plaque is a good physical and dosimetric match to the 10 mm major axis of the tumor base . in fig . 6a the silicone seed carrier of the coms 14 mm plaque has been fully loaded with 13 uniformly weighted i-125 seeds ( model iai-125a , isoaid llc , port richey , florida , usa ) . in fig . 6b only the portion of the silicone seed carrier that conforms to the shape of the tumor base has been loaded with 7 uniformly weighted i-125 seeds . fig . ru plaques are a viable alternative to i-125 and pd-103 plaques for tumors less than 4 mm tall . the ru plaques are widely used in europe [ 2 , 3 , 18 , 19 ] . 6d is similar to 6b except that the i-125 seed closest to the macula has been removed and replaced by a pair of seeds in the two carrier positions adjacent to the position that was just vacated . possible advantages of this spatially semi - conformal , non uniform redistribution of radionuclide are that the two half - weighted seeds are further away from the macula than the seed they replaced , and they also happen to be oriented with their axes of maximum self - attenuation directed at the macula . in fig . 6e we have a coms 14 mm shell with a modified seed carrier that has been semi - conformally loaded using the same seed pattern and radionuclide weighting distribution as fig . b ) coms 14 mm plaque with its carrier rotated to enable a partial loading of the carrier with i-125 seeds to dosimetrically conform to the shape of the tumor and its 2 mm retinal margin . d ) similar to b ) except that the i-125 seed closest to the macula has been removed and replaced by two adjacent seeds of half weighting . e ) coms 14 mm shell modified with a gold insert in place of the silicone seed carrier and semi - conformally loaded as in d ) except with pd-103 seeds . in all cases the plaques were automatically centered under the tumor and a dose of 85 gy was prescribed to the apex of the tumor . from left to right , the columns illustrate the radionuclide loading of the plaque , isodose lines on the retina , and isodose lines on a meridian plane bisecting the eye through the tumor apex . the yellow 300 gy isodose line is unlabeled the plaque shells were all automatically centered below the geometric center , or centroid , of the tumor base and automatically rotated to balance the distance of the suture eyelets from the limbus in order to simplify the surgery . the seed carriers of the coms plaques were manually rotated within the shell so that a roughly elliptically shaped subset of the seed positions in the carrier would spatially conform to the elliptical shape of the tumor base and retinal margin . in all cases the american brachytherapy society ( abs ) recommended minimum dose of 85 gy was prescribed to the apex of the tumor . from left to right , the columns in fig . 6 illustrate the radionuclide loading of the plaque , isodose lines on the retina , and lastly isodose lines on a meridian plane bisecting the eye through the tumor apex . the gray shaded hexagonal patches lining the concave surface of the ru plaque model illustrate beta - ray patch - source modeling of the relative distribution of the radionuclide embedded in the ru plaque which enables the software to reproduce the central axis and surface dose - rate measurements provided by the manufacturer to within a few percent . 7 wherein the curves plot the relative area as a function of dose to the objects of interest . normal tissue tolerance for retina and the prescription dose of 85 gy to the tumor apex are specifically marked along the top of the graphs . 6a ) , and the intermittent dash - dot lines are the ru-106 beta - ray plaque ( fig . the intermittent dash - dot lines are the nearly equivalent modified coms plaque ( fig . the intermittent dash - dot lines are the model cca ru-106 beta - ray plaque ( fig . the blue curves are the areal dose to the photosensitive portion of the retina that lies outside the margin that surrounds the tumor base . b ) retina dose - area histograms for the plans presented in figs . 6b , 6d and 6e . the heavy solid lines represent the coms plaque conformally loaded with i-125 seeds ( fig . 6d ) with the i-125 seed closest to the macula relocated to the adjacent carrier positions as two half - strength seeds . the intermittent dash - dot lines represent the gold - insert - modified coms plaque ( fig . the threshold dose for retinal damage is generally considered to be about 30 - 35 gy . emami et al . report 5%/5yr normal tissue tolerance for retina to be 45 gy and 50%/5yr tolerance to be 65 gy . animal studies have shown damage to rods with 20 gy and to cones with 100 gy . although damage to photoreceptors has been reported , it is the inner retinal layers and retinal vascular endothelial cells that are the most affected , causing vessel closure and ischemic ( capillary dropout ) retinopathy which , in turn , leads to additional forms of damage including neovascularization which can induce secondary glaucoma , retinal detachment , and vitreous hemorrhage . when the macula and fovea are involved , radiation maculopathy typically results in blindness [ 8 , 9 ] . in figs . 7a and b , the orange and blue colored rdah curves are of particular interest , because they plot relative area as a function of dose to the macular region and to the photosensitive portion of the retina that lies outside the ptv - like margin that surrounds the tumor base . these are the regions where one would like to reduce radiation dose with particular attention to areas which exceed normal tissue tolerance ( e.g. 45 gy ) . with regard to the retina outside the margin ( blue lines ) , the coms plaque conformally loaded with i-125 sources ( fig . 6b ) , plotted as the thickest blue line , irradiates the least area to doses above 40 gy . the ru beta - ray plaque irradiates the least area for doses below about 25 gy . with regard to the macula ( orange curves ) , which happens to lie just above the posterior lip of the plaques in this particular example , the conformally loaded plaque ( fig . it delivers a much higher maximum dose to the macula and consistently irradiates a greater area of the macula as a function of dose . this occurs because it uses the fewest number of seeds of the highest individual activity and one of those seeds happens to be positioned very close to the macula . in figs . 6d and 7b observe that by removing the i-125 seed closest to the macula and redistributing its original activity between the two adjacent positions in the carrier , we get a spatially semi - conformal loading that significantly reduces the maximum dose in the macular region at the cost of only a small loss of coverage at the periphery of the margin . in figs . 6e and 7b we see that substituting pd-103 ( dash - dot lines ) for the i-125 ( dashed lines ) in fig . 6d reduces the area receiving doses below 50 gy at the cost of higher dose to about 15% of the tumor base and 5% of the margin . this is to be expected because the radial dose function is steeper for pd-103 than for i-125 and the dose is prescribed to the apex of the tumor . in all of these rival plans , adequate dosimetric coverage of the tumor base and margin is achieved . the ru beta - ray plaque ( fig . 6c ) delivered good coverage of the tumor base and margin , and irradiated the least area at the low end of the dose range . arguably , it might be a good option for a circularly shaped tumor further away from the macula . in fig . 6a delivers a slightly higher maximum dose to the macula and irradiates a noticeably larger area of retina outside the margin ( dashed blue lines in fig . 7a ) in the dose range above 25 gy compared to its i-125 rivals , the plaques of figs . 6b and 6d . among rivals , the plan that delivers the prescribed dose to the tumor and margin while minimizing the area of macula ( orange curves in fig . 7 ) receiving doses greater than normal tissue tolerance ( e.g. 45 gy ) will likely be the plan of choice . reducing the area of non - specific retina outside the margin ( blue curves ) receiving doses greater than normal tissue tolerance 6 , the two plaques that are semi - conformally loaded ( figs . 6d and 6e ) deliver the least macula dose , as well as the least area outside the margin for doses above 40 gy , with the i-125 version irradiating slightly less area to doses greater than about 100 gy , and the pd-103 version slightly less area to doses below about 50 gy . the radiation dose prescription recommendations for episcleral plaque therapy which are currently in use are based on clinical experience gained at a time when there was great uncertainty regarding plaque dosimetry and surgical placement . considering the advances in plaque design , treatment planning and delivery over the past decade , and the observation that vision - impairing complications appear to be a topic of great concern in the ophthalmology literature the retina dose - area histogram provides a quantitative metric by which rival episcleral plaque treatment plan options may be quickly compared with regard to tumor and margin coverage , and possibly reducing the severity of subsequent radiation retinopathy and maculopathy . as with the dose - volume histogram , spatial location is lost in this method of analysis , so it must always be used as a companion to 2d and 3d isodose maps when making decisions regarding patient treatments .

purposeepiscleral plaques have a history of over a half century in the delivery of radiation therapy to intraocular tumors such as choroidal melanoma . although the tumor control rate is high , vision - impairing complications subsequent to treatment remain an issue . notable , late complications are radiation retinopathy and maculopathy . the obvious way to reduce the risk of radiation damage to the retina is to conform the prescribed isodose surface to the tumor base and to reduce the dose delivered to the surrounding healthy retina , especially the macula . using a fusion of fundus photography , ultrasound and ct images , tumor size , shape and location within the eye can be accurately simulated as part of the radiation planning process . in this work an adaptation of the dose - volume histogram ( dvh ) , the retina dose - area histogram ( rdah ) is introduced as a metric to help compare rival plaque designs and conformal treatment planning options with the goal of reducing radiation retinopathy.material and methodsthe rdah is calculated by transforming a digitized fundus - photo collage of the tumor into a rasterized polar map of the retinal surface known as a retinal diagram ( rd ) . the perimeter of the tumor base is digitized on the rd and its area computed . area and radiation dose are calculated for every pixel in the rd.resultsthe areal resolution of the rdah is a function of the pixel resolution of the raster image used to display the rd and the number of polygon edges used to digitize the perimeter of the tumor base . a practical demonstration is presented.conclusionsthe rdah provides a quantitative metric by which episcleral plaque treatment plan options may be evaluated and compared in order to confirm adequate dosimetric coverage of the tumor and margin , and to help minimize dose to the macula and retina .

Purpose Material and methods Results Discussion Conclusions

the documented use of brachytherapy in the treatment of choroidal melanoma dates back to the use of radon seeds in the late 1930s , and episcleral plaques embedded with the radionuclide co-60 were described by stallard in the 1960s . 1 , intraocular tumors such as choroidal melanoma typically appear as small dome - shaped mounds rising inward from the scleral shell of the eye . the plaque containing the radionuclide is surgically positioned on the sclera below the base of the tumor and then sutured to the sclera to affix the plaque at that location for the 3 to 7 day duration of the implant . the photosensitive portion of the retina , drawn as the thick black line , is modeled as being inset 1 mm from the exterior of the sclera and extending from the posterior pole to the ora serata the most notable vision - impairing complications subsequent to episcleral plaque therapy are cataract , radiation maculopathy ( rm ) and to a lesser extent radiation retinopathy ( rr ) , optic neuropathy and neovascular glaucoma . the severity is dependent on the dose rate , the total dose of radiation and the amount of retina irradiated . the obvious way to reduce vision - impairing complications is , to the extent practical , to conform the prescribed isodose surface to the target volume and reduce the dose delivered to those photosensitive portions of the retina that fall outside of the target volume , with particular attention to the macula . therefore , the objective of conformal plaque therapy is to enclose the tumor apex and base + margin within the prescribed isodose surface while sparing as much of the surrounding tissue as possible . fortunately , the gold shell of the plaque absorbs virtually all primary radiation emanating from the radionuclide seeds that is initially directed away from the eye , and because the dome portion of the tumor is surrounded by radiobiologically inert vitreous humor , it is not critical for the isodose surface to closely conform to the shape of the dome in order to achieve the radiobiological goals of conformal therapy . it is primarily the dose delivered to the target volume and to the retina that are of radiobiological concern , and therefore it is sufficient that the prescribed isodose surface covers , and preferably conforms to , the shape of the base ( + margin ) of the tumor . this software fuses fundus photography , ultrasound , and either computed tomography ( ct ) or magnetic resonance ( mr ) images to build interactive three - dimensional ( 3d ) virtual models of each patient 's ocular anatomy , the plaques and the radiation sources . a conformal isodose plan with a steep dose gradient outside the target volume is of little clinical value if it can not be reliably delivered if the plaque is not positioned on the eye at the location and orientation intended by the plan , then portions of the tumor may receive less dose than intended , and the healthy retina could receive more dose than intended . historically , the information regarding tumor size , shape and location within the eye that was often provided for plaque treatment planning purposes consisted of approximations and hand - drawn sketches . occasionally , at the time of surgery , a plaque might be found to be too small , perhaps due to intervening tumor growth , requiring the plaque to be reconfigured while the patient remained under sedation or risk inadequate coverage of the tumor base . the position on the eye at which the plaque would be affixed was not determined until the time of surgery during which a bright light was used to transilluminate the eye and cast a shadow of the tumor base onto the underlying translucent sclera . under these conditions , using image - based 3d treatment simulation , the tumor location can be accurately determined and plaque position can be planned in advance of surgery . to facilitate accurate positioning at the planned position , the simulation software provides targeting support in the form of suture eyelet coordinates on the sclera in a format familiar to the ophthalmic surgeon . in practice , the meridian clock hours can be estimated to with a few minutes by the surgeon , and the chord distance on the meridian precisely measured using an adjustable castroviejo caliper . providing suture coordinates also frees the plaque shell from having to physically conform to the shape of the tumor base , it is sufficient that the radiation sources mounted within the shell provide the conformal isodose coverage when the shell is positioned as planned . although both the hardware , software and a targeting system now exist for planning and delivering conformal treatment of intraocular tumors , until now a means of quantitatively comparing rival treatment plan options has been lacking . in conventional radiation therapy treatment planning , the dose - volume histogram ( dvh ) has proven itself a useful tool for comparing rival treatment plans . in this work an adaptation of the dvh concept , the retina dose - area histogram ( rdah ) is introduced as a quantitative tool for the comparison of rival plaque designs and treatment plan options with the ultimate goal of reducing radiation retinopathy . in this version of the software histograms are calculated for the photosensitive region of the retina between the posterior pole and the ora serrata , the macula , the tumor base , the base plus retinal margin and for photosensitive retina outside the base and margin . in the ps software , the sclera posterior to the equator sclera anterior to the equator is modeled as a 1 mm thick oblate spheroidal shell which intersects the dome - shaped cornea . 1 , the photosensitive portion of the retina is modeled as a lining covering most of the interior surface of the sclera . the determination of the size , shape and location of the tumor base on the retina begins with a digitized fundus - photo collage of the retina which must include the macula , the optic disc , and as much of the tumor as can be photographed . the center of the optic disk and the posterior pole near the macula are readily identifiable landmarks on the collage . 2a , the 3d coordinate of every pixel in the collage can be calculated relative to those landmarks once the 3d coordinates of those landmarks are determined . the fundus collage can be calibrated and fused with 3d - ct imaging studies by finding the posterior pole and the center of the optic disc on the collage and in 3d - ct space . b ) the size and shape of the eye , and the center of the optic disc are derived from a transverse axial ct slice that bisects the eye through the poles and the optic nerve retinal diagram ( rd ) of a left eye , illustrating a 3.4 mm apical height , 10 6 mm elliptically shaped tumor and a 2 mm retinal margin surrounding the base of the tumor . also projected onto the diagram are generic approximations of where the six extraocular muscle insert into the underlying sclera , the shell of a coms 14 mm plaque and its suture eyelets , the surgical suture coordinates of those eyelets expressed as meridian clock hour and chord distance from the limbus , and placeholder markers for the radionuclide seeds in the silicone carrier of the plaque . all objects , including the fundus - photo collage have been circumferentially warped as a function of latitude for correct mapping to the polar rd the ps coordinates of the posterior pole , the center of the optic disc , and the radius of curvature of the retina initially default to the coms recommended standard model of the eye . if available , a transverse axial ct image which bisects the eye through the anterior pole at the cornea , the lens , the posterior pole at the macula , and which shows the optic nerve entering the eye can be used to customize the ps model to match the spherical or oblate curvature of the actual patient eye as illustrated in fig . in general , the slice spacing is too great , and the resolution of ct and mri is too low to reliably determine tumor size and shape within the eye . however , by finding a common pair of landmarks in both the fundus photos and ct images , the pixels of the fundus collage can be transformed to 3d space and fused with the ct or mri study to reveal the tumor location for a specific patient . ct and mri are also very useful companions to ultrasound for estimating the size , shape and location of anterior tumors which can not be photographed . the tumor location is determined by projecting the digitized fundus collage onto a two dimensional ( 2d ) map of the retinal surface and then manually outlining the tumor base . the retinal diagram ( rd ) is a polar map of the retinal surface from the posterior pole to the limbus . for example , the rd circle representing the great circle of latitude at the equator of the eye is drawn with a smaller diameter , and the circumferential distance between 1 mm tic marks on the equatorial circle of the rd is smaller than for the circle on the rd which represents the circle of latitude at the ora serrata which lies in the anterior hemisphere of the eye . there is an exact transformation between the retinal surface in 3d space and the rd , and most objects of interest near the retina , such as the tumor apex , the extraocular muscle insertion locations , the plaque perimeter and the radiation sources can be projected onto the rd as illustrated in fig . note that all 3d objects mapped onto the retinal diagram , including the fundus photo collage , must be circumferentially stretched according to their latitude as was illustrated for the circle of latitude at the ora . after the perimeter of the tumor base has been outlined on the rd , the points defining the perimeter polygon can be transformed back into 3d coordinates on the retina , and a ptv - like margin added by expanding the perimeter polygon on the spherical and/or oblately shaped surface of the retina as determined from the ct images . the 3d model of the tumor is completed by using ultrasound imaging to determine the shape and height of the tumor dome resulting in an interactive 3d model that includes the ocular anatomy , the tumor , the plaque , the suture coordinates and the calculated radiation dosimetry as illustrated in fig . 3d fusion of ct imaging , fundus photography , ultrasound measurements , plaque , suture eyelets , surgical coordinates and radiation dosimetry once the 3d model and its rd projection have been created , rival treatment planning options , such as the choice of radionuclide , and the number and location of radiation sources , may be quantitatively compared using a retina dose - area histogram . the dose - area histogram for the surface of the retina is calculated by first plotting a raster image analogous to the rd seen in fig . the area on the spherical surface of the retina represented by a pixel on the rasterized rd varies with its latitude , so the area of each pixel in the rd image is calculated as a function of latitude , and then the coordinates of the center of that that pixel are transformed from rd space to 3d space and the dose delivered at that point is calculated . the areas are binned as a function of dose for the object they represent on the rd , and plotted in a manner analogous to the ubiquitous dose - volume histogram . the objects for which dose - area will be calculated are : ( 1 ) the entire photosensitive surface of the retina represented by rd pixels that fall within the ora circle , ( 2 ) pixels that fall within the tumor perimeter anywhere on the diagram , ( 3 ) pixels that fall outside of the tumor perimeter and within the ora circle , ( 4 ) pixels that fall within the margin surrounding the tumor base , ( 5 ) pixels that fall outside of the margin and within the ora circle and ( 6 ) pixels that fall within the macular region which is approximated as a 5 mm diameter disc surrounding the posterior pole . in practice , this is easily accomplished by filling the tumor base and margin perimeter polygons on the raster image with different colors which encode what objects those pixels belong to and simply sorting according to the color of each pixel when binning the object areas . the spatial resolution of the histogram will increase , as will the calculation time , as a function of the number of pixels that comprise the raster image . the height h can be calculated from the chord diameter d of the dome at its base and the curvature of the sphere as h = rc ( 1 cos(asin((d/2)/rc ) ) ) diagram illustrating the terms used in the analytical solution of the spherical surface of a tumor with a circular base of chord diameter ( d ) , spherical radius of curvature ( rc ) and dome height ( h ) for a 24 mm equatorial diameter spherical eye , and assuming that the retina is inset 1 mm from the external surface of the eye , the radius of curvature of the retina r c = 11 mm . the spacial resolution of the histogram , and hence , the visual smoothness of the plotted dose - area curves , increases with the number of pixels that comprise the rd on the raster image . the third column in table 1 is the number of colored pixels that filled the polygon on the rd image . recall that although pixels on the rasterized rd image are all identical squares , the area of spherical surface that each pixel represents varies with its radial distance , or latitude , from the pole of the rd . the rightmost column is the area on the spherical surface of the retina occupied by the tumor base . it is calculated by numerically integrating the areas of the pixels that filled the tumor polygon . the leftmost column is the diameter , measured in pixels , of the rd in the rasterized image that is used to calculate the rdah . the second column is the number of polygon edges that were used to approximate the circular base of a 10 mm chord diameter tumor . the third column is the number of pixels that filled the polygon on the rd image . note that although pixels on the rd image are all identical squares , the area that each pixel represents on the spherical surface varies with its radial distance from the pole of the rd . the rightmost column is the area on the spherical surface of the retina occupied by the tumor base . it is calculated by numerically integrating the areas of the pixels that filled the tumor polygon . the analytical solution for the area of this tumor on the spherical surface is 83.08 mm in table 2 the number of polygon edges used to draw the tumor on the rd image is varied from 24 to 180 . as expected , the numerical integration approaches the analytical solution as the perimeter of the polygon approaches a circle . the dose - area histograms in the remainder of this work were calculated using a rd diameter of 1024 pixels which provides very smooth dose - area curves . the description of table 2 is the same as table 1 except that here the number of polygon edges used to draw the tumor on the rd image is varied from 24 to 180 . as expected , the numerical integration approaches the analytical solution as the perimeter of the polygon approaches a circle the 14 mm diameter coms plaque is a good physical and dosimetric match to the 10 mm major axis of the tumor base . 6b only the portion of the silicone seed carrier that conforms to the shape of the tumor base has been loaded with 7 uniformly weighted i-125 seeds . b ) coms 14 mm plaque with its carrier rotated to enable a partial loading of the carrier with i-125 seeds to dosimetrically conform to the shape of the tumor and its 2 mm retinal margin . in all cases the plaques were automatically centered under the tumor and a dose of 85 gy was prescribed to the apex of the tumor . from left to right , the columns illustrate the radionuclide loading of the plaque , isodose lines on the retina , and isodose lines on a meridian plane bisecting the eye through the tumor apex . the yellow 300 gy isodose line is unlabeled the plaque shells were all automatically centered below the geometric center , or centroid , of the tumor base and automatically rotated to balance the distance of the suture eyelets from the limbus in order to simplify the surgery . the seed carriers of the coms plaques were manually rotated within the shell so that a roughly elliptically shaped subset of the seed positions in the carrier would spatially conform to the elliptical shape of the tumor base and retinal margin . 6 illustrate the radionuclide loading of the plaque , isodose lines on the retina , and lastly isodose lines on a meridian plane bisecting the eye through the tumor apex . 7 wherein the curves plot the relative area as a function of dose to the objects of interest . normal tissue tolerance for retina and the prescription dose of 85 gy to the tumor apex are specifically marked along the top of the graphs . the blue curves are the areal dose to the photosensitive portion of the retina that lies outside the margin that surrounds the tumor base . 7a and b , the orange and blue colored rdah curves are of particular interest , because they plot relative area as a function of dose to the macular region and to the photosensitive portion of the retina that lies outside the ptv - like margin that surrounds the tumor base . with regard to the retina outside the margin ( blue lines ) , the coms plaque conformally loaded with i-125 sources ( fig . with regard to the macula ( orange curves ) , which happens to lie just above the posterior lip of the plaques in this particular example , the conformally loaded plaque ( fig . it delivers a much higher maximum dose to the macula and consistently irradiates a greater area of the macula as a function of dose . this occurs because it uses the fewest number of seeds of the highest individual activity and one of those seeds happens to be positioned very close to the macula . 6d and 7b observe that by removing the i-125 seed closest to the macula and redistributing its original activity between the two adjacent positions in the carrier , we get a spatially semi - conformal loading that significantly reduces the maximum dose in the macular region at the cost of only a small loss of coverage at the periphery of the margin . 6d reduces the area receiving doses below 50 gy at the cost of higher dose to about 15% of the tumor base and 5% of the margin . this is to be expected because the radial dose function is steeper for pd-103 than for i-125 and the dose is prescribed to the apex of the tumor . in all of these rival plans , adequate dosimetric coverage of the tumor base and margin is achieved . 6c ) delivered good coverage of the tumor base and margin , and irradiated the least area at the low end of the dose range . 6a delivers a slightly higher maximum dose to the macula and irradiates a noticeably larger area of retina outside the margin ( dashed blue lines in fig . among rivals , the plan that delivers the prescribed dose to the tumor and margin while minimizing the area of macula ( orange curves in fig . considering the advances in plaque design , treatment planning and delivery over the past decade , and the observation that vision - impairing complications appear to be a topic of great concern in the ophthalmology literature the retina dose - area histogram provides a quantitative metric by which rival episcleral plaque treatment plan options may be quickly compared with regard to tumor and margin coverage , and possibly reducing the severity of subsequent radiation retinopathy and maculopathy . as with the dose - volume histogram , spatial location is lost in this method of analysis , so it must always be used as a companion to 2d and 3d isodose maps when making decisions regarding patient treatments .

approximately 225,000 new cases of ovarian cancer are diagnosed worldwide , and ~140,200 women die of the disease each year.1 when compared to other solid tumors , ovarian cancer has a strong proclivity for early peritoneal dissemination and a disproportionately high percentage of women present with advanced stage disease . a combination of maximal surgical cytoreduction and platinum taxane - based chemotherapy comprises the mainstay of primary therapy . approximately 75% of patients experience an initial complete clinical response , but the majority recur , with a median time to first recurrence of 16 months.2 the behavior of recurrent ovarian cancer is variable . recurrent tumors can be isolated or widely metastatic and relatively indolent , or rapidly progressing . the primary goals of therapy are to prolong disease - free intervals and to improve the quality of life . responsiveness to chemotherapy and length of remission are predictors of overall prognosis.3 patients with recurrent ovarian cancer are risk stratified based on the time to recurrence after the completion of platinum - based chemotherapy : patients with a treatment - free interval of > 6 months after the completion of primary platinum - based therapy are considered as platinum sensitive and patients with a treatment - free interval of < 6 months are considered as platinum resistant . patients who progress while on therapy or within 4 weeks of platinum - based therapy are generally considered as platinum refractory . with successive lines of chemotherapy , several chemotherapeutic options exist for the treatment of platinum - resistant ovarian cancer , including pegylated liposomal doxorubicin ( pld),4,5 gemcitabine,5,6 topotecan,4 and etoposide.7,8 the overall response to any of these therapies is ~10%20% , with a median progression - free survival ( pfs ) of 34 months and a median overall survival ( os ) of 912 months.3 selection of therapy depends on prior treatment history , patient characteristics , and the side effect profile of each drug . bevacizumab ( avastin , genentech , san francisco , ca , usa ) , a monoclonal anti - vascular endothelial growth factor ( vegf)-a antibody targeting tumor angiogenesis , has been investigated and widely adopted for the treatment of recurrent ovarian cancer for the last several years . on november 14 , 2014 , following the publication of phase iii avastin use in platinum - resistant epithelial ovarian cancer ( aurelia ) trial,9 the us food and drug administration ( fda ) approved bevacizumab for use in recurrent , platinum - resistant ovarian cancer . bevacizumab is the first new drug to receive fda approval for the treatment of ovarian cancer since gemcitabine ( in combination with carboplatin ) in 2006 . bevacizumab , in combination with pld , weekly paclitaxel , or topotecan , is currently approved for patients with platinum - resistant disease who have received no more than two previous lines of chemotherapy . this review focuses on the efficacy , safety , acceptability , and therapeutic role of bevacizumab for the treatment of recurrent , platinum - resistant ovarian cancer . tumor angiogenesis plays a pivotal role in the growth and metastasis of ovarian cancer , because of the unique pattern of early dissemination of free - floating cells that form tumor implants in the peritoneal cavity . in order for a tumor to grow > 12 mm in size , it must recruit a blood supply from surrounding host tissue.10 tumors that fail to develop an adequate blood supply may remain quiescent for many years . an angiogenic switch , which is associated with an increased growth and metastatic potential , occurs when tumors become vascularized.11 angiogenic switch occurs as a result of alterations in the tumor - stromal microenvironment induced by the activation of tumor oncogenes , tissue hypoxia , and increased tumor expression of multiple proangiogenic factors , including vegf , fibroblast growth factor ( fgf ) , platelet - derived growth factor ( pdgf ) , angiopoietins ( ang1 and ang2 ) , and others . vegf - a is one of the most important regulators of angiogenesis , which was first isolated in 198312 and independently verified in 1989.13 we now know that vegf - a is one of a family of seven soluble vegf ligands ( vegf - a e and placental growth factor-1 and -2 ) that bind and signal through cell surface receptor tyrosine kinases vegf receptor ( vegfr)-1 , vegfr-2 , and vegfr-3 with variable affinities and effects depending on the biological context.14,15 vegf - a preferentially binds vegfr-1 and vegfr-2 , which are the most important receptors for vegf - a - mediated angiogenesis.16 vegf - a signaling through vegfr-2 promotes endothelial cell proliferation and resistance to apoptosis through c - raf - mapk / erk kinase and pi3k / akt signaling pathway activation17,18 and induces microvascular permeability , contributing to the extravasation of plasma proteins and water into the tumor microenvironment.19 this in turn creates a favorable environment for cell migration and proliferation , enhancing endothelial cell migration and neovascularization of the tumor - associated stroma , as well as tumor growth and metastasis . increased capillary permeability also contributes to the formation of malignant ascites , and vegf concentrations in malignant ascites , which is common in ovarian cancer , exceed the 95th percentile of concentrations observed in benign diseases.20 in ovarian cancers , vegf is detectable by immunohistochemistry in ovarian tumors , as well as in ascites and serum samples , and the expression levels are cancer specific , with no expression in benign ovarian tumors , intermediate expression in borderline tumors , and high levels of expression in invasive cancers.21,22 vegf expression also correlates with ovarian cancer stage at diagnosis and shorter pfs and os.2325 mouse monoclonal anti - vegf - a antibodies were shown to dramatically reduce tumor growth and malignant ascites formation in vivo , leading to the development of bevacizumab , a humanized monoclonal anti - vegf - a antibody , in 1997.26 bevacizumab demonstrated a promising activity against advanced colorectal cancers , which quickly led to multiple studies in breast , renal , cervical , and ovarian carcinomas . bevacizumab was first demonstrated to have an activity as monotherapy in recurrent ovarian cancer in 2007 . there are now four published phase iii randomized controlled trials9,2729 examining bevacizumab in ovarian cancer : two in the frontline setting , one in platinum - sensitive recurrent disease , and one in platinum - resistant disease . in gynecologic oncology group ( gog ) 218 , 1,873 women with stage iii or iv previously untreated and incompletely resected ovarian cancer were randomized to the first - line adjuvant therapy in one of the three arms after primary surgical cytoreduction : control ( carboplatin area under the concentration [ auc ] 6 plus paclitaxel 175 mg / m plus placebo ) , control with bevacizumab 15 mg / kg , or control with bevacizumab followed by bevacizumab maintenance.27 combination of bevacizumab therapy with bevacizumab maintenance therapy resulted in an improved pfs ( median pfs 14.1 months vs 10.3 months without bevacizumab , hazard ratio [ hr ] 0.717 , 95% confidence interval [ ci ] 0.6250.824 , p<0.001 ) , but there was no difference in os . icon7 , a very similar trial , evaluated bevacizumab for the first - line adjuvant therapy after primary surgery in women with stages i iia high - grade or clear cell ovarian carcinoma or stages iib iv epithelial ovarian cancer , primary peritoneal cancer , or fallopian tube cancer . ( epithelial ovarian cancer , primary peritoneal cancer , and fallopian tube cancer share similar carcinogenesis , pattern of spread , staging , and treatment and are frequently combined with clinical trials . icon7 randomized 1,528 women for first - line adjuvant therapy with carboplatin ( auc 5 or 6 ) plus paclitaxel 175 mg / m every 3 weeks for six cycles or this regimen plus bevacizumab 7.5 mg / kg given concurrently.28 a subset of 470 women in the bevacizumab arm received an additional 12 cycles of bevacizumab maintenance therapy . in updated analysis after 28 months of median follow - up , pfs was significantly improved with the addition of bevacizumab therapy ( median pfs 19.8 months vs 17.4 months , hr 0.87 , 95% ci 0.770.99 , p=0.04 ) , but there was no statistically significant improvement in os . with two large randomized trials demonstrating modest improvement in pfs with no difference in os , bevacizumab was not widely adopted for the first - line therapy outside of clinical trials . further discussion on the role of bevacizumab in the first - line therapy is beyond the scope of this review and can be found elsewhere . gog 218 and icon7 were followed by two trials evaluating bevacizumab in the recurrent setting . ovarian cancer study comparing efficacy and safety of chemotherapy and anti - angiogenic therapy in platinum - sensitive recurrent disease ( oceans ) randomized 484 women with recurrent , platinum - sensitive ovarian , fallopian tube , and primary peritoneal cancer to carboplatin ( auc 4 ) on day 1 with gemcitabine 1,000 mg / m on days 1 and 8 , with bevacizumab 15 mg / kg or placebo . bevacizumab was significantly associated with an increased pfs ( median pfs 12.4 months vs 8.4 months , hr 0.484 , 95% ci 0.3880.605 , p<0.0001),29 but there was no significant difference in os.30 most recently , the aurelia trial demonstrated a significant improvement in pfs for women with platinum - resistant ovarian cancer ( table 1).9 gog 170d was the first of two concurrent phase ii trials published in 2007 that demonstrated a promising bevacizumab activity in ovarian cancer . in this study , burger et al reported an objective response rate ( orr ) of 21% among 62 evaluable patients with recurrent ovarian cancer who received bevacizumab 15 mg / kg every 3 weeks until disease progression or unacceptable toxicity.31 of note , there were no gastrointestinal ( gi ) perforations observed in this study . gog 170d was originally designed to evaluate 6 months pfs but was reopened to evaluate orr after an unanticipated high rate of clinical responses . there were two complete responses and eleven partial responses , with a median duration of response of 10.3 months , and 31 patients maintained stable disease . response rates among platinum - resistant patients , who accounted for 58% of the study group , were not specifically reported . an exploratory analysis of prognostic factors predicting response found no significant association between response and platinum sensitivity.31 shortly after gog 170d , cannistra et al reported an orr of 15.9% among 44 women with recurrent , platinum - resistant ovarian cancer who received bevacizumab 15 mg / kg every 3 weeks.32 an additional 61.4% experienced stable disease . despite early termination due to an unexpectedly high rate of gi perforation ( 11.4% ) , cannistra et al reported a response rate comparable to other single - agent therapies in the recurrent , platinum - resistant setting . median pfs was 4.4 months , and median os was 10.7 months at the time of study termination . following upon the burger et al and cannistra et al studies , multiple phase ii clinical trials have evaluated bevacizumab as a doublet with other cytotoxic drugs that are frequently used for recurrent ovarian cancer . pfs ranges from 6 months to 8 months , and os ranges from 13.8 months to 33.2 months ( table 1).3342 the majority of these trials include both platinum - sensitive and platinum - resistant patients ; however , there are five trials specifically evaluating platinum - resistant patients with similar response rates and survival outcomes.36,37,39,41,42 in addition to the encouraging orrs observed with bevacizumab , phase ii studies also demonstrate high rates of disease stabilization , which is an important consideration for the platinum - resistant population , where therapeutic options are limited and where prolonged stable disease allows patients to maintain a reasonable quality of life while managing their cancer as a chronic illness . in all phase ii studies of bevacizumab combination therapy , orrs , pfs , and os compare favorably with the previous studies of cytotoxic chemotherapy for recurrent , platinum - resistant ovarian cancer . the aurelia trial is the first phase iii clinical trial to directly compare bevacizumab combined with chemotherapy to chemotherapy alone in the recurrent , platinum - resistant setting.9 a total of 361 patients with recurrent ovarian , fallopian tube , and primary peritoneal cancer who progressed within 6 months of receiving platinum - based chemotherapy were assigned a single - agent chemotherapy from the following options based on the investigator choice : 1 ) weekly paclitaxel 80 mg / m , 2 ) pld 40 mg / m every 4 weeks , or 3 ) topotecan 4 mg / m on days 1 , 8 , and 15 every 4 weeks or 1.25 mg / m on days 15 every 3 weeks ( table 1 ) . patients were stratified by chemotherapy regimen prior to randomization to bevacizumab combination therapy or chemotherapy alone . the addition of bevacizumab resulted in an orr of 30.9% , compared to 12.6% for chemotherapy alone ( p<0.001 ) , and a median pfs of 6.7 months vs 3.4 months ( hr 0.48 , 95% ci 0.380.60 , p<0.001 ) . there was a nonsignificant trend toward an improved os ( 16.6 months vs 13.3 months , hr 0.85 , 95% ci 0.661.08 , p<0.174 ) ; however , the true effect of bevacizumab on os is obscured and likely diluted by a 40% crossover to bevacizumab monotherapy among patients in the chemotherapy arm at the time of disease progression . subgroup analysis of the aurelia trial confirmed that bevacizumab consistently improves orr and pfs across chemotherapy cohorts ( table 1 ) . interestingly , the orr associated with bevacizumab combination therapy was the highest in the paclitaxel cohort ( 53.3% vs 30.2% ) compared to topotecan ( 17% vs 0% ) and the pld ( 13.7% vs 7.8% ) . median pfs followed the same trend for paclitaxel ( 10.4 months vs 3.9 months , hr 0.46 , 95% ci 0.30.71 ) , topotecan ( 5.8 months vs 2.1 months , hr 0.32 95% ci 0.210.49 ) , and pld ( 5.4 months vs 3.5 months , hr 0.57 , 95% ci 0.930.83 ) . it would be erroneous to conclude from these subgroup analyses of aurelia that there is a superiority of one of these chemotherapies given that the trial design did not randomize the chemotherapeutic agent . this trend is also present among phase ii studies of bevacizumab combination therapy ( table 1 ) and may reflect a higher activity of frequently dosed taxanes in epithelial ovarian cancer . such a metronomic schedule of some chemotherapies may also provide antiangiogenic effects not seen with conventional dosing schedules.43,44 additionally , metronomic dosing , such as with oral daily cyclophosphamide , may also impart immunostimulatory effects.45,46 a few studies have examined bevacizumab in combination with other targeted biologic therapies in recurrent ovarian cancer . epidermal growth factor receptor activation has been shown to promote vegf secretion ; therefore , nimeiri et al reported a small phase ii clinical trial of combination of bevacizumab and erlotinib , which demonstrated no increased activity compared to the previous studies of bevacizumab alone , and an increased toxicity with a 15% rate of gi perforation and high rates of nausea and diarrhea.47 in a phase i study by azad et al , partial response was observed in 6/13 patients with ovarian cancer with a combination of bevacizumab 5 mg / kg every 2 weeks and sorafenib , a receptor tyrosine kinase inhibitor ( tki ) known to target vegfr-2 and raf , which is known to be a downstream signaling target of vegf.48 rapid dose reductions for fatigue , anorexia , weight loss , hypertension , and proteinuria suggest that this combination may not be tolerable for long - term therapy , and 2/13 patients developed grade 2 fistulas . gog 170d was the first of two concurrent phase ii trials published in 2007 that demonstrated a promising bevacizumab activity in ovarian cancer . in this study , burger et al reported an objective response rate ( orr ) of 21% among 62 evaluable patients with recurrent ovarian cancer who received bevacizumab 15 mg / kg every 3 weeks until disease progression or unacceptable toxicity.31 of note , there were no gastrointestinal ( gi ) perforations observed in this study . gog 170d was originally designed to evaluate 6 months pfs but was reopened to evaluate orr after an unanticipated high rate of clinical responses . there were two complete responses and eleven partial responses , with a median duration of response of 10.3 months , and 31 patients maintained stable disease . response rates among platinum - resistant patients , who accounted for 58% of the study group , were not specifically reported . an exploratory analysis of prognostic factors predicting response found no significant association between response and platinum sensitivity.31 shortly after gog 170d , cannistra et al reported an orr of 15.9% among 44 women with recurrent , platinum - resistant ovarian cancer who received bevacizumab 15 mg / kg every 3 weeks.32 an additional 61.4% experienced stable disease . despite early termination due to an unexpectedly high rate of gi perforation ( 11.4% ) , cannistra et al reported a response rate comparable to other single - agent therapies in the recurrent , platinum - resistant setting . median pfs was 4.4 months , and median os was 10.7 months at the time of study termination . following upon the burger et al and cannistra et al studies , multiple phase ii clinical trials have evaluated bevacizumab as a doublet with other cytotoxic drugs that are frequently used for recurrent ovarian cancer . pfs ranges from 6 months to 8 months , and os ranges from 13.8 months to 33.2 months ( table 1).3342 the majority of these trials include both platinum - sensitive and platinum - resistant patients ; however , there are five trials specifically evaluating platinum - resistant patients with similar response rates and survival outcomes.36,37,39,41,42 in addition to the encouraging orrs observed with bevacizumab , phase ii studies also demonstrate high rates of disease stabilization , which is an important consideration for the platinum - resistant population , where therapeutic options are limited and where prolonged stable disease allows patients to maintain a reasonable quality of life while managing their cancer as a chronic illness . in all phase ii studies of bevacizumab combination therapy , orrs , pfs , and os compare favorably with the previous studies of cytotoxic chemotherapy for recurrent , platinum - resistant ovarian cancer . the aurelia trial is the first phase iii clinical trial to directly compare bevacizumab combined with chemotherapy to chemotherapy alone in the recurrent , platinum - resistant setting.9 a total of 361 patients with recurrent ovarian , fallopian tube , and primary peritoneal cancer who progressed within 6 months of receiving platinum - based chemotherapy were assigned a single - agent chemotherapy from the following options based on the investigator choice : 1 ) weekly paclitaxel 80 mg / m , 2 ) pld 40 mg / m every 4 weeks , or 3 ) topotecan 4 mg / m on days 1 , 8 , and 15 every 4 weeks or 1.25 mg / m on days 15 every 3 weeks ( table 1 ) . patients were stratified by chemotherapy regimen prior to randomization to bevacizumab combination therapy or chemotherapy alone . the addition of bevacizumab resulted in an orr of 30.9% , compared to 12.6% for chemotherapy alone ( p<0.001 ) , and a median pfs of 6.7 months vs 3.4 months ( hr 0.48 , 95% ci 0.380.60 , p<0.001 ) . there was a nonsignificant trend toward an improved os ( 16.6 months vs 13.3 months , hr 0.85 , 95% ci 0.661.08 , p<0.174 ) ; however , the true effect of bevacizumab on os is obscured and likely diluted by a 40% crossover to bevacizumab monotherapy among patients in the chemotherapy arm at the time of disease progression . subgroup analysis of the aurelia trial confirmed that bevacizumab consistently improves orr and pfs across chemotherapy cohorts ( table 1 ) . interestingly , the orr associated with bevacizumab combination therapy was the highest in the paclitaxel cohort ( 53.3% vs 30.2% ) compared to topotecan ( 17% vs 0% ) and the pld ( 13.7% vs 7.8% ) . median pfs followed the same trend for paclitaxel ( 10.4 months vs 3.9 months , hr 0.46 , 95% ci 0.30.71 ) , topotecan ( 5.8 months vs 2.1 months , hr 0.32 95% ci 0.210.49 ) , and pld ( 5.4 months vs 3.5 months , hr 0.57 , 95% ci 0.930.83 ) . it would be erroneous to conclude from these subgroup analyses of aurelia that there is a superiority of one of these chemotherapies given that the trial design did not randomize the chemotherapeutic agent . this trend is also present among phase ii studies of bevacizumab combination therapy ( table 1 ) and may reflect a higher activity of frequently dosed taxanes in epithelial ovarian cancer . such a metronomic schedule of some chemotherapies may also provide antiangiogenic effects not seen with conventional dosing schedules.43,44 additionally , metronomic dosing , such as with oral daily cyclophosphamide , may also impart immunostimulatory effects.45,46 a few studies have examined bevacizumab in combination with other targeted biologic therapies in recurrent ovarian cancer . epidermal growth factor receptor activation has been shown to promote vegf secretion ; therefore , nimeiri et al reported a small phase ii clinical trial of combination of bevacizumab and erlotinib , which demonstrated no increased activity compared to the previous studies of bevacizumab alone , and an increased toxicity with a 15% rate of gi perforation and high rates of nausea and diarrhea.47 in a phase i study by azad et al , partial response was observed in 6/13 patients with ovarian cancer with a combination of bevacizumab 5 mg / kg every 2 weeks and sorafenib , a receptor tyrosine kinase inhibitor ( tki ) known to target vegfr-2 and raf , which is known to be a downstream signaling target of vegf.48 rapid dose reductions for fatigue , anorexia , weight loss , hypertension , and proteinuria suggest that this combination may not be tolerable for long - term therapy , and 2/13 patients developed grade 2 fistulas . the infusion can be completed within 30 minutes,49 and infusion - related side effects are rare , with serious infusion reactions occurring in < 0.2% based on the postmarketing data.50 the toxicities most attributed to bevacizumab include gi perforation , hypertension , proteinuria , venous thromboembolism ( vte ) , impaired wound healing , and bleeding . rates of grade 3 toxicity among patients with recurrent ovarian cancer in phase ii trials are summarized in table 1 . despite the development of bevacizumab - related toxicites and recommendations for holding or discontinuing therapy in certain scenarios ( such as described later in this section ) , it is recognized that the decision to do so may be complicated by the response to treatment , the prognosis and disease setting of the patient , and , often , the lack of reasonably effective alternatives . hypertension is the most common side effect of bevacizumab , followed by proteinuria , but these rarely lead to discontinuation of therapy . the rate of grade 3 hypertension varies from 2% to 23% in ovarian cancer studies,51 with grade 2 hypertension occurring in 20% of patients in the aurelia trial.9 proteinuria occurs in up to 64% of patients receiving bevacizumab , with the highest rates not surprisingly among patients with renal carcinoma.52 management of hypertension and proteinuria can be guided by the national cancer institute grading system.51 proteinuria can be monitored until reaching grade 3 severity ( > 3.5 g/24 h or urine protein creatinine ratio > 3.5 ) , at which point bevacizumab may be held until protein levels return to grade 2 ( 1.03.5 g/24 h or urine protein creatinine ratio 13.5 ) . grade 2 hypertension ( systolic blood pressure [ bp ] 140159 mmhg and diastolic bp 9099 mmhg ) may be addressed with antihypertensive medication . grade 3 hypertension ( bp 160 mmhg systolic or 100 mmhg diastolic ) may require escalation of antihypertensive medication , including multidrug therapy , and many doctors choose to hold bevacizumab until bps return to grade 2 levels . grade 4 hypertension describes life - threatening consequences requiring emergent management and discontinuation of bevacizumab . the mechanism behind bevacizumab - related hypertension is incompletely understood but may be related to the lower endogenous levels of nitric oxide , interaction with angiotensins i and ii receptors , or functional loss of capillary vascular beds leading to an increased systemic vascular resistance.53,54 proteinuria secondary to anti - vegf therapy is thought to result from the disruption of vegf - dependent interactions between glomerular endothelial cells and podocytes , which in turn disrupts the filtration barrier.55 renal thrombotic microangiopathy has also been described on renal biopsy , but systemic manifestations , such as thrombocytopenia and schistocytosis , are rare.55,56 the sequelae of uncontrolled hypertension include malignant hypertension , stroke , and posterior reversible leukoencephalopathy syndrome ( pres ) . pres is a neurologic disorder characterized by the loss of cerebral vascular autoregulation and vasogenic edema . signs and symptoms include headache , seizures , lethargy , altered mental status , blindness , and other visual changes or alterations in neurologic function . there are at least 15 case reports of pres associated with bevacizumab , some with fatal outcome , although many cases resolve with supportive care.57 gi perforation is probably the most publicized and concerning complication of bevacizumab therapy . the reported rate of gi perforation in recurrent ovarian cancer varies from 0% to 11.4% ; however , in large phase iii clinical trials , the rate of perforation is 0%2.6% . the rate of bowel perforation reported in clinical trials has decreased over time , which is believed to be , at least in part , due to the efforts to screen out patients at higher risk . in one of the earliest trials , cannistra et al reported the highest rate of perforation ( 11.4% ) , resulting in early termination of their study and a fda investigational drug action letter reporting the gi perforation risk of bevacizumab among patients with ovarian cancer . in an unplanned retrospective subgroup analysis , the authors found that exposure to three or more prior chemotherapy regimens was the only significant characteristic associated with gi perforation , with 23.8% of patients in this group experiencing a perforation ( p<0.01).32 retrospective studies associate bowel obstruction , bowel wall thickening or suspected bowel involvement , inflammatory bowel disease , and prior bowel surgery with increased rates of perforation.51,58,59 if patients are screened for high - risk features , some gi perforations may be prevented . studies reporting 0% perforation rates include < 100 patients , and the absence of perforation is likely related to sample size . in the aurelia trial , patients with more than two prior lines of chemotherapy , bowel obstruction , or evidence of rectosigmoid tumor involvement were excluded , resulting in a gi perforation rate of 2%.9 patients without risk factors should be closely monitored . gi perforations carry a high rate of mortality , especially in the setting of recurrent , platinum - resistant ovarian cancer . many such patients have been heavily pretreated and carry a significant burden of peritoneal disease , with marginal nutritional and functional status , and may not tolerate or benefit from surgical exploration and repair . therefore , a bowel perforation is a sentinel event that often leads to the discontinuation of further anticancer therapy and a transition to comfort care . thromboembolic adverse events are reported in most trials of bevacizumab . however , the true elevation in the risk above the baseline risk associated with metastatic carcinoma is somewhat controversial . based on a pooled analysis of 1,745 patients with metastatic colorectal , breast and non - small - cell lung cancers , the addition of bevacizumab to standard chemotherapy is associated with an increased risk of arterial thromboembolic events ( hr 2.0 , 95% ci 1.053.75 , p=0.031 ) , but there was no increase in vte ( hr 0.89 , 95% ci 0.661.20 , p=0.44).60 the rates of arterial thromboembolic events , including transient ischemic attacks , ischemic stroke , and myocardial infarction , are low among patients with ovarian cancer , but all patients receiving bevacizumab should be considered at risk and monitored for symptoms . in the aurelia trial , chemotherapy plus bevacizumab vs chemotherapy alone led to grade 3 arterial or venous thromboembolic events in 2% vs 0% and 3% vs 4% of patients , respectively.9 many oncologists believe that patients who experience a vte ( deep vein thrombosis or pulmonary embolus ) may continue bevacizumab as long as therapeutic anticoagulation has been initiated . wound - healing complications related to bevacizumab are less commonly reported in ovarian cancer trials because initiation of bevacizumab is delayed at least 46 weeks after surgery ( as in gog 218 and icon7 when bevacizumab was not initiated until cycle 2 ) . in the brite registry , which monitors postmarket outcomes among patients with colorectal cancer who receive bevacizumab in the community , 23/622 ( 3.7% ) of patients who reported having a major surgery within 60 days of bevacizumab experienced wound complications.61 because of bleeding risk and impaired wound healing , the manufacturer recommends at least 4 weeks between bevacizumab and any major operative procedures.50 mucosal bleeding and epistaxis are commonly reported by patients receiving bevacizumab , but serious gi bleeding and perioperative hemorrhage have also been reported . in combination with cytotoxic chemotherapy , hematologic , gi , dermatologic , and mucosal verschraegen et al reported a concern for synergistic amplification of mucosal , cutaneous , and cardiac toxicity in a phase ii trial of bevacizumab with pld 30 mg / m given every 3 weeks.37 a total of 16% of patients experienced some degree of cardiac insufficiency . bowel perforation occurred in 4/100 heavily pretreated patients with ovarian cancer at 714 days after paclitaxel infusion,62 but this was not recapitulated in subsequent clinical trials , and rates of perforation with combination therapy fall within the range reported for bevacizumab alone and in combination with many different chemotherapies ( table 2 ) . because chemotherapy for platinum - resistant recurrent ovarian cancer is rarely curative , the quality of life is an important consideration in the choice of therapy . the quality of life outcomes from icon7 and gog 218 , two large phase iii trials evaluating bevacizumab in the primary adjuvant setting , suggest no difference during chemotherapy , with a slight decrement in the quality of life related to continuation of maintenance bevacizumab in icon7 among patients who remained progression free after adjuvant therapy.63,64 in contrast , patient - reported outcomes were improved with the addition of bevacizumab to standard chemotherapy for patients with recurrent , platinum - resistant disease in the aurelia trial.65 the disparity in these findings may relate to differences in disease burden and pretreatment quality of life among patients receiving primary therapy after debulking surgery when compared to patients with recurrent disease . gi symptoms , including nausea , bloating , constipation , and early satiety , related to carcinomatosis are among the most burdensome symptoms associated with recurrent metastatic ovarian cancer . significantly more patients in the bevacizumab arm of the aurelia trial reported at least a 15% improvement in their abdominal / gi symptoms ( 21.9% vs 9.3% , p=0.002).65 the treatment effect was particularly notable in the subset of 113 patients with ascites at baseline , with 44% of patients in the bevacizumab arm reporting at least 15% improvement in their gi symptoms ( vs 4.1% with standard chemotherapy alone , p<0.001).65 this observation is likely directly related to the reduction in ascites production . only 2% of patients who received bevacizumab required a paracentesis after entering the study when compared to 17% of patients receiving chemotherapy alone.9 data from the aurelia trial provide additional support for small case series reporting rapid , complete resolution of previously intractable malignant ascites.66,67 low - dose intraperitoneal bevacizumab has been suggested for the management of malignant ascites . a chinese randomized controlled trial compared the addition of intraperitoneal bevacizumab 300 mg/20 ml to every 2 weeks intraperitoneal cisplatin 40 mg / m , with every 3 weeks carboplatin ( auc 5 ) and paclitaxel ( 135 mg / m ) in the frontline setting.68 after 6 weeks , 58% ( 18/31 ) of the bevacizumab group experienced a complete resolution of ascites when compared to 40.7% of patients who received cisplatin alone . more significantly , 23/31 patients required no further peritoneal drainage during the 6-week study when compared to 7/27 patients who received cisplatin alone . peritoneal drainage times were used as proxy for ascites volume and were significantly reduced in the group who received bevacizumab , and intraperitoneal vegf and ca-125 levels were also significantly lower . while this treatment regimen is somewhat unconventional , intraperitoneal bevacizumab seems to be well tolerated , and there were no grade 3 or 4 toxicities reported . there have been a number of other antiangiogenic agents under investigation for the treatment of ovarian cancer . aflibercept , also known as vegf - trap , is a heterodimer composed of binding domains of vegfr-1 and vegfr-2 with an immunoglobulin g fc domain , which promotes trapping and immune clearance of vegf . this small molecule has a high affinity for multiple vegf family ligands , including vegf - a , -b , and placental growth factor , and was anticipated to have an enhanced activity over bevacizumab . in 2011 , coleman et al reported an orr of 54% in a phase i / ii trial of aflibercept with docetaxel for recurrent ovarian cancer.69 tew et al reported a phase ii randomized trial comparing 2 mg / kg vs 4 mg / kg aflibercept monotherapy in 215 women with recurrent , platinum - resistant ovarian cancer who had already progressed on pld or topotecan . unfortunately , the orr was very low in both groups ( 0.9% vs 4.6% ) and did not meet primary endpoints for response . reported grades 3 and 4 toxicities included hypertension , proteinuria , fatigue , and gi perforation , with rates similar to those reported for bevacizumab.70 in light of the role of vegf in promoting malignant ascites formation , gotlieb et al designed a third phase ii trial to investigate the potential for aflibercept for palliative ascites reduction . this double blind , placebo control trial of aflibercept 4 mg / kg every 2 weeks demonstrated a significant prolongation of intervals between palliative paracenteses ( 55.1 days vs 23.3 days , p=0.0019 ) but did not confirm a difference in os . while nonsignificant , the os in the treatment group was actually shorter ( 12.9 months vs 16 months ) , and there was an increased grades 34 toxicity , including a gi perforation rate of 10%.71 although the two have not been directly compared , there does not appear to be a compelling advantage of aflibercept over bevacizumab , despite the enhanced vegf inhibition , and the effect on ascites reduction is not unique . because the majority of proangiogenic cell signaling is transmitted through receptor tyrosine kinases , including the vegfr-1 , -2 , and -3 , pdgf receptor , and fgf receptor , receptor tkis represent another class of targeted antiangiogenic therapy undergoing intense investigation . clinical trials examining tkis and other antiangiogenic drugs are summarized in table 3.7284 based on the small studies , tkis appear to be associated with comparable or somewhat increased rates of grade 3 hypertension and rare gi perforations . however , most studies describe dose reductions and drug discontinuation related to other toxicities , including fatigue , diarrhea , mucositis , and dermatologic conditions . among the tkis currently under investigation , cediranib and pazopanib cediranib is a tki targeting vegfr-1 , -2 , and -3 as well as c - kit . cediranib was initially tested for the treatment of colorectal and renal cancers with disappointing results . phase ii trials in recurrent ovarian cancer , however , demonstrate orrs from 12% to 17% in a mixed platinum - sensitive and platinum - resistant population,80,81 leading to the development of icon6 , a phase iii trial testing the effect of cediranib combined with carboplatin and paclitaxel with and without maintenance for platinum - sensitive recurrent ovarian cancer , similar in design to the three - arm upfront gog 218 trial for bevacizumab . final results of icon6 are not yet published , but preliminary outcomes were quite exciting . as compared with standard carboplatin and paclitaxel therapy , the addition of cediranib followed by cediranib maintenance resulted in a significantly improved pfs ( 12.6 months vs 9.4 months , hr 0.57 , 95% ci 0.450.74 , p=0.00001 ) , and this is the first anti - angiogenic drug with an improvement in os in ovarian cancer ( 20.3 months vs 17.6 months , hr 0.70 , p=0.0419).82 the role of cediranib for platinum - resistant ovarian cancer has not yet been defined , but subgroup data from phase ii studies revealed no orr among platinum - resistant patients . pazopanib is a tki targeting all three vegfrs , pdgf receptor , fgf receptor , and c - kit , with additional targets , including colony - stimulating factor 1 , lymphocyte - specific kinase , and il-2 inducible t - cell kinase . friedlander et al reported an 18% orr among 17 patients with platinum - sensitive and -resistant recurrent ovarian cancer.83 more exciting , however , are the recently published results from pignata et al describing a randomized phase ii trial comparing pazopanib 800 mg daily combined with weekly paclitaxel to weekly paclitaxel alone . combined pazopanib and paclitaxel resulted in an orr of 56% with a significant improvement in both pfs ( 6.35 months vs 3.49 months , hr 0.42 , 95% ci 0.250.69 , p=0.0002 ) and a trend toward improved os ( 19.1 months vs 13.7 months , hr 0.60 , 95% ci 0.321.13 , p=0.56).84 the pacovar trial,85 a phase i / ii study investigating daily metronomic cyclophosphamide in combination with pazopanib , is highly anticipated , and multiple other studies are currently underway . as an alternative distinct from the vegf signaling pathway , trebananib ( amg 386 ) is an antiangiogenic agent targeting angiopoietins ang1 and ang2 , which signal through their receptor tie-2 , to mediate and stabilize endothelial and tumor vascular integrity . trebananib was recently evaluated in trinova-1 , a phase iii trial that randomized 919 women with recurrent ovarian cancer to trebananib 15 mg / kg vs placebo in combination with weekly paclitaxel 80 mg / m.86 the trial included patients with both platinum - sensitive and platinum - resistant diseases , which accounted for just > 50% of the placebo and treatment groups . trebananib resulted in a prolonged pfs ( median pfs 7.2 months vs 5.4 months , hr 0.66 , 95% ci 0.570.77 , p<0.0001 ) , but there was no difference in os . unlike bevacizumab , the toxicity profile of trebananib includes edema , ascites , and pleural effusions ; whereas adverse events typically associated with anti - vegf therapy , including hypertension , proteinuria , and gi perforation , are rare . trebananib is also being evaluated in combination with pld in trinova-2 ( nct01281254 ) and for the frontline setting in combination with carboplatin and paclitaxel in trinova-3 ( nct01493505 ) . in phase iii trials in all ovarian cancer treatment settings , the addition of bevacizumab to conventional chemotherapy results in ~24 months increase in median pfs , without a statistically significant improvement in os . in the recurrent , platinum - resistant setting , bevacizumab monotherapy is well tolerated and appears to have a comparable activity to other nonplatinum chemotherapeutic agents currently in use . when combined with other chemotherapies routinely used for platinum - resistant ovarian cancer , bevacizumab appears to double the expected response rate , with only a modest increase in toxicity . the most common side effects of hypertension and proteinuria can be medically managed with a minimal effect on quality of life . in appropriately screened patients , the role of bevacizumab in controlling ascites and the impact on symptom management and quality of life in the palliative setting are intriguing . data from the recent aure - lia trial lend support to previous case reports and small case series describing a palliative benefit from reduction in paracentesis - dependent ascites . while not traditionally included in clinical trial outcomes , diminished ascites production on bevacizumab may decrease the need for invasive procedures , and the rate of hospitalization for anorexia and failure to thrive related to symptomatic ascites . an examination of the frontline gog 218 trial suggested that ascites may act as a predictor of the population of women more likely to have a long - term benefit from bevacizumab.87 aflibercept has been studied for reduction in ascites , but the modest antitumor activity reported suggests no real advantage to bevacizumab . aflibercept may be a reasonable second - line antiascites therapy for symptomatic patients who do not respond to bevacizumab . a single optimal dose regimen for bevacizumab has not been established for the treatment of ovarian cancer . reported dose regimens for bevacizumab include 7.5 mg / kg every 3 weeks ( icon7 ) , 15 mg / kg every 3 weeks ( gog 218 , oceans , and aurelia ) , and 10 mg / kg every 2 weeks ( aurelia).9 bevacizumab has not been tested in humans in doses > 20 mg / kg.50 both doses evaluated in the aurelia trial have been approved by the fda for platinum - resistant ovarian cancer , and we are not aware of any data demonstrating the superiority of one over the other . as the half - life of bevacizumab is ~21 days,50 we favor 15 mg / kg every 3 weeks . this regimen also coordinates well with 21-day chemotherapy cycles , which are common in the treatment of ovarian cancer ; however , every 2 weeks dosing may be more practical for 4-week long cycles , such as with pld . as chemotherapy in the recurrent , platinum - resistant setting is palliative in nature , the duration of therapy should be governed by individual patient goals , response to therapy and/or ability to maintain stable disease , and the toxicities experienced . in the aurelia trial , therapy with both bevacizumab and chemotherapy was intended to be continued until progression or unacceptable toxicity . we suggest reevaluating response to therapy after three cycles , with ongoing discussions on the duration of therapy . if toxicities emerge that are either chemotherapy or bevacizumab specific , we recommend continuation of the tolerated agent until progression . bevacizumab may often be continued for many months , or in some cases , years . the optimal partner to bevacizumab has not been rigorously examined ; however , combination with weekly paclitaxel appears to result in the highest orr , median pfs , and a trend toward improved os . although not included in the aurelia trial , combination therapy with metronomic daily oral cyclophosphamide is a well - tolerated and convenient dosing alternative that may share similar antiangiogenic properties with weekly paclitaxel and may add immunotherapeutic activity as well . based on the current evidence , choice of cytotoxic chemotherapy for combination with bevacizumab in the recurrent , platinum - resistant setting should be based on the prior treatment history and patient ability to tolerate anticipated toxicities . for patients who may not tolerate combination therapy , bevacizumab monotherapy is a reasonable alternative , with response rates at least comparable to other monotherapy in the platinum - resistant setting and fewer toxicities affecting day - to - day quality of life . there remain incompletely answered questions regarding bevacizumab in the treatment of platinum - resistant ovarian cancer . one question is the benefit of treating with bevacizumab - based therapy after previous bevacizumab therapy . in colorectal cancer , bevacizumab plus standard second - line chemotherapy in patients with metastatic colorectal cancer progressing after standard first - line bevacizumab - based treatment improved os ( hr = 0.81 , p=0.0062).88 whether this is true in ovarian cancer remains to be seen . however , a retrospective review at a single institution in recurrent ovarian cancer looked at 46 patients who received bevacizumab with cytotoxic chemotherapy after having received prior bevacizumab suggested an improved pfs compared to those who did not receive bevacizumab with their cytotoxic therapy.89 the ongoing mito16mango2b trial ( nct01802749 ) aims to evaluate whether patients who have received bevacizumab during the first - line therapy will benefit from bevacizumab in the second - line chemotherapy in platinum - sensitive patients . another question is whether sequential therapy of bevacizumab and chemotherapy might offer a similarly efficacious strategy in terms of clinical benefit . also , that the use of ca-125 in defining progression for patients on bevacizumab may be more limited has been suggested by the findings that ~10%15% of patients in the single - agent bevacizumab trial for platinum - resistant ovarian cancer ( gog 170d ) would have stopped treatment based on ca-125 rather than radiologic evidence of disease progression criteria.90 finally , there remains active , but unestablished , investigation into biomarkers that may be used to define populations most likely to be considered or excluded from bevacizumab therapy . there are a number of ongoing phase iii clinical trials evaluating bevacizumab for ovarian cancer ; however , these focus on the primary adjuvant setting or platinum - sensitive disease . at present , bevacizumab continues to be the most thoroughly studied antiangiogenic therapy for the treatment of recurrent , platinum - resistant ovarian cancer ; however , intensive research in the past several years has yielded several promising alternatives . recent studies of pazopanib have been particularly exciting , and outcomes of ongoing trials are highly anticipated . combination of bevacizumab with other targeted agents has demonstrated an enhanced antitumor activity at the cost of increased toxicity ; however , previously studied targeted therapies have had limited activity on their own , until recently . parp inhibitors , such as olaparib , represent a significant breakthrough for targeted therapy in ovarian cancer and have demonstrated orrs of 41% in brca1/2 carriers and 24% in patients with sporadic tumors.91 a phase ii trial evaluating the combination of bevacizumab and olaparib for platinum - sensitive ovarian cancer is currently ongoing . , bevacizumab has added to the arsenal of available treatments for women with epithelial ovarian cancer . this includes the fda - approved use with chemotherapy in women with recurrent , platinum - resistant ovarian cancer who have had up to two prior chemotherapy regimens . in addition , it appears reasonable to consider the use as a single agent in selected patients of the same population who may not be good candidates for cytotoxic chemotherapy . data also appear to support a possible benefit in suboptimally cytoreduced patients with high - risk ovarian cancer in the frontline setting . the role in platinum - sensitive disease is less compelling at this time , given the cost and commitment in time for treatment for a relatively modest improvement in pfs without prolonging the survival . there will be more to learn about the appropriate settings and combinations for bevacizumab in the upcoming years .

patients with platinum - resistant ovarian cancer have progression of disease within 6 months of completing platinum - based chemotherapy . while several chemotherapeutic options exist for the treatment of platinum - resistant ovarian cancer , the overall response to any of these therapies is ~10% , with a median progression - free survival of 34 months and a median overall survival of 912 months . bevacizumab ( avastin ) , a humanized , monoclonal antivascular endothelial growth factor antibody , has demonstrated antitumor activity in the platinum - resistant setting and was recently approved by us food and drug administration for combination therapy with weekly paclitaxel , pegylated liposomal doxorubicin , or topotecan . this review summarizes key clinical trials investigating bevacizumab for recurrent , platinum - resistant ovarian cancer and provides an overview of efficacy , safety , and quality of life data relevant in this setting . while bevacizumab is currently the most studied and clinically available antiangiogenic therapy , we summarize recent studies highlighting novel alternatives , including vascular endothelial growth factor - trap , tyrosine kinase inhibitors , and angiopoietin inhibitor trebananib , and discuss their application for the treatment of platinum - resistant ovarian cancer .

Introduction Bevacizumab Efficacy of bevacizumab in recurrent, platinum-resistant ovarian cancer Safety and tolerability Patient focused perspectives and quality of life Other antiangiogenic agents Conclusions and role in therapy

responsiveness to chemotherapy and length of remission are predictors of overall prognosis.3 patients with recurrent ovarian cancer are risk stratified based on the time to recurrence after the completion of platinum - based chemotherapy : patients with a treatment - free interval of > 6 months after the completion of primary platinum - based therapy are considered as platinum sensitive and patients with a treatment - free interval of < 6 months are considered as platinum resistant . with successive lines of chemotherapy , several chemotherapeutic options exist for the treatment of platinum - resistant ovarian cancer , including pegylated liposomal doxorubicin ( pld),4,5 gemcitabine,5,6 topotecan,4 and etoposide.7,8 the overall response to any of these therapies is ~10%20% , with a median progression - free survival ( pfs ) of 34 months and a median overall survival ( os ) of 912 months.3 selection of therapy depends on prior treatment history , patient characteristics , and the side effect profile of each drug . bevacizumab ( avastin , genentech , san francisco , ca , usa ) , a monoclonal anti - vascular endothelial growth factor ( vegf)-a antibody targeting tumor angiogenesis , has been investigated and widely adopted for the treatment of recurrent ovarian cancer for the last several years . on november 14 , 2014 , following the publication of phase iii avastin use in platinum - resistant epithelial ovarian cancer ( aurelia ) trial,9 the us food and drug administration ( fda ) approved bevacizumab for use in recurrent , platinum - resistant ovarian cancer . bevacizumab , in combination with pld , weekly paclitaxel , or topotecan , is currently approved for patients with platinum - resistant disease who have received no more than two previous lines of chemotherapy . this review focuses on the efficacy , safety , acceptability , and therapeutic role of bevacizumab for the treatment of recurrent , platinum - resistant ovarian cancer . increased capillary permeability also contributes to the formation of malignant ascites , and vegf concentrations in malignant ascites , which is common in ovarian cancer , exceed the 95th percentile of concentrations observed in benign diseases.20 in ovarian cancers , vegf is detectable by immunohistochemistry in ovarian tumors , as well as in ascites and serum samples , and the expression levels are cancer specific , with no expression in benign ovarian tumors , intermediate expression in borderline tumors , and high levels of expression in invasive cancers.21,22 vegf expression also correlates with ovarian cancer stage at diagnosis and shorter pfs and os.2325 mouse monoclonal anti - vegf - a antibodies were shown to dramatically reduce tumor growth and malignant ascites formation in vivo , leading to the development of bevacizumab , a humanized monoclonal anti - vegf - a antibody , in 1997.26 bevacizumab demonstrated a promising activity against advanced colorectal cancers , which quickly led to multiple studies in breast , renal , cervical , and ovarian carcinomas . icon7 , a very similar trial , evaluated bevacizumab for the first - line adjuvant therapy after primary surgery in women with stages i iia high - grade or clear cell ovarian carcinoma or stages iib iv epithelial ovarian cancer , primary peritoneal cancer , or fallopian tube cancer . ovarian cancer study comparing efficacy and safety of chemotherapy and anti - angiogenic therapy in platinum - sensitive recurrent disease ( oceans ) randomized 484 women with recurrent , platinum - sensitive ovarian , fallopian tube , and primary peritoneal cancer to carboplatin ( auc 4 ) on day 1 with gemcitabine 1,000 mg / m on days 1 and 8 , with bevacizumab 15 mg / kg or placebo . bevacizumab was significantly associated with an increased pfs ( median pfs 12.4 months vs 8.4 months , hr 0.484 , 95% ci 0.3880.605 , p<0.0001),29 but there was no significant difference in os.30 most recently , the aurelia trial demonstrated a significant improvement in pfs for women with platinum - resistant ovarian cancer ( table 1).9 gog 170d was the first of two concurrent phase ii trials published in 2007 that demonstrated a promising bevacizumab activity in ovarian cancer . an exploratory analysis of prognostic factors predicting response found no significant association between response and platinum sensitivity.31 shortly after gog 170d , cannistra et al reported an orr of 15.9% among 44 women with recurrent , platinum - resistant ovarian cancer who received bevacizumab 15 mg / kg every 3 weeks.32 an additional 61.4% experienced stable disease . despite early termination due to an unexpectedly high rate of gi perforation ( 11.4% ) , cannistra et al reported a response rate comparable to other single - agent therapies in the recurrent , platinum - resistant setting . pfs ranges from 6 months to 8 months , and os ranges from 13.8 months to 33.2 months ( table 1).3342 the majority of these trials include both platinum - sensitive and platinum - resistant patients ; however , there are five trials specifically evaluating platinum - resistant patients with similar response rates and survival outcomes.36,37,39,41,42 in addition to the encouraging orrs observed with bevacizumab , phase ii studies also demonstrate high rates of disease stabilization , which is an important consideration for the platinum - resistant population , where therapeutic options are limited and where prolonged stable disease allows patients to maintain a reasonable quality of life while managing their cancer as a chronic illness . in all phase ii studies of bevacizumab combination therapy , orrs , pfs , and os compare favorably with the previous studies of cytotoxic chemotherapy for recurrent , platinum - resistant ovarian cancer . the aurelia trial is the first phase iii clinical trial to directly compare bevacizumab combined with chemotherapy to chemotherapy alone in the recurrent , platinum - resistant setting.9 a total of 361 patients with recurrent ovarian , fallopian tube , and primary peritoneal cancer who progressed within 6 months of receiving platinum - based chemotherapy were assigned a single - agent chemotherapy from the following options based on the investigator choice : 1 ) weekly paclitaxel 80 mg / m , 2 ) pld 40 mg / m every 4 weeks , or 3 ) topotecan 4 mg / m on days 1 , 8 , and 15 every 4 weeks or 1.25 mg / m on days 15 every 3 weeks ( table 1 ) . epidermal growth factor receptor activation has been shown to promote vegf secretion ; therefore , nimeiri et al reported a small phase ii clinical trial of combination of bevacizumab and erlotinib , which demonstrated no increased activity compared to the previous studies of bevacizumab alone , and an increased toxicity with a 15% rate of gi perforation and high rates of nausea and diarrhea.47 in a phase i study by azad et al , partial response was observed in 6/13 patients with ovarian cancer with a combination of bevacizumab 5 mg / kg every 2 weeks and sorafenib , a receptor tyrosine kinase inhibitor ( tki ) known to target vegfr-2 and raf , which is known to be a downstream signaling target of vegf.48 rapid dose reductions for fatigue , anorexia , weight loss , hypertension , and proteinuria suggest that this combination may not be tolerable for long - term therapy , and 2/13 patients developed grade 2 fistulas . an exploratory analysis of prognostic factors predicting response found no significant association between response and platinum sensitivity.31 shortly after gog 170d , cannistra et al reported an orr of 15.9% among 44 women with recurrent , platinum - resistant ovarian cancer who received bevacizumab 15 mg / kg every 3 weeks.32 an additional 61.4% experienced stable disease . despite early termination due to an unexpectedly high rate of gi perforation ( 11.4% ) , cannistra et al reported a response rate comparable to other single - agent therapies in the recurrent , platinum - resistant setting . pfs ranges from 6 months to 8 months , and os ranges from 13.8 months to 33.2 months ( table 1).3342 the majority of these trials include both platinum - sensitive and platinum - resistant patients ; however , there are five trials specifically evaluating platinum - resistant patients with similar response rates and survival outcomes.36,37,39,41,42 in addition to the encouraging orrs observed with bevacizumab , phase ii studies also demonstrate high rates of disease stabilization , which is an important consideration for the platinum - resistant population , where therapeutic options are limited and where prolonged stable disease allows patients to maintain a reasonable quality of life while managing their cancer as a chronic illness . in all phase ii studies of bevacizumab combination therapy , orrs , pfs , and os compare favorably with the previous studies of cytotoxic chemotherapy for recurrent , platinum - resistant ovarian cancer . the aurelia trial is the first phase iii clinical trial to directly compare bevacizumab combined with chemotherapy to chemotherapy alone in the recurrent , platinum - resistant setting.9 a total of 361 patients with recurrent ovarian , fallopian tube , and primary peritoneal cancer who progressed within 6 months of receiving platinum - based chemotherapy were assigned a single - agent chemotherapy from the following options based on the investigator choice : 1 ) weekly paclitaxel 80 mg / m , 2 ) pld 40 mg / m every 4 weeks , or 3 ) topotecan 4 mg / m on days 1 , 8 , and 15 every 4 weeks or 1.25 mg / m on days 15 every 3 weeks ( table 1 ) . epidermal growth factor receptor activation has been shown to promote vegf secretion ; therefore , nimeiri et al reported a small phase ii clinical trial of combination of bevacizumab and erlotinib , which demonstrated no increased activity compared to the previous studies of bevacizumab alone , and an increased toxicity with a 15% rate of gi perforation and high rates of nausea and diarrhea.47 in a phase i study by azad et al , partial response was observed in 6/13 patients with ovarian cancer with a combination of bevacizumab 5 mg / kg every 2 weeks and sorafenib , a receptor tyrosine kinase inhibitor ( tki ) known to target vegfr-2 and raf , which is known to be a downstream signaling target of vegf.48 rapid dose reductions for fatigue , anorexia , weight loss , hypertension , and proteinuria suggest that this combination may not be tolerable for long - term therapy , and 2/13 patients developed grade 2 fistulas . gi perforations carry a high rate of mortality , especially in the setting of recurrent , platinum - resistant ovarian cancer . based on a pooled analysis of 1,745 patients with metastatic colorectal , breast and non - small - cell lung cancers , the addition of bevacizumab to standard chemotherapy is associated with an increased risk of arterial thromboembolic events ( hr 2.0 , 95% ci 1.053.75 , p=0.031 ) , but there was no increase in vte ( hr 0.89 , 95% ci 0.661.20 , p=0.44).60 the rates of arterial thromboembolic events , including transient ischemic attacks , ischemic stroke , and myocardial infarction , are low among patients with ovarian cancer , but all patients receiving bevacizumab should be considered at risk and monitored for symptoms . bowel perforation occurred in 4/100 heavily pretreated patients with ovarian cancer at 714 days after paclitaxel infusion,62 but this was not recapitulated in subsequent clinical trials , and rates of perforation with combination therapy fall within the range reported for bevacizumab alone and in combination with many different chemotherapies ( table 2 ) . because chemotherapy for platinum - resistant recurrent ovarian cancer is rarely curative , the quality of life is an important consideration in the choice of therapy . the quality of life outcomes from icon7 and gog 218 , two large phase iii trials evaluating bevacizumab in the primary adjuvant setting , suggest no difference during chemotherapy , with a slight decrement in the quality of life related to continuation of maintenance bevacizumab in icon7 among patients who remained progression free after adjuvant therapy.63,64 in contrast , patient - reported outcomes were improved with the addition of bevacizumab to standard chemotherapy for patients with recurrent , platinum - resistant disease in the aurelia trial.65 the disparity in these findings may relate to differences in disease burden and pretreatment quality of life among patients receiving primary therapy after debulking surgery when compared to patients with recurrent disease . in 2011 , coleman et al reported an orr of 54% in a phase i / ii trial of aflibercept with docetaxel for recurrent ovarian cancer.69 tew et al reported a phase ii randomized trial comparing 2 mg / kg vs 4 mg / kg aflibercept monotherapy in 215 women with recurrent , platinum - resistant ovarian cancer who had already progressed on pld or topotecan . while nonsignificant , the os in the treatment group was actually shorter ( 12.9 months vs 16 months ) , and there was an increased grades 34 toxicity , including a gi perforation rate of 10%.71 although the two have not been directly compared , there does not appear to be a compelling advantage of aflibercept over bevacizumab , despite the enhanced vegf inhibition , and the effect on ascites reduction is not unique . phase ii trials in recurrent ovarian cancer , however , demonstrate orrs from 12% to 17% in a mixed platinum - sensitive and platinum - resistant population,80,81 leading to the development of icon6 , a phase iii trial testing the effect of cediranib combined with carboplatin and paclitaxel with and without maintenance for platinum - sensitive recurrent ovarian cancer , similar in design to the three - arm upfront gog 218 trial for bevacizumab . as compared with standard carboplatin and paclitaxel therapy , the addition of cediranib followed by cediranib maintenance resulted in a significantly improved pfs ( 12.6 months vs 9.4 months , hr 0.57 , 95% ci 0.450.74 , p=0.00001 ) , and this is the first anti - angiogenic drug with an improvement in os in ovarian cancer ( 20.3 months vs 17.6 months , hr 0.70 , p=0.0419).82 the role of cediranib for platinum - resistant ovarian cancer has not yet been defined , but subgroup data from phase ii studies revealed no orr among platinum - resistant patients . friedlander et al reported an 18% orr among 17 patients with platinum - sensitive and -resistant recurrent ovarian cancer.83 more exciting , however , are the recently published results from pignata et al describing a randomized phase ii trial comparing pazopanib 800 mg daily combined with weekly paclitaxel to weekly paclitaxel alone . trebananib was recently evaluated in trinova-1 , a phase iii trial that randomized 919 women with recurrent ovarian cancer to trebananib 15 mg / kg vs placebo in combination with weekly paclitaxel 80 mg / m.86 the trial included patients with both platinum - sensitive and platinum - resistant diseases , which accounted for just > 50% of the placebo and treatment groups . in the recurrent , platinum - resistant setting , bevacizumab monotherapy is well tolerated and appears to have a comparable activity to other nonplatinum chemotherapeutic agents currently in use . when combined with other chemotherapies routinely used for platinum - resistant ovarian cancer , bevacizumab appears to double the expected response rate , with only a modest increase in toxicity . in appropriately screened patients , the role of bevacizumab in controlling ascites and the impact on symptom management and quality of life in the palliative setting are intriguing . reported dose regimens for bevacizumab include 7.5 mg / kg every 3 weeks ( icon7 ) , 15 mg / kg every 3 weeks ( gog 218 , oceans , and aurelia ) , and 10 mg / kg every 2 weeks ( aurelia).9 bevacizumab has not been tested in humans in doses > 20 mg / kg.50 both doses evaluated in the aurelia trial have been approved by the fda for platinum - resistant ovarian cancer , and we are not aware of any data demonstrating the superiority of one over the other . as chemotherapy in the recurrent , platinum - resistant setting is palliative in nature , the duration of therapy should be governed by individual patient goals , response to therapy and/or ability to maintain stable disease , and the toxicities experienced . the optimal partner to bevacizumab has not been rigorously examined ; however , combination with weekly paclitaxel appears to result in the highest orr , median pfs , and a trend toward improved os . although not included in the aurelia trial , combination therapy with metronomic daily oral cyclophosphamide is a well - tolerated and convenient dosing alternative that may share similar antiangiogenic properties with weekly paclitaxel and may add immunotherapeutic activity as well . based on the current evidence , choice of cytotoxic chemotherapy for combination with bevacizumab in the recurrent , platinum - resistant setting should be based on the prior treatment history and patient ability to tolerate anticipated toxicities . for patients who may not tolerate combination therapy , bevacizumab monotherapy is a reasonable alternative , with response rates at least comparable to other monotherapy in the platinum - resistant setting and fewer toxicities affecting day - to - day quality of life . there remain incompletely answered questions regarding bevacizumab in the treatment of platinum - resistant ovarian cancer . also , that the use of ca-125 in defining progression for patients on bevacizumab may be more limited has been suggested by the findings that ~10%15% of patients in the single - agent bevacizumab trial for platinum - resistant ovarian cancer ( gog 170d ) would have stopped treatment based on ca-125 rather than radiologic evidence of disease progression criteria.90 finally , there remains active , but unestablished , investigation into biomarkers that may be used to define populations most likely to be considered or excluded from bevacizumab therapy . at present , bevacizumab continues to be the most thoroughly studied antiangiogenic therapy for the treatment of recurrent , platinum - resistant ovarian cancer ; however , intensive research in the past several years has yielded several promising alternatives . parp inhibitors , such as olaparib , represent a significant breakthrough for targeted therapy in ovarian cancer and have demonstrated orrs of 41% in brca1/2 carriers and 24% in patients with sporadic tumors.91 a phase ii trial evaluating the combination of bevacizumab and olaparib for platinum - sensitive ovarian cancer is currently ongoing . this includes the fda - approved use with chemotherapy in women with recurrent , platinum - resistant ovarian cancer who have had up to two prior chemotherapy regimens .

the micronucleus ( mn ) test as a short and easy assay for the evaluation of the effect of mutagenic action ( schmid 1975 ) is a suitable substitution for the time - consuming analysis chromosome aberrations ( countryman and heddle 1976 ) . recently , the mn test with fluorescent stains proved to be more sensitive in detecting small micronuclei compared to the mn test using traditional methods , such as feulgen s reaction ( dias et al . 2005 ) . therefore , the use of fluorescence has been proposed as an accurate method for the detection of micronuclei . interphase cytogenetics using fluorescence in situ hybridization ( fish ) with specific dna probes not only provides a sensitive tool to detect small chromosome rearrangements , but also offers the possibility of better understanding the origin of the micronuclei ( maluszynska et al . the micronucleus test applied with fish employing chromosome or chromosome region specific dna probes is widely applied in human cytogenetics , toxicological and radiation studies . in plant cytogenetics the repetitive dna sequences recognizing a specific chromosome region , such as centromeres and telomeres , as well as rdna are used most extensively as probes for fish for plant chromosomes ( bolzan and bianchi 2006 ) . there is only one morphological type of micronuclei which differs in size , however they can originate from different chromosomes or chromosome fragments . it was proved that the mn assay using fish with telomeric and/or centromeric dna sequences is able to detect the clastogenic or aneugenic effect ( acar et al . . studies concerning the evaluation of the origin of chemically induced micronuclei by maleic hydrazide ( mh ) and n - nitroso - n - methylurea ( mnu ) in barley cells were previously done by our group ( juchimiuk et al . the cytogenetic effects of a gamma ray in the root tips of many plant species were measured previously ( evans and hof 1975 ) . the specific localization of the radiation induced chromosome aberrations using traditional chromosome staining has been the subject of studies in barley and other plant species ( natarajan and ahnstrom 1970 ; kunzel et al . a gamma ray , causing breaks in one or two chains of dna , is routinely used in plant mutagenesis ( hagberg and persson 1968 ) and most barley mutant varieties were developed by applying this type of radiation . here , we quantitatively analyze the gamma ray - induced micronuclei in order to examine the involvement of specific chromosomes or chromosome fragments in their formation . fish with different dna probes ( 5s and 25s rdna , telomere- and centromere - specific dna sequences ) was applied in the analysis of the micronuclei . the next aim was a comparison of the possible origin of the micronuclei induced by physical and chemical treatment ( mh and mnu ) in hordeum vulgare cells . vulgare , a model plant , was chosen as the majority of large chromosomes can be distinguished because of the specific localization of rdna . barley ( hordeum vulgare l. , 2n = 14 ) seeds of the cv . the irradiation was carried out in the international atomic energy agency , seibersdorf laboratory , austria . after irradiation the seeds were presoaked in distilled water for 8 h and germinated in petri dishes at 21c in the dark . roots of m1 seedlings were used as the source of meristems for the investigations of aberrations . the material was fixed in ethanol : glacial acetic acid ( 3:1 ) at 60 h of germination . fluorescence in situ hybridization was applied according to the method described by maluszynska and heslop - harrison ( 1991 ) with some minor modifications . four dna probes were used in two fish experiments : clone ht100.3 containing 30 copies of arabidopsis - type telomeric repeats ( ( tttaggg)n ) labelled with rhodamine-5-dutp by pcr ( roche ) , clone ccs1 containing a part of the centromeric retrotranspozons isolated from brachypodium sylvaticum ( aragon - alcaide et al . 2000 ) labelled with digoxigenin-11-dutp using pcr ( roche ) , clone pta-794 containing 5s rdna from triticum aestivum ( gerlach and dyer 1980 ; hasterok et al . 2002 ) labelled with rhodamine-5-dutp using a pcr labeling kit ( amersham life sciences ) , and clone pclai containing 25s rdna isolated from arabidopsis thaliana ( unfriend and gruendler 1990 ) labelled with digoxigenin-11-dutp by nick translation ( roche ) . prior to fish , pretreatment with rnase , washing , dehydration of the chromosome preparations were applied as in a previous work of our group ( juchimiuk et al . the hybridization mixture containing 2.5 g / ml of labelled dna , 50% formamide , 10% dextran sulphate and 0.1 mg/l salmon testes dna in 2 ssc was denaturated at 75c for 10 min and immediately placed on ice for a few minutes . the hybridization mixture ( 38 l ) was added to the chromosome preparations and covered with a plastic coverslip . the chromosomes and dna probes were denatured for 5 min at 70c on a hot plate ( hybaid thermal cycler pcr in situ ) . hybridization was carried out at 37c in a moist chamber for 20 h. after hybridization , slides were subsequently washed for 4 min in 2 ssc at 42c , 2 4 min in 0.1 ssc at 42c , 3 3 min in 2 ssc at 42c , 3 3 min in 2 ssc at room temperature and for 5 min in 0.2% tween in 4 ssc at room temperature . the digoxigenin - labelled probe was detected using fitc - conjugated anti - digoxigenin antibodies ( roche ) and then the signal was amplified by a fitc - conjugated secondary antibody ( dako ) . after three washes for 8 min in 0.2% tween in 4 ssc at 37c and dehydration in the ethanol series , the slides were mounted in a vectashield medium ( vector laboratories ) containing 6 g/ ml dapi ( sigma ) . images were captured using a hamamatsu c5810 ccd camera and processed using adobe photoshop 4.0 . the frequency of micronuclei with specific dna signals and without signals was calculated . for each experimental group 100 cells with micronuclei analysed on three slides , each made from three meristems , were evaluated . the total frequencies of dapi - stained micronuclei in 3000 interphase cells were analyzed on the same slides before the fish experiments . barley ( hordeum vulgare l. , 2n = 14 ) seeds of the cv . the irradiation was carried out in the international atomic energy agency , seibersdorf laboratory , austria . after irradiation the seeds were presoaked in distilled water for 8 h and germinated in petri dishes at 21c in the dark . roots of m1 seedlings were used as the source of meristems for the investigations of aberrations . the material was fixed in ethanol : glacial acetic acid ( 3:1 ) at 60 h of germination . fluorescence in situ hybridization was applied according to the method described by maluszynska and heslop - harrison ( 1991 ) with some minor modifications . four dna probes were used in two fish experiments : clone ht100.3 containing 30 copies of arabidopsis - type telomeric repeats ( ( tttaggg)n ) labelled with rhodamine-5-dutp by pcr ( roche ) , clone ccs1 containing a part of the centromeric retrotranspozons isolated from brachypodium sylvaticum ( aragon - alcaide et al . 2000 ) labelled with digoxigenin-11-dutp using pcr ( roche ) , clone pta-794 containing 5s rdna from triticum aestivum ( gerlach and dyer 1980 ; hasterok et al . 2002 ) labelled with rhodamine-5-dutp using a pcr labeling kit ( amersham life sciences ) , and clone pclai containing 25s rdna isolated from arabidopsis thaliana ( unfriend and gruendler 1990 ) labelled with digoxigenin-11-dutp by nick translation ( roche ) . prior to fish , pretreatment with rnase , washing , dehydration of the chromosome preparations were applied as in a previous work of our group ( juchimiuk et al . the hybridization mixture containing 2.5 g / ml of labelled dna , 50% formamide , 10% dextran sulphate and 0.1 mg/l salmon testes dna in 2 ssc was denaturated at 75c for 10 min and immediately placed on ice for a few minutes . the hybridization mixture ( 38 l ) was added to the chromosome preparations and covered with a plastic coverslip . the chromosomes and dna probes were denatured for 5 min at 70c on a hot plate ( hybaid thermal cycler pcr in situ ) . hybridization was carried out at 37c in a moist chamber for 20 h. after hybridization , slides were subsequently washed for 4 min in 2 ssc at 42c , 2 4 min in 0.1 ssc at 42c , 3 3 min in 2 ssc at 42c , 3 3 min in 2 ssc at room temperature and for 5 min in 0.2% tween in 4 ssc at room temperature . the digoxigenin - labelled probe was detected using fitc - conjugated anti - digoxigenin antibodies ( roche ) and then the signal was amplified by a fitc - conjugated secondary antibody ( dako ) . after three washes for 8 min in 0.2% tween in 4 ssc at 37c and dehydration in the ethanol series , the slides were mounted in a vectashield medium ( vector laboratories ) containing 6 g/ ml dapi ( sigma ) . images were captured using a hamamatsu c5810 ccd camera and processed using adobe photoshop 4.0 . the frequency of micronuclei with specific dna signals and without signals was calculated . for each experimental group 100 cells with micronuclei analysed on three slides , each made from three meristems , were evaluated . the total frequencies of dapi - stained micronuclei in 3000 interphase cells were analyzed on the same slides before the fish experiments . dapi staining applied before fish experiment allowed analysis of the total frequency of gamma ray - induced micronuclei in barley root meristematic cells ( fig . 1 ) . the frequencies of micronuclei are shown for 60 h of postincubation as they were the highest during this time . the frequency of micronuclei after gamma irradiation varied from 4.5% to 18.2% , depending on dose and postincubation time ( data not presented ) . 225 gy of gamma ray was a stronger inducer of micronuclei than 175 gy . no published data on gamma ray - induced micronuclei are known for barley ( only chromosome aberrations ) . in the present study , the frequencies of gamma ray - induced micronuclei in barley cells showed that they were higher than the frequencies of chemically ( mh- and mnu- ) induced micronuclei in applied and estimated doses earlier ( juchimiuk et al . 2007 ) . 1frequencies of gamma ray - induced micronuclei in meristematic cells of barley roots after 60 h of postincubation frequencies of gamma ray - induced micronuclei in meristematic cells of barley roots after 60 h of postincubation fish was used for a detailed characterization of the micronuclei induced by gamma ray in barley cells . ht100.3 and ccs1 were used as probes in the first fish experiments , whereas in the second one 25s and 5s rdna were used . the analysis of the number of rdna signals in micronuclei makes the identification of individual chromosome / chromatid engaged in their formation possible ( juchimiuk et al . this was possible due to the large number of rdna sites in this species : only one chromosome pair is characterized by a lack of rdna signals , two chromosome pairs with 25s rdna signals ( nor chromosomes ) , four chromosomes pairs with 5s rdna ( fig . 2 ) . the application of fish with centromeric and telomeric probes allowed the question of whether interstitial or distal chromosome / chromatid fragments or whole chromosomes are involved in micronuclei formation to be answered . in this study micronuclei with different fish signals were observed . examples of interphase control and irradiated cells with signals of different dna sequences with the examples of probable chromosome origin of gamma ray - induced micronuclei are presented in figs . 3 and 4 . it is well known that a gamma ray can produce chromosome and chromatid aberrations ; however , it is difficult to distinguish between them on the basis of the size of the signals of probes applied in interphase nuclei due to their three - dimensional structure . after the first fish experiment with ht100.3 and ccs1 as probes , the micronuclei with telomeric signals or both telomeric and centromeric signals were observed . 3b does not contain signals of the probes applied , and the chromosome origin of this micronuclei is unknown ; however , the involvement of the interstitial chromosome region in its formation might be confirmed . 3c f , because they possess only a telomere - specific dna , are examples of the involvement of a different number of acentric and distal chromosome fragments in their formation . g contains two telomeric and two centromeric signals and thus indicates its origin from one incomplete chromosome and one centric fragment , or from three chromosome fragments : two centric and one acentric terminal fragment . 3h has four telomeric signals and one centric signal and thus there are few possible origins of its complex origin : from one complete , laggard chromosome , chromosome without telomeres , acentric terminal fragment , or from 3 fragments including the centric one . surprisingly , the presence of micronuclei with telomeric and centromeric sequences proved that a gamma ray can cause spindle fibre defects . telomeric sequences - red , centromeric sequences - green , dapi staining - blue . control cell , without micronucleus ( a ) ; the cell with : micronucleus without specific signals ( b ) , micronucleus with one telomere specific signal ( c ) , micronucleus with two telomere specific signals ( d ) , micronucleus with three telomere specific signals ( e ) , micronucleus with four telomere specific signals ( f ) , micronucleus with two telomere and two centromere specific signals ( g ) , micronucleus with four telomere and one centromere specific signals ( h ) . 5s rdna sequences - red , 25s rdna sequences - green , dapi staining - blue . control cell , without micronucleus ( a ) ; the cell with : micronucleus without specific signals ( b ) , micronucleus with one 5s rdna specific signal ( c ) , micronucleus with two 5s rdna specific signals ( d ) , micronucleus with four 5s rdna specific signals ( e ) , micronucleus with one 25s rdna specific signal ( f ) , micronucleus with two 25s rdna specific signals ( g ) , micronucleus with two 5s rdna and two 25s rdna specific signals ( h ) . bars represent 10 m hordeum vulgare cv . start idiogram with the distribution of the rrna genes and chromosome numbering . 5s rdna red , 25s rdna green hordeum vulgare interphase cells and probable origin of micronuclei after gamma irradiation of seeds . telomeric sequences - red , centromeric sequences - green , dapi staining - blue . control cell , without micronucleus ( a ) ; the cell with : micronucleus without specific signals ( b ) , micronucleus with one telomere specific signal ( c ) , micronucleus with two telomere specific signals ( d ) , micronucleus with three telomere specific signals ( e ) , micronucleus with four telomere specific signals ( f ) , micronucleus with two telomere and two centromere specific signals ( g ) , micronucleus with four telomere and one centromere specific signals ( h ) . bars represent 10 m hordeum vulgare interphase cells and probable origin of micronuclei after gamma irradiation of seeds . 5s rdna sequences - red , 25s rdna sequences - green , dapi staining - blue . control cell , without micronucleus ( a ) ; the cell with : micronucleus without specific signals ( b ) , micronucleus with one 5s rdna specific signal ( c ) , micronucleus with two 5s rdna specific signals ( d ) , micronucleus with four 5s rdna specific signals ( e ) , micronucleus with one 25s rdna specific signal ( f ) , micronucleus with two 25s rdna specific signals ( g ) , micronucleus with two 5s rdna and two 25s rdna specific signals ( h ) . bars represent 10 m the use of rdna makes the analysis of the involvement of specific chromosomes and/or specific chromosome fragments in the micronuclei formation possible . the origin of the micronucleus in fig . 4b is unknown due to the lack of rdna signals : it could originate from an interstitial fragment ( without rrna genes ) of any chromosome or from whole chromosome no . 4c contains one signal of 5s rdna and it probably originated from the chromatid fragment with 5s rrna genes of chromosome no . 1 , 2 , 3 or 4 . 4d that possesses two signals of 5s rdna is an example of an aberration formed after a double - chromatid break of one of the chromosome no . 1 , 2 , 3 or 4 or from single - chromatid breaks of two of these chromosomes . the presence of four signals of 5s rdna in the micronucleus indicates that two chromosomes from among chromosomes no . 1 , 2 , 3 or 4 might be involved in the formation of this aberration ( fig . 4f clearly proved its origin from a whole chromatid or a chromatid fragment of chromosome no . 6 or 7 . the total number of 25s rdna sites in this cell is five , indicating the probability of a duplication event of chromosome fragment including 25s rdna region . chromosome / chromosome fragments no . 6 or 7 and chromosome / chromosome fragments no . 1 , 2 , 3 , or 4 may have created the micronucleus in fig . the applied doses of gamma ray did not influence the frequencies of micronuclei with particular signals ( data not presented ) . figure 5 shows a lack of correlation between the postincubation times used in the study and the frequency of micronuclei with specific signals , thus all of the data obtained were pooled ( diagrams ) . fish with telomeric and centromeric dna as probes showed that the micronuclei with telomeric dna were observed most frequently ( 81% ) , and rarely were micronuclei without signals ( 14% ) . only 5% of the gamma - induced micronuclei are characterized by the presence of both : telomeric and centromeric dna sequences . calculating the centromere signal - positive and centromere signal - negative micronuclei is well known in plant cells ; however , it is used more often in human cells ( schuler et al . 1997 ; jovtchev et al . although , individual chromosome painting of plants is difficult , the rdna has become a useful chromosome marker for a few plant species , including arabidopsis and barley ( weiss and maluszynska 2000 ) . in this work applying rdna - fish to barley chromosomes enabled an analysis of the engaging of rrna - bearing chromosomes in micronuclei formation . the relatively high frequency ( 50% ) of micronuclei including 5s rdna signals after hybridization and micronuclei without signals ( 34% ) was found . 12% of micronuclei are characterized by the presence of 25s rdna sites , and only 4% with both rdna signals . therefore , even taking into consideration the higher number of 5s rdna - bearing chromosomes ( 5 pairs ) than 25s rdna bearing chromosomes ( 2 pairs ) in the barley diploid genome , it can be concluded that chromosomes no . 1 , 2 , 3 or 4 were involved in formation of gamma ray - induced chromosome aberrations more often than chromosomes no . 5 , 6 or 7 . theoretically , we can state that the mean percent of micronuclei per one pair of 5s rdna - bearing chromosomes is 12.5% , whereas per one pair of 25s rdna - bearing chromosomes only 6% . fish applied in a similar way in relation to the involvement of chromosomes with known markers was done in humans and has shown that different human chromosomes are not randomly involved in mn ( guttenbach and schmidt 1994 ) . 5the frequency of micronuclei with signals of specific dna sequences in barley cells after gamma irradiation in 36 h , 48 h , 60 h of postincubation . ( a ) fish with telomeric and centromeric dna as probes ( b ) fish with rdna as probes . the pie charts show data , which were pooled for all postincubation times the frequency of micronuclei with signals of specific dna sequences in barley cells after gamma irradiation in 36 h , 48 h , 60 h of postincubation . ( a ) fish with telomeric and centromeric dna as probes ( b ) fish with rdna as probes . the pie charts show data , which were pooled for all postincubation times a similar study concerning a comparison of the frequency of micronuclei with specific dna sequences was performed for mh- and nmu - treated barley cells ( juchimiuk et al . a comparison of the possible origin of the micronuclei induced by physical ( done in this work ) and chemical treatment ( mh and mnu ) in cells of hordeum vulgare was possible . the comparison of the effect of mutagenic treatment showed that a gamma ray in applied doses induced micronuclei with almost a 2 times higher frequency than mh and mnu . however , gamma ray , mh and mnu caused terminal deletions most often and with a similar frequency in barley cells . ( 2002 ) reported that most mnu - induced micronuclei showed telomere - specific signals . surprisingly , no micronuclei with only centromere - specific signal were observed in the case of treatment with gamma ray , which indicates that large interstitial fragments including only centromeric dna are not generated by physical treatment , but formed by chemical treatment . the comparison showed that micronuclei without centromeric- and telomeric - specific fish signals , probably arising form interstitial fragments , were observed with similar frequencies ( about 15% ) after gamma ray treatment and after chemical treatment . it should be underlined that not all initially radiation - induced chromatin breaks are observed as they can remain open and rejoin with another break or restitute . our studies confirm earlier observations that some chromosomes and chromosome regions are more prone to the formation of aberrations than others ( kumar and natarajan 1965 ; natarajan and ahnstrom 1970 ) . it was confirmed that small chromosome regions in the median and distal arm positions were identified as preferred sites for translocation breakpoints induced by gamma radiation in barley ( kunzel et al . many factors could be responsible for distributions of chromosome aberrations that are not random : interphase organization , the transcriptional activity of different chromosome regions and chromosome size ( natarajan 2002 ) . fish with rdna and ht100.3/ccs1 sequences as probes confirmed its usefulness in the characterization of micronuclei content , as well as in understanding and comparing the mechanisms of the actions of mutagens applied in genetic toxicology . based on the number of centromeric and telomeric signals in micronuclei the possible mechanism of gamma - ray induced micronuclei formation was stated : the micronuclei originated from the acentric fragments as a result of chromosome breakage or whole lagging chromosomes as a result of the dysfunction of the mitotic apparatus . an application of rdna as probes allowed it to be stated that 5s rdna - bearing chromosomes are involved in micronuclei formation more often than nor - bearing chromosomes . the recent work allowed the origin of physically- and chemically - induced micronuclei in barley cells to be compared . even though the mode of action of chemical and physical mutagens is different , the origin of micronuclei was most often from terminal fragments . molecular cytogenetics techniques gives us insight into the mechanisms of the formation of chromosome aberrations during genotoxicity studies . in future studies , the combination of multiple dna probes labelled with various fluorochromes can make fish a more powerful tool in plant mutagenesis .

a micronucleus test in combination with fluorescent in situ hybridization ( fish ) using telomere- , centromere - specific probes and 5s and 25s rdna was used for a detailed analysis of the effects of gamma ray irradiation on the root tip meristem cells of barley , hordeum vulgare ( 2n = 14 ) . fish with four dna probes was used to examine the involvement of specific chromosomes or chromosome fragments in gamma ray - induced micronuclei formation and then to explain their origin . additionally , a comparison of the possible origin of the micronuclei induced by physical and chemical treatment : maleic hydrazide ( mh ) and n - nitroso - n - methylurea ( mnu ) was done . the micronuclei induced by gamma ray could originate from acentric fragments after chromosome breakage or from whole lagging chromosomes as a result of a dysfunction of the mitotic apparatus . no micronuclei containing only centromeric signals were found . an application of rdna as probes allowed it to be stated that 5s rdna bearing chromosomes are involved in micronuclei formation more often than nor chromosomes . this work allowed the origin of physically- and chemically - induced micronuclei in barley cells to be compared : the origin of micronuclei was most often from terminal fragments . fish confirmed its usefulness in the characterization of micronuclei content , as well as in understanding and comparing the mechanisms of the actions of mutagens applied in plant genotoxicity .

Introduction Material and methods Material and treatment Fluorescence in situ hybridization (FISH) Results and discussion Conclusions

the micronucleus ( mn ) test as a short and easy assay for the evaluation of the effect of mutagenic action ( schmid 1975 ) is a suitable substitution for the time - consuming analysis chromosome aberrations ( countryman and heddle 1976 ) . recently , the mn test with fluorescent stains proved to be more sensitive in detecting small micronuclei compared to the mn test using traditional methods , such as feulgen s reaction ( dias et al . interphase cytogenetics using fluorescence in situ hybridization ( fish ) with specific dna probes not only provides a sensitive tool to detect small chromosome rearrangements , but also offers the possibility of better understanding the origin of the micronuclei ( maluszynska et al . the micronucleus test applied with fish employing chromosome or chromosome region specific dna probes is widely applied in human cytogenetics , toxicological and radiation studies . in plant cytogenetics the repetitive dna sequences recognizing a specific chromosome region , such as centromeres and telomeres , as well as rdna are used most extensively as probes for fish for plant chromosomes ( bolzan and bianchi 2006 ) . there is only one morphological type of micronuclei which differs in size , however they can originate from different chromosomes or chromosome fragments . studies concerning the evaluation of the origin of chemically induced micronuclei by maleic hydrazide ( mh ) and n - nitroso - n - methylurea ( mnu ) in barley cells were previously done by our group ( juchimiuk et al . the cytogenetic effects of a gamma ray in the root tips of many plant species were measured previously ( evans and hof 1975 ) . the specific localization of the radiation induced chromosome aberrations using traditional chromosome staining has been the subject of studies in barley and other plant species ( natarajan and ahnstrom 1970 ; kunzel et al . a gamma ray , causing breaks in one or two chains of dna , is routinely used in plant mutagenesis ( hagberg and persson 1968 ) and most barley mutant varieties were developed by applying this type of radiation . here , we quantitatively analyze the gamma ray - induced micronuclei in order to examine the involvement of specific chromosomes or chromosome fragments in their formation . fish with different dna probes ( 5s and 25s rdna , telomere- and centromere - specific dna sequences ) was applied in the analysis of the micronuclei . the next aim was a comparison of the possible origin of the micronuclei induced by physical and chemical treatment ( mh and mnu ) in hordeum vulgare cells . vulgare , a model plant , was chosen as the majority of large chromosomes can be distinguished because of the specific localization of rdna . barley ( hordeum vulgare l. , 2n = 14 ) seeds of the cv . the irradiation was carried out in the international atomic energy agency , seibersdorf laboratory , austria . after irradiation the seeds were presoaked in distilled water for 8 h and germinated in petri dishes at 21c in the dark . fluorescence in situ hybridization was applied according to the method described by maluszynska and heslop - harrison ( 1991 ) with some minor modifications . four dna probes were used in two fish experiments : clone ht100.3 containing 30 copies of arabidopsis - type telomeric repeats ( ( tttaggg)n ) labelled with rhodamine-5-dutp by pcr ( roche ) , clone ccs1 containing a part of the centromeric retrotranspozons isolated from brachypodium sylvaticum ( aragon - alcaide et al . 2002 ) labelled with rhodamine-5-dutp using a pcr labeling kit ( amersham life sciences ) , and clone pclai containing 25s rdna isolated from arabidopsis thaliana ( unfriend and gruendler 1990 ) labelled with digoxigenin-11-dutp by nick translation ( roche ) . prior to fish , pretreatment with rnase , washing , dehydration of the chromosome preparations were applied as in a previous work of our group ( juchimiuk et al . the hybridization mixture ( 38 l ) was added to the chromosome preparations and covered with a plastic coverslip . the chromosomes and dna probes were denatured for 5 min at 70c on a hot plate ( hybaid thermal cycler pcr in situ ) . the digoxigenin - labelled probe was detected using fitc - conjugated anti - digoxigenin antibodies ( roche ) and then the signal was amplified by a fitc - conjugated secondary antibody ( dako ) . the total frequencies of dapi - stained micronuclei in 3000 interphase cells were analyzed on the same slides before the fish experiments . barley ( hordeum vulgare l. , 2n = 14 ) seeds of the cv . fluorescence in situ hybridization was applied according to the method described by maluszynska and heslop - harrison ( 1991 ) with some minor modifications . four dna probes were used in two fish experiments : clone ht100.3 containing 30 copies of arabidopsis - type telomeric repeats ( ( tttaggg)n ) labelled with rhodamine-5-dutp by pcr ( roche ) , clone ccs1 containing a part of the centromeric retrotranspozons isolated from brachypodium sylvaticum ( aragon - alcaide et al . prior to fish , pretreatment with rnase , washing , dehydration of the chromosome preparations were applied as in a previous work of our group ( juchimiuk et al . the chromosomes and dna probes were denatured for 5 min at 70c on a hot plate ( hybaid thermal cycler pcr in situ ) . the digoxigenin - labelled probe was detected using fitc - conjugated anti - digoxigenin antibodies ( roche ) and then the signal was amplified by a fitc - conjugated secondary antibody ( dako ) . the frequency of micronuclei with specific dna signals and without signals was calculated . the total frequencies of dapi - stained micronuclei in 3000 interphase cells were analyzed on the same slides before the fish experiments . dapi staining applied before fish experiment allowed analysis of the total frequency of gamma ray - induced micronuclei in barley root meristematic cells ( fig . the frequencies of micronuclei are shown for 60 h of postincubation as they were the highest during this time . 225 gy of gamma ray was a stronger inducer of micronuclei than 175 gy . no published data on gamma ray - induced micronuclei are known for barley ( only chromosome aberrations ) . in the present study , the frequencies of gamma ray - induced micronuclei in barley cells showed that they were higher than the frequencies of chemically ( mh- and mnu- ) induced micronuclei in applied and estimated doses earlier ( juchimiuk et al . 1frequencies of gamma ray - induced micronuclei in meristematic cells of barley roots after 60 h of postincubation frequencies of gamma ray - induced micronuclei in meristematic cells of barley roots after 60 h of postincubation fish was used for a detailed characterization of the micronuclei induced by gamma ray in barley cells . ht100.3 and ccs1 were used as probes in the first fish experiments , whereas in the second one 25s and 5s rdna were used . the analysis of the number of rdna signals in micronuclei makes the identification of individual chromosome / chromatid engaged in their formation possible ( juchimiuk et al . this was possible due to the large number of rdna sites in this species : only one chromosome pair is characterized by a lack of rdna signals , two chromosome pairs with 25s rdna signals ( nor chromosomes ) , four chromosomes pairs with 5s rdna ( fig . the application of fish with centromeric and telomeric probes allowed the question of whether interstitial or distal chromosome / chromatid fragments or whole chromosomes are involved in micronuclei formation to be answered . examples of interphase control and irradiated cells with signals of different dna sequences with the examples of probable chromosome origin of gamma ray - induced micronuclei are presented in figs . it is well known that a gamma ray can produce chromosome and chromatid aberrations ; however , it is difficult to distinguish between them on the basis of the size of the signals of probes applied in interphase nuclei due to their three - dimensional structure . after the first fish experiment with ht100.3 and ccs1 as probes , the micronuclei with telomeric signals or both telomeric and centromeric signals were observed . 3b does not contain signals of the probes applied , and the chromosome origin of this micronuclei is unknown ; however , the involvement of the interstitial chromosome region in its formation might be confirmed . 3c f , because they possess only a telomere - specific dna , are examples of the involvement of a different number of acentric and distal chromosome fragments in their formation . g contains two telomeric and two centromeric signals and thus indicates its origin from one incomplete chromosome and one centric fragment , or from three chromosome fragments : two centric and one acentric terminal fragment . surprisingly , the presence of micronuclei with telomeric and centromeric sequences proved that a gamma ray can cause spindle fibre defects . control cell , without micronucleus ( a ) ; the cell with : micronucleus without specific signals ( b ) , micronucleus with one telomere specific signal ( c ) , micronucleus with two telomere specific signals ( d ) , micronucleus with three telomere specific signals ( e ) , micronucleus with four telomere specific signals ( f ) , micronucleus with two telomere and two centromere specific signals ( g ) , micronucleus with four telomere and one centromere specific signals ( h ) . 5s rdna sequences - red , 25s rdna sequences - green , dapi staining - blue . control cell , without micronucleus ( a ) ; the cell with : micronucleus without specific signals ( b ) , micronucleus with one 5s rdna specific signal ( c ) , micronucleus with two 5s rdna specific signals ( d ) , micronucleus with four 5s rdna specific signals ( e ) , micronucleus with one 25s rdna specific signal ( f ) , micronucleus with two 25s rdna specific signals ( g ) , micronucleus with two 5s rdna and two 25s rdna specific signals ( h ) . bars represent 10 m hordeum vulgare cv . start idiogram with the distribution of the rrna genes and chromosome numbering . 5s rdna red , 25s rdna green hordeum vulgare interphase cells and probable origin of micronuclei after gamma irradiation of seeds . bars represent 10 m hordeum vulgare interphase cells and probable origin of micronuclei after gamma irradiation of seeds . 5s rdna sequences - red , 25s rdna sequences - green , dapi staining - blue . control cell , without micronucleus ( a ) ; the cell with : micronucleus without specific signals ( b ) , micronucleus with one 5s rdna specific signal ( c ) , micronucleus with two 5s rdna specific signals ( d ) , micronucleus with four 5s rdna specific signals ( e ) , micronucleus with one 25s rdna specific signal ( f ) , micronucleus with two 25s rdna specific signals ( g ) , micronucleus with two 5s rdna and two 25s rdna specific signals ( h ) . bars represent 10 m the use of rdna makes the analysis of the involvement of specific chromosomes and/or specific chromosome fragments in the micronuclei formation possible . the origin of the micronucleus in fig . 4b is unknown due to the lack of rdna signals : it could originate from an interstitial fragment ( without rrna genes ) of any chromosome or from whole chromosome no . 4d that possesses two signals of 5s rdna is an example of an aberration formed after a double - chromatid break of one of the chromosome no . the presence of four signals of 5s rdna in the micronucleus indicates that two chromosomes from among chromosomes no . 1 , 2 , 3 or 4 might be involved in the formation of this aberration ( fig . the total number of 25s rdna sites in this cell is five , indicating the probability of a duplication event of chromosome fragment including 25s rdna region . the applied doses of gamma ray did not influence the frequencies of micronuclei with particular signals ( data not presented ) . figure 5 shows a lack of correlation between the postincubation times used in the study and the frequency of micronuclei with specific signals , thus all of the data obtained were pooled ( diagrams ) . fish with telomeric and centromeric dna as probes showed that the micronuclei with telomeric dna were observed most frequently ( 81% ) , and rarely were micronuclei without signals ( 14% ) . only 5% of the gamma - induced micronuclei are characterized by the presence of both : telomeric and centromeric dna sequences . calculating the centromere signal - positive and centromere signal - negative micronuclei is well known in plant cells ; however , it is used more often in human cells ( schuler et al . although , individual chromosome painting of plants is difficult , the rdna has become a useful chromosome marker for a few plant species , including arabidopsis and barley ( weiss and maluszynska 2000 ) . in this work applying rdna - fish to barley chromosomes enabled an analysis of the engaging of rrna - bearing chromosomes in micronuclei formation . the relatively high frequency ( 50% ) of micronuclei including 5s rdna signals after hybridization and micronuclei without signals ( 34% ) was found . 12% of micronuclei are characterized by the presence of 25s rdna sites , and only 4% with both rdna signals . therefore , even taking into consideration the higher number of 5s rdna - bearing chromosomes ( 5 pairs ) than 25s rdna bearing chromosomes ( 2 pairs ) in the barley diploid genome , it can be concluded that chromosomes no . 1 , 2 , 3 or 4 were involved in formation of gamma ray - induced chromosome aberrations more often than chromosomes no . theoretically , we can state that the mean percent of micronuclei per one pair of 5s rdna - bearing chromosomes is 12.5% , whereas per one pair of 25s rdna - bearing chromosomes only 6% . fish applied in a similar way in relation to the involvement of chromosomes with known markers was done in humans and has shown that different human chromosomes are not randomly involved in mn ( guttenbach and schmidt 1994 ) . 5the frequency of micronuclei with signals of specific dna sequences in barley cells after gamma irradiation in 36 h , 48 h , 60 h of postincubation . ( a ) fish with telomeric and centromeric dna as probes ( b ) fish with rdna as probes . the pie charts show data , which were pooled for all postincubation times the frequency of micronuclei with signals of specific dna sequences in barley cells after gamma irradiation in 36 h , 48 h , 60 h of postincubation . ( a ) fish with telomeric and centromeric dna as probes ( b ) fish with rdna as probes . the pie charts show data , which were pooled for all postincubation times a similar study concerning a comparison of the frequency of micronuclei with specific dna sequences was performed for mh- and nmu - treated barley cells ( juchimiuk et al . a comparison of the possible origin of the micronuclei induced by physical ( done in this work ) and chemical treatment ( mh and mnu ) in cells of hordeum vulgare was possible . the comparison of the effect of mutagenic treatment showed that a gamma ray in applied doses induced micronuclei with almost a 2 times higher frequency than mh and mnu . however , gamma ray , mh and mnu caused terminal deletions most often and with a similar frequency in barley cells . ( 2002 ) reported that most mnu - induced micronuclei showed telomere - specific signals . surprisingly , no micronuclei with only centromere - specific signal were observed in the case of treatment with gamma ray , which indicates that large interstitial fragments including only centromeric dna are not generated by physical treatment , but formed by chemical treatment . the comparison showed that micronuclei without centromeric- and telomeric - specific fish signals , probably arising form interstitial fragments , were observed with similar frequencies ( about 15% ) after gamma ray treatment and after chemical treatment . it should be underlined that not all initially radiation - induced chromatin breaks are observed as they can remain open and rejoin with another break or restitute . it was confirmed that small chromosome regions in the median and distal arm positions were identified as preferred sites for translocation breakpoints induced by gamma radiation in barley ( kunzel et al . fish with rdna and ht100.3/ccs1 sequences as probes confirmed its usefulness in the characterization of micronuclei content , as well as in understanding and comparing the mechanisms of the actions of mutagens applied in genetic toxicology . based on the number of centromeric and telomeric signals in micronuclei the possible mechanism of gamma - ray induced micronuclei formation was stated : the micronuclei originated from the acentric fragments as a result of chromosome breakage or whole lagging chromosomes as a result of the dysfunction of the mitotic apparatus . an application of rdna as probes allowed it to be stated that 5s rdna - bearing chromosomes are involved in micronuclei formation more often than nor - bearing chromosomes . the recent work allowed the origin of physically- and chemically - induced micronuclei in barley cells to be compared . even though the mode of action of chemical and physical mutagens is different , the origin of micronuclei was most often from terminal fragments . molecular cytogenetics techniques gives us insight into the mechanisms of the formation of chromosome aberrations during genotoxicity studies . in future studies , the combination of multiple dna probes labelled with various fluorochromes can make fish a more powerful tool in plant mutagenesis .

biomineralized tissues , such as bone and dentin , manifest a remarkable stability and stiffness , which is attributed to their heterogeneous organic / inorganic composite nature . the organic matrix mainly involves fibrous collagen and to a lesser extent ( glyco)proteins and citrate , whereas the inorganic part constitutes ca - deficient hydroxy - carbonate apatite ( hca ; 39 wt % carbonate ) , substituted with additional ions , for example , na , mg , and f. repairing bone / tooth defects or fractures demands an implant with the minimum requirement of biocompatibility , that is , that it is nontoxic and will not be expelled by the human body . whereas the first generation of biomaterials was essentially inert resorbable and bioactive ceramics and glasses have found applications mainly in orthopedic , spine , and periodontal surgery . on contact with body fluids , they possess tissue - bonding abilities because of the formation of a biomimetic layer of amorphous calcium phosphate ( acp ) that subsequently converts into nanocrystalline hca . however , despite massive efforts for their elucidation , many details of these biomimetic processes remain largely unknown . whereas the composition , structure , and evolution of the biomimetic phosphate layer generated in vitro at the surface of bioactive glasses ( bgs ) have been addressed by a diversity of characterization techniques , primarily powder x - ray diffraction , electron microscopy ( em ) coupled to energy - dispersive x - ray ( edx ) spectroscopy , and infrared and raman spectroscopy , very few studies employed solid - state nuclear magnetic resonance ( nmr ) . yet , several structural investigations using h and p nmr spectroscopy are reported on naturally biomineralized tissues , such as bone and tooth . however , whereas their inorganic phosphate content is substantial ( 60 wt % ) , the biomimetically grown surface layer of bioglasses constitutes a minor fraction out of the entire bg - dominated specimen , thereby compromising nmr signal sensitivity and complicating the investigations . more progress is required in this area to improve the structural understanding of the bioactive layer that interfaces the implant and the living organism . by utilizing si and p magic - angle spinning ( mas ) nmr , we recently reported a study(28 ) of the reactions leading to hca formation at the surface of an ordered mesoporous bioactive glass ( mbg ) of composition 10cao85sio25p2o5 in a simulated body fluid ( sbf).(43 ) here , we examine the characteristics of the biomimetically grown acp and hca constituents of the surface layer generated either in sbf or buffered water media from a very similar mbg specimen , by using transmission electron microscopy ( tem ) , and particularly p and h mas nmr . we contrast the hp cross - polarization ( cp ) dynamics observed from the amorphous and structurally ordered components of the phosphate layer with that of a synthetic crystalline hydroxyapatite sample ( labeled haref ) as well as with the amorphous calcium phosphate clusters ( henceforth referred to as cap ) initially present in the mbg pore wall . such experimentation provides qualitative information about the ( shortest ) h p internuclear distances in a structure , here revealing very similar h p spatial contacts in each pair of amorphous cap / acp and crystalline hca / haref phases : this underscores the close structural similarity between the orthophosphate component of the mbg structure and the biomimetic acp formed in vitro , on one hand , and the biomimetic apatite compared with the crystalline reference , on the other . an mbg sample of nominal molar composition 10cao85sio25p2o5 , onward labeled s85 according to its mol % of sio2 , was prepared at 40 c using an evaporation - induced self - assembly ( eisa ) process(46 ) with the p123 triblock copolymer as structure - directing agent , as described in ref ( 23 ) . tetraethyl orthosilicate ( teos ) , triethyl phosphate ( tep ) , and ca(no3)24h2o served as precursors for introducing each of the elements si , p , and ca , respectively . the reaction produced homogeneous membranes that were calcined at 700 c for 6 h to remove organic species and nitrate ions . x - ray fluorescence ( xrf ) spectroscopy employing a philips panalytical axios spectrometer ( philips electronics nv ) and x - rays generated by the rh k line at = 0.614 yielded a charge - balanced analyzed composition of ca0.098si0.838p0.064o1.93 . an sbf solution was prepared according to kokubo et al.(43 ) by dissolving nacl , kcl , nahco3 , k2hpo43h2o , mgcl26h2o , cacl2 , and na2so4 in distilled water . the solution was buffered at ph 7.45 by using tris(hydroxymethyl)-aminomethane / hcl ( tris ) and passed through a 0.22 m millipore filter to avoid bacterial contamination . additionally , the in vitro studies involved a medium of distilled water , buffered by tris / hcl to provide the same ph value as the sbf . the in vitro apatite formation was investigated by immersing 1.00 g of s85 grains in 50 ml of either sbf or tris solutions for 16 h : the resulting specimens are henceforth referred to as sbf16h and tris16h respectively . the sbf / tris - soaking was performed at 37 c , employing a sealed polyethylene container under continuous orbital stirring ( 100 rpm ) in an ecotron ht incubator . each sample was filtered and washed with water to quench the surface reactions and removing potentially precipitated salts . finely ground specimens were sprinkled on a carbon film with holes , supported by a copper grid . they were examined in a field - emission gun transmission electron microscope ( jem 2100f ; jeol , japan ) at 200 kv acceleration voltage . cation compositions were determined on selected fragments by edx , employing a retractable detector with an ultrathin window , and using the jeol jed-2300 software for data analysis . all nmr experimentation employed finely ground samples filled in 6 mm zirconia pencil rotors undergoing mas at 9.0 khz . an agilent / varian / chemagnetics infinity 400 spectrometer was utilized at a magnetic field of 9.4 t , giving larmor frequencies of 162.0 mhz for p and 400.1 mhz for h in the frequency - sign convention recommended by levitt.(47 ) single - pulse ( bloch - decay ) p nmr experiments employed 70 flip - angles at a nutation frequency of nutp/2 = 48 khz . typically , 340 signal transients ( 36 transients for haref ) were coadded , using relaxation delays of 600 s. h nmr spectra were recorded by hahn spin echoes(48 ) by employing nuth/2 = 48 khz ( used for hard h pulses throughout all experimentation ) , an echo delay of echo = 111 s , 5.0 s of relaxation delay , and 1024 transients / acquisition . hahn condition nuth nutp = 2r , giving nutation frequencies of 20 and 38 khz for p and h , respectively , at the spinning frequency r/2 = 9.0 khz . between 4096 and 14 336 transients were accumulated for variable contact intervals ( cp ) , using relaxation delays of 4.0 s. h p heteronuclear correlation ( hetcor ) 2d nmr spectra were acquired from the s85 , sbf16h , and tris16h specimens using hp cp for magnetization transfer ( cp = 1.5 ms ) and 800 accumulated signal transients / t1-value ( except for s85 : 1616 transients / t1 ) . the spectral window was 22 khz both for the direct ( p ; horizontal ) and indirect ( h ; vertical ) spectral dimensions , respectively , except for sbf16h ( 16 khz for the h dimension ) . time - proportional phase incrementation(48 ) arranged absorptive 2d nmr spectra with frequency sign discrimination along the indirect dimension , thereby reducing its effective range by 1/2 . we acquired 110(t1 ) 250(t2 ) data points , which were zero - filled to a ( 256 1024 ) grid before 2d fourier transformation . we verified that the application of high - power h decoupling did not affect the p nmr peak widths perceptibly , and all experimentation was performed without h decoupling , including the nmr relaxation measurements discussed in section . the processing of the 1d nmr spectra did not involve signal apodization , whereas for the 2d hetcor data set , 80 hz gaussian and 20 hz ( 40 hz for s85 ) lorentzian broadening was applied along the p and h dimensions , respectively . chemical shifts are quoted relative to 85% h3po4 ( p ) and neat tetramethylsilane ( h ) . high - resolution tem was employed to investigate the nanostructure and composition of the s85 mbg before and after its soaking in sbf and tris solutions . electron diffraction ( ed ) verified an amorphous material , whereas elemental compositions probed by edx measurements at selected spots accorded very well with that of the xrf analysis . tem images recorded from fragments of the ( a ) pristine s85 , ( b ) sbf16h , and ( c , d ) tris16h specimens , with accompanying ed patterns displayed as insets in image a and d. the dark areas in panels b and c correspond to the si - rich mbg , whereas lighter domains represent the phosphate layer . ( d ) zoom of the image in c , revealing an elongated ha crystal . cation compositions measured by edx over the displayed areas are reported in at % at the bottom left corner of the images a and d. representative tem images recorded from fragments of the sbf16h and tris16h specimens are displayed in figure 1b and 1c , respectively : they evidence a large number of 1050 nm sized elongated particles grown at the mbg surface . their crystalline character is evident from both the tem image shown in figure 1d that reveals needle - shaped nanocrystals and the accompanying ed pattern ( inset ) . cation compositions probed by tem / edx over a collection of fragments from the sbf16h and tris16h specimens revealed nca / np molar ratios scattered around the ha characteristic value of 1.7 . results from both powder x - ray diffraction and scanning electron microscopy ( sem ) coupled to edx further confirmed the presence of ha in the mbg surface layer.(29 ) onward , we will employ the label h(c)a when referring collectively to the biomimetic apatite phase forming in either sbf or tris solutions : note that acp crystallizes into ha in tris / hcl - buffered water , whereas the presence of carbonate ions in the sbf solution(43 ) leads to the formation of hca , analogously to bone and tooth formed in vivo . the simultaneous incorporation of na and co32 ions into the sbf - induced calcium phosphate layer at the s85 surface was recently evidenced by sem / edx,(29)na mas , and c cpmas nmr.(28 ) figure 2 shows p mas nmr spectra acquired directly by single pulses that quantitatively reflect the various p populations of each sample , as opposed to those recorded by hp cp ( discussed below ) that draw their magnetization from neighboring h species , thereby emphasizing p sites in close proximity to protons . figure 2a displays the results from the pristine s85 mbg sample and the crystalline hydroxyapatite reference specimen , hence , accompanying their common orthophosphate structural building blocks , both haref and the cap component of s85 produce a p nmr peak maximum around 3 ppm . however , the very different ordering of the two structures is reflected by their distinct peak shapes and full width at half - maximum ( fwhm ) height of the corresponding p nmr signals : the nmr response from the well - ordered haref structure features a narrow lorentzian peak ( 0.67 ppm ) , whereas that from the mbg is gaussian - shaped and an order of magnitude broader ( 6.3 ppm ) . as previously highlighted and explored further in this contribution , the cap clusters of s85 share many nmr and hence local structural features with the acp layer initially forming at the mbg surface on its contact with ( simulated ) body fluids . p mas nmr spectra acquired by single pulses from the ( a ) pristine s85 ( black trace ) and crystalline haref ( gray line ) samples . the gray rectangle in panel a marks a minor resonance assigned to p o si moieties . ( b ) after mbg exposure to sbf ( black trace ) or tris ( gray trace ) solutions for 16 h. figure 2b displays the p nmr spectra resulting after soaking the s85 mbg for 16 h in either tris / hcl or sbf solutions . both reveal a significant signal - narrowing and the presence of two p sources in the specimens , reflecting one ordered phase ( i.e. , biomimetic h(c)a ) and two amorphous components , that is , cap and acp . the latter can not readily be distinguished by their 1d nmr - associated parameters ; however , as the surface - associated cap clusters tend to rapidly dissolve during the initial stages of the mbg soaking , their contributions to the p nmr spectra of the sbf16h and tris16h samples are expected to be minor . these peak assignments were motivated further by gunawidjaja et al.(28 ) spectral deconvolution results ( discussed in detail elsewhere(29 ) ) yielded very close values of the chemical shift ( 3.5 ppm ) and peak widths ( fwhm 5.4 ppm ) for biomimetic acp in both sbf16h and tris16h specimens , whereas the corresponding parameters for h(c)a were as follows : = 3.18 ppm , fwhm = 1.94 ppm for sbf16h ; = 3.08 ppm , fwhm = 1.80 ppm for tris16h . the relative amounts of apatite out of all p - bearing phases were estimated to be 43 % and 50% for the sbf16h and tris16h specimens , respectively . the identifications of broad and narrow p nmr signal portions in figure 2b to indeed stem from amorphous cap / acp and h(c)a phases are proven by the h p hetcor 2d nmr spectra of figure 3 , which were recorded from the ( a ) s85 , ( b ) sbf16h , and ( c ) tris16h specimens . this experiment incorporates hp cp for magnetization transfers ( as discussed in section ) ; an nmr peak appearing at the 2d shift - coordinate ( h , p ) evidences a close spatial proximity between the h and p sites associated with each respective chemical shift.(48 ) the left panel of figure 3 shows the 2d nmr spectra as well as their projections along the p ( horizontal ) and h ( vertical ) dimensions . the mid panel comprises directly excited h mas nmr spectra . because each of the latter quantitatively reflects all h environments of the sample , a comparison with its respective hetcor projection reveals which proton sites are associated with which respective phosphate- and silicate - bearing mbg components , respectively . this is most transparently appreciated from figure 4 that displays the h mas spectra and hetcor projections in a larger format . p 2d hetcor nmr spectra recorded from the ( a ) s85 , ( b ) sbf16h , and ( c ) tris16h specimens . they are shown together with sum projections along each spectral dimension , as well as directly excited h mas nmr spectra ( mid - panel ) . the latter reveal a primary resonance around 4 ppm from water molecules , a narrow peak at 2.1 ppm from isolated silanols ( labeled sioh ) , and signals from minor remainings of organic templating moieties ( marked by asterisks ) . in c right panel : slices along the p dimension , taken through the as - indicated h chemical shifts around 0 ( top trace ) and 4 ppm ( bottom trace ) of each 2d spectrum : they either comprise a narrow ( top ) or broad ( bottom ) p nmr peak , stemming from biomimetic h(c)a and amorphous acp / cap components , respectively . directly excited h mas nmr spectra ( left panel ) and projections along the h dimension of 2d hetcor spectra ( right panel ) . for comparison , the directly excited h spectrum from haref is also displayed at the bottom of the right panel . the present hetcor 2d nmr spectra appear similar to our previously presented results from analogous pristine(44 ) and sbf - treated(28 ) s85 samples . each amorphous ( acp / cap ) and crystalline ( h(c)a ) phosphate phase is primarily associated with proton environments stemming from water molecules and hydroxyl groups , respectively , which resonates at h 4 ppm ( broad peak ) and 0 ppm ( narrow signal ) , respectively . the latter shift is a fingerprint of hydroxyapatite , cf . the h mas nmr spectrum from haref shown in figure 4 . because of the very low amount of such phosphate - associated oh moieties relative to the entire proton ensemble in the s85-deriving specimens , the accompanying signal h 0 is visible solely in the hetcor projections , whereas it remains undetectable in directly excited h nmr spectra ( figure 4 ) . the dual presence of amorphous and crystalline phosphates in the sbf / tris - exposed samples is unambiguously evidenced from the 2d nmr spectra of figure 3b , c that primarily reveal two nmr correlations , centered at the pairs of shifts { h , p } around { 4 , 3 } ppm and { 0 , 3 } ppm : each respective slice along the p spectral dimension ( right panel ) comprises one broad and one narrow p nmr peak , as indeed suggested by the 1d p nmr spectra of figure 2 . given these observations , it is noteworthy that the broad p nmr signal from the cap clusters of the pristine s85 mbg correlates with those of both the h resonances from water molecules and the oh groups . this proves that whereas the structural properties of the cap clusters are predominantly shared by those of acp , they exhibit some features associated with ha , such as the presence of minor amounts of its characteristic hydroxy groups . previously , we suggested the presence of such oh moieties in the cap clusters of mbgs , observed tentatively from a 2d hetcor spectrum of an s85 sample associated with a composition close to the current one;(44 ) however , this oh nmr signal is sufficiently resolved in the present hetcor 2d spectrum to eliminate any ambiguity . furthermore , it is interesting that the slice along the p dimension taken at h = 0 ( figure 3a ; right panel ) hints two distinctp nmr signals : one broad and one narrow . the latter indicates a local ordering in a minor population of the phosphate / hydroxyl groups of the cap clusters , presumably induced by the calcination process . ( see section . ) whereas the h dimension of the hetcor spectrum from s85 is very broad , a weak nmr signal is discernible in the h region around 810 ppm , which may signify minor amounts of acidic p oh moieties associated with the cap clusters . yet , the 2d hetcor nmr spectra in figure 3 , particularly those from sbf16h and tris16h , reveal that if present , the population of such acidic protons must be very low . all other resonances present in the directly excited h nmr spectra shown in the left panel of figure 4 were assigned and discussed by leonova et al.(44 ) this includes the sharp peak at 2.1 ppm arising from isolated ( non - hydrogen - bonded ) silanols at the silica surface as well as those from minute remainings of organic precursor / templating molecules that appear at 3.6 ppm and 1.1 ppm in the nmr spectra . a hp cpmas experiment excites p nmr signals by transferring magnetization from nearby protons at a rate that depends exponentially on tcp1 : for an isolated h p spin - pair , this cp time constant ( tcp ) is proportional to the inverse square of the heteronuclear through - space dipolar coupling constant b , that is , to the sixth power of the h p internuclear distance r : tcp b r. the efficiency of the magnetization transfer , that is , the observed p nmr signal strength , is dictated by the ratio cp / tcp , where the contact interval cp represents the duration of the cp process . hence , by employing small cp values , one may experimentally arrange the sole excitation of nmr signals from the p sites being in close spatial proximity to protons ( within a few angstroms ) , whereas prolonged cpmas application will effect long - range hp magnetization transfers . however , in relatively dense structures , such as the calcium phosphates of the present work , a multitude of distinct through - space h whereas tcp is extractable as a phenomenological parameter by variable-cp cpmas experimentation , it may only be interpreted in terms of an effective dipolar coupling constant , beff , associated with an internuclear distance , reff . yet , the cp kinetics is dominated by the set of largest coupling constants of each p site , that is , those involving the shortest h ( where each subscript denotes the spin - pair index ) , their contributions are roughly additiveherein , we will use the terminology of strong ( or weak ) h p contact when referring to a short ( long ) effective internuclear distance , which is associated with a low ( high ) tcp - value . figure 5 conveys the dependence of the p nmr peakshapes acquired by using three distinct hp cp contact intervals for each mbg specimen . note that the nmr signals from the p sites in closest proximity to protons are emphasized in cpmas acquisitions employing the shortest value cp = 0.5 ms . as opposed to the spectra of figure 2 , which quantitatively reveal the various phosphate populations , the peakshape alterations in figure 5 for variable cp values suggest distinct h p contacts in the different phosphate - bearing phases present in the sbf / tris - soaked s85 samples . striking differences are observed when cp is varied for each of the sbf16h and tris16h specimens : the shortest contact interval cp = 0.5 ms emphasizes the broad nmr signal from acp relative to its crystalline counterpart . the contribution from the latter grows for increasing cp values , which accounts for the concomitant narrowing of the net p nmr peak . the most likely reason for this observation is enhanced h p contacts stemming from a larger total proton content in the acp / cap phases ( compared with that in the more ordered h(c)a structure ) , whereas the respective shortest internuclear distance is comparable within all structures . cpmas nmr spectra acquired from the as - indicated samples ( see right spectral portion ) using three distinct cp contact intervals according to 0.5 ms ( black ) , 2.0 ms ( red ) , and 4.0 ms ( green ) . the full width at half - maximum ( fwhm ; in ppm ) height of each p peak is specified at the left bottom part of each spectrum . on the other hand , the absence of line shape alterations observed from the pristine s85 mbg throughout the entire range of cp values evidences uniform p h contacts in the cap clusters of its pore walls . furthermore , these nmr spectra ( figure 5 ) are also essentially identical to that obtained by single pulses in figure 2a . these observations for the amorphous cap clusters mirror analogous reports by single - pulse and cpmas p nmr on synthetic acp . to gain more quantitative insight into the cp dynamics and h p contacts in the various phosphorus - bearing phases grown from the s85-surface , we conducted a larger series of cpmas nmr experiments with variable cp values for the s85 , tris16h , and haref samples . because of their single - phase character , integration of each p peak from the haref and s85 specimens provided directly the nmr signal - buildup representative for the p sites of each respective crystalline and amorphous component . for tris16h , on the other hand , its p nmr peak required deconvolution into two signals stemming from the acp and ha constituents , respectively . these results are plotted in figure 6a , together with best - fit curves ( discussed in section ) that reveal each of the acp and ha contributions . as suggested from figure 5 , the ha phase indeed manifests a significantly slower p nmr signal buildup compared with its amorphous counterparts . integrated hp cpmas nmr - signal intensities plotted for variable contact intervals ( cp ) and shown together with best fits to eqs 4 or 5 . ( a ) results obtained from the tris16h sample . the total signal buildup ( tot ; black symbols ) is shown together with its respective amorphous ( acp ; gray symbols ) and crystalline ( ha ; red ) components . ( b ) integrated signal intensities and best - fit curves obtained from the as - indicated samples of s85 , haref , and the tris16h(acp ) and tris16h(ha ) components displayed in panel a. for facilitating comparison , each signal buildup curve is normalized to the same maximum amplitude . ( c ) zoom of diagram b over 0 cp 1.55 ms , emphasizing the distinct initial rates of the magnetization transfers . to improve visualization , the experimental uncertainties are indicated only in diagram c. figures 6b , c compare the cp kinetics observed for the apatite p environments in haref and tris16h as well as the amorphous phosphates , cap ( of s85 ) and acp ( of tris16h ) . we conclude that both amorphous components display similar initial p nmr signal buildup , which is faster than that observed from the two crystalline phases . the latter pair also exhibits essentially identical cp dynamics . before analyzing the hp cp dynamics in detail , we inspect the associated relaxation ( decay ) processes of the transverse h and p magnetization in the presence of an applied rf field ( t1 ) for each relevant h2o and oh resonance as well as that of each po43 site of the amorphous and crystalline phosphate constituents of the s85 , haref , and tris16h specimens . the rate of magnetization decay is dictated by t11 , where the time - constant t1 depends on both the external magnetic field and the mas frequency , but is primarily dictated by molecular motions over a time scale of the inverse rf nutation frequency ( nut1 ) that ( for instance ) may modulate heteronuclear dipolar interactions at the spin site to drive relaxation . the nmr signal decays were probed by appending a spin - lock pulse of variable duration ( sl ) either to the excitation pulse ( for h ) , or to the contact interval in a cp - based experiment ( for p ) . the nutation frequency of the spin - lock pulse was identical to that employed during cp for each respective h and p spin species , except for the absence of a ramped p rf amplitude . except for the apatite oh groups present in the tris16h sample , whose signal is not discernable in directly excited h spectra ( see section ) , all proton relaxation time constants were obtained by direct integration of the well - resolved nmr peaks from oh groups in haref and from the h2o molecules in s85 and tris16h . however , note that for the s85-deriving samples , the latter measurements reflect the entire water reservoir , which mainly derives from protons associated with the silicate portion of the mbg surface . to circumvent the slow p spin lattice ( t1 ) relaxation discussed in section ( table 2 ) , the p t1 values were estimated by numerically fitting the directly integrated po43 nmr signals acquired by h spin - locking followed by hp cp . utilizing cpmas is advantageous because it ensures the sole probing of the h and p sites present in the phosphate phases . however , the analysis of some of these experiments ( as well as in section ) relies on exclusive magnetization transfers within the following h and p pairs : h2opo43 ( amorphous ) and ohpo43 ( crystalline ) : this assumption is justified by the 2d hetcor nmr results in figure 3 ( see section ) . the integrated peak intensities , i(sl ) , were fit to the following expressions that involve either one ( eq 2 ) or two ( a and b ; eq 3 ) componentswhere i0 is proportional to the h or p population , and t1h and t1p represent the respective h and p relaxation time constants . for the cases of biexponential decays in eq 3 , the signal fractions { xa , xb } associated with the respective parameters { t1x , a , t1x , b } obey xa + xb = 1 . biexponential decays were generally observed for both h and p when sl increased ( figure 7 ) , except for the acp component of tris16h [ tris16h(acp ) ] ; its h2o magnetization displayed a monoexponential damping , whereas the orthophosphate signal - decay fitted equally well to eq 2 , thereby giving the single parameter t1p = 4.74 ms ( r = 0.989 ) . transverse magnetization - decay of ( a ) h and ( b , c ) p due to relaxation during spin - locking by an rf field , plotted for variable spin - lock intervals ( sl ) and the integrated nmr signals of the following nuclei ( indicated by bold letters ) : ( a ) oh of haref and h2o in s85 and tris16h , respectively ; ( b ) po43 groups in the cap clusters of pristine s85 , and the acp phase of tris16h ; and ( c ) po43 of haref and the ha component of tris16h . the resulting time constants { t1x , a , t1x , b } and weights { xa , xb } are given in table 2 , with the component displaying the largest fraction listed topmost . in general , we obtained a pair involving one rapidly ( low t1 value ) and one slowly ( large t1 ) relaxing component . because cp invariably underestimates the contributions from rapidly decaying signals , the relative populations accounting for the biexponential decays of the p sites may only be interpreted semiquantitatively . the biexponential relaxation observed for the two s85-deriving specimens is not surprising , given their complex multicomponent character , whereas for the case of haref , they may stem from hydroxy and phosphate ions present at the surface and interior of the crystallites , respectively , as discussed by isobe et al.(56 ) and jger et al.(57 ) in the context of nanocrystalline ha . to determine both the underlying time constant ( tcp ) and damping parameters ( t1h , t1p ) of each cp buildup curve in figure 6 , we fitted the integrated signal intensity of each nmr peak , i(cp ) , to either of the following expressionsequations 46 are valid when ( i ) tcp(t1h , t1p ) and assume ( ii ) the absence of spin - diffusion among protons and that ( iii ) the h population grossly outnumbers that of p. the first two requirements are generally met under our experimental conditions ( a spinning rate of 9.0 khz and low h contents of the samples ) , but the third assumption is more questionable . furthermore , to improve the nmr spectral signal - to - noise and the cp - transfer stability , all of our cpmas experimentation involved ramping of the p rf field,(49 ) which decelerates the magnetization transfers relative to traditional constant amplitude we focus primarily on qualitatively comparing the cp kinetics and best - fit tcp , t1h , and t1p parameters among the three samples of the present study , which were all recorded under identical experimental conditions . the ( large ) values of t1h and t1p from the tris16h(ha ) and haref components ( see table 2 ) were not possible to estimate within the range cp 10 ms available to us to avoid compromising the integrity of our old mas probehead ; consequently , these data were fitted to eq 4 . the directly measured p nmr signal decays of the amorphous cap and acp constituents of the s85 and tris16h specimens reveal significantly damped curves ( section ) . hence , their cp kinetics data were fitted to both eqs 5 and 6 for the latter case assuming a single tcp parameter and fixed value(s ) of t1p , as obtained from the independent fitting of each respective spin - lock decay , by using eq 2 for tris16h(acp ) and eq 3 for s85 . the two damping constants { t1p , a , t1p , b } and their accompanying fractions { xa , xb } associated with s85 ( from table 2 ) were employed to fit the experimental data to the weighted sum i(cp ; t1p , a , t1p , b ) = xai(cp ; t1p , a ) + xbi(cp ; t1p , b ) , with each term evaluated from eq 6 . employing eq 6 with freely varying t1p values provided ill - defined best - fit parameters . table 3 lists the best - fit parameters . the tcp values are indeed very similar for the two crystalline ha phases ( tcp 1.5 ms ) being roughly twice as large as those of the two amorphous cap / acp phosphates ( 0.69 ms to 0.80 ms ) . besides differences in the cp rate constants , the primary distinction between the crystalline and amorphous p - bearing phases is their nmr relaxation in the presence of rf fields : both the cap ( of pristine s85 ) and acp ( of tris16h ) phases display significantly faster nmr signal decays through their much smaller values of t1h and t1p . table 3 evidences very similar best - fit tcp - values extracted from either eq 5 or 6 , that is , regardless if the t1p relaxation was explicitly accounted for . the p sites of the s85 structure display closest internuclear distances to water molecules ( figure 3 ) . the most evident h p contact in the sbf / tris - exposed samples is that from the biomimetic apatite - like phase , which becomes emphasized because of its very narrow h resonance . yet , its associated h and p nmr peak widths from the oh and po43 environments of the sbf / tris exposed samples are significantly larger than those of the well - ordered haref sample ( see table 1 ) : the p fwhm from the latter is 0.67 ppm , whereas the tris16h and sbf16h counterparts amount to 2.50 ppm and 2.81 ppm , respectively . their hetcor - observed h signals are also relatively broad , revealing fwhm values around 0.8 ppm ( tris16h ) and 1.2 ppm ( sbf16h ) , which may be contrasted with that of 0.5 ppm observed from haref . this confirms a lower structural order of both the oh and po4 environments of the in - vitro - formed h(c)a phases . peak maximum pmax ( whose uncertainty of 0.05 ppm is primarily dictated by the external chemical shift referencing ) and full width at half - maximum ( fwhm ) of the p peak obtained by direct excitation ( figure 2 ) . chemical shift coordinates ( h , p ) at the center of gravity of the 2d nmr peak from the apatite environments ( figure 3 ) . fwhm of the h and p signals , respectively ( separated by semicolon ) , obtained from the corresponding 2d slices in figure 3 . note that the slightly narrower p nmr signal observed in the single - pulse acquisitions ( figure 2 ) from the tris16h sample compared with its sbf - soaked counterpart is unambiguously established from the hetcor slices ( figure 3b , c ; right panel ) that separate the p resonance of the apatite phase from the others . hence , whereas the signal narrowing in figure 2 is primarily attributed to a larger h(c)a contribution out of the total amount of p - bearing phases in the tris16h specimen relative to its sbf16h counterpart ( as discussed in detail elsewhere for a larger set of samples(29 ) ) , table 2 evidences an inherently narrower p signal from h(c)a grown in tris solution compared with sbf . this is attributed to an overall higher ordering of the former ha phase , stemming partially from its absence of co32 , na , and other substituted ions , as compared with the hca structure generated from sbf.(28)table 2h and p nmr relaxation propertiessample / componentt1h ( ms)abt1p ( ms)act1 ( s)dharef28 2 ( 0.79)66 4 ( 0.70)8.8 0.61.1 0.2 ( 0.21)0.65 0.04 ( 0.30)tris16h(ha / oh)en.d.80 30 ( 0.83)79 10.83 0.3 ( 0.17)tris16h(acp / h2o)f1.8 0.15.9 0.3 ( 0.78)79 10.96 0.2 ( 0.22)s851.1 0.1 ( 0.69)27 3 ( 0.73)128 415 2 ( 0.31)0.28 0.07 ( 0.27)abest - fit time constants ( using either eq 2 or eq 3 ) for h ( t1h ) and p ( t1p ) responsible for the decay of transverse magnetization during a spin - lock rf field . the relative weight of each contribution to the biexponential decay is given within parentheses.bmeasured using a 90 pulse , followed by a spin - lock pulse.cmeasured using hp cp ( cp = 1.5 ms ) , followed by a spin - lock pulse.ddetermined from a saturation - recovery experiment . note that for the case of tris16h , only the t1-value representative for the netp peak was measured.econcerns the ordered apatite - like component giving the ohpo43 correlation established by 2d hetcor.fconcerns the acp component responsible for the h2opo43 correlation . best - fit time constants ( using either eq 2 or eq 3 ) for h ( t1h ) and p ( t1p ) responsible for the decay of transverse magnetization during a spin - lock rf field . measured using hp cp ( cp = 1.5 ms ) , followed by a spin - lock pulse . determined from a saturation - recovery experiment . note that for the case of tris16h , only the t1-value representative for the netp peak was measured . concerns the ordered apatite - like component giving the ohpo43 correlation established by 2d hetcor . concerns the present investigation involves to our knowledge the first detailed account of the h and p nmr relaxation properties and cp time constants associated with two such phases : biomimetically grown acp and the cap clusters of mbgs . p hetcor 2d nmr spectra in figure 3b , c , the po43 groups are in close spatial proximity solely to water molecules but neither to oh groups nor acidic protons , regardless if acp is formed in sbf or tris - buffered water . betts(60 ) structural model of acp being built by aggregated ca9(po4)6 clusters , with water molecules occupying their interstices . our results confirm several observations from previous h and p mas nmr studies on synthetic acp , except for the presence of acidic protons in the structure , as either inferred from nmr or implied by generalized p contacts in synthetic and biomimetic hydroxyapatite as well as evidence closely related p nmr characteristics of the cap clusters in the s85 mbg relative to the acp surface layer formed in vitro . the latter pair of phases exhibits similar degrees of structural disorder , as inferred from their comparable chemical shift - distributions , that is , p nmr fwhm ( see figure 3 and section ) . the roughly twice as large tcp values observed from the two ordered apatite structures ( ha and haref ) relative to the amorphous phosphates ( acp and cap ) reflect a higher proton density in the latter . equation 1 then implies the following relationship between the respective effective dipolar coupling constantsequation 7 is consistent with a multitude of distinct h however , h(c)a solely comprises oh groups , whereas water molecules constitute the proton source in the acp structure ( figure 3 ) ; then , one plausible and natural structural scenario consistent with eq 7 is the replacement of each oh group in the apatite structure by one h2o molecule ( accompanied by removal of one ca ion ) such that each shortest h this provides a doubled proton density in the latter structure and the following stoichiometric relationshipwhere the left - hand side is formulated by assuming the posner cluster as structural unit . note that the postulated h2o for oh substitutions naturally leads to the previously suggested ( but thus far not proven ) interstitial locations of the water molecules in acp . we stress that whereas eq 8 is consistent with our nmr data , it must not be taken too literally , and many other structural scenarios may also provide h both h and p ( table 2 ) , each very large relaxation time constant associated with the dominating nmr signal fraction observed from the two crystalline apatites , that is , haref and the ordered phosphate component of tris16h , rationalizes the observed absence of p signal - damping during cp ( figure 6 and table 3 ) . furthermore , the markedly faster decays for both h and p of the amorphous phosphates , acp in tris16h and cap in s85 , are also mirrored in their significantly lower values of t1h and t1p , relative to haref and h(c)a ( table 2 ) . the main distinguishing property between the two amorphous components is the accelerated signal damping of the p sites in the acp phase of the tris16h sample , as compared with that of the cap clusters of the pristine mbg . this likely stems from a higher mobility of the water molecules of biomimetic acp relative to the cap clusters present in the mbg pore walls . kaflak and kolodziejski highlighted a strong dependence of the p t1 relaxation on the water content in synthetic and biological apatites,(36 ) which correlates very well with our observed data.table 3best - fit parameters from variable-cph p cp experimentssample / componenttcp ( ms)t1h ( ms)i0 ( a.u)rharefa1.77 0.04n.d.0.99 0.010.998tris16h(ha / oh)ab1.52 0.08n.d.1.00 0.020.993tris16h(acp / h2o)cd0.69 0.09 ( 0.80 0.12)3.3 0.4 ( 3.3 0.4)1.62 0.12 ( 1.89 0.17)0.976 ( 0.976)s85d0.69 0.03 ( 0.80 0.03)45 8 ( 40 8)1.05 0.02 ( 1.37 0.03)0.994 ( 0.996)abest - fit { tcp , t1h , t1p } results and correlation coefficients r obtained by fitting the experimental integrated signal intensity to eq 4.bconcerns the ordered apatite - like component giving the ohpo43 correlation established by 2d hetcor.cconcerns the acp component responsible for the h2opo43 correlation.dobtained by fitting experimental data to eq 5 . values within parentheses were obtained by instead employing eq 6 and the fixed value t1p = 4.74 ms [ for tris16h(acp ) ] , whereas for s85 , the biexponential parameters { t1x , a = 27 , t1x , b = 0.28 } ms and signal fractions { xa = 0.73 , xb = 0.27 } were used from table 2 . best - fit { tcp , t1h , t1p } results and correlation coefficients r obtained by fitting the experimental integrated signal intensity to eq 4 . concerns the ordered apatite - like component giving the ohpo43 correlation established by 2d hetcor . concerns the acp component responsible for the h2opo43 correlation . obtained by fitting experimental data to eq 5 . values within parentheses were obtained by instead employing eq 6 and the fixed value t1p = 4.74 ms [ for tris16h(acp ) ] , whereas for s85 , the biexponential parameters { t1x , a = 27 , t1x , b = 0.28 } ms and signal fractions { xa = 0.73 , xb = 0.27 } were used from table 2 . equation 3 , which involves a t1-driven nmr signal - decay also of p , has to our knowledge hitherto only been noted recently in one related study in the context of crystalline calcium phosphates and bone.(36 ) in general , p relaxation during spin - locking has been ignored in the analysis of hp cp - dynamics . furthermore , the biexponential character of the p relaxation generally observed from our samples has hitherto not been found from synthetic or biological apatites . however , previous reports typically employed cp during either static conditions or slower mas rates than our experimentation carried out at 9.0 khz , which may quench an otherwise active spin - diffusion . yet , our tcp values for the ohpo43 and h2opo43 contacts in the crystalline and amorphous phases agree well with the corresponding results observed from natural bone specimens by maltsev et al.,(39 ) which were obtained at 12 khz mas . table 2 also lists the p t1 values obtained by saturation - recovery experimentation(48 ) from the s85 , haref , and tris16h samples . lattice nmr relaxation compared with the much less ordered apatite - like phosphate environments of bone and tooth , which kaflak and kolodziejski attributed to the higher oh content present in ha relative to the natural tissues.(36 ) interestingly , the amorphous ( and oh - poor ) cap clusters of s85 indeed display much slower spin lattice relaxation ( t1p = 128 s ; table 2 ) compared with haref ( t1p = 8.8 s ) : the latter value agrees well with previous studies . furthermore , the relaxation of the net p nmr peak of the intermediately ordered phosphates in the tris16h and sbf16h samples reveals t1p values of 79 1 s and 96 3 s , respectively . hence , these time constants fall in - between those observed from the crystalline ( haref ) and amorphous ( cap ) structures , which supports the proposition that the spin we have investigated structural features of the phosphate environments in amorphous ( acp ) and crystalline ( ha ) phases generated in vitro at the surface of an s85 tem / edx results obtained from s85 before and after its soaking in sbf or tris - solutions for 16 h revealed a biomimetically grown surface layer of calcium phosphate comprising domains of nanocrystalline h(c)a . h p 2d hetcor nmr spectra recorded from these sbf16h and tris16h specimens verified the coexistence of acp and h(c)a : each respective component phase displayed the pairs of correlations h2opo43(acp ) and ohpo43(apatite ) , meaning that the orthophosphate ions are in close spatial proximity solely to water molecules and hydroxyl groups in the acp and h(c)a phases , respectively . neither our acp formed in sbf nor tris - buffered water revealed any significant amounts of acidic protons . the hp cpmas kinetics was monitored for each relevant po43 , oh , and h2o moiety in the s85 and tris16 h specimens as well as those in a synthetic ha sample haref : we contrasted the underlying rate constants obtained by analyzing a series of nmr experiments involving variable contact intervals . despite that the in - vitro - generated ha structure is less ordered than its haref counterpart , both structures feature very closely related sets of h p internuclear distances , as evidenced by their essentially identical rates of cp p nmr signal growth . the structure of the two amorphous phases , biomimetic acp and the cap clusters of the mbg pore walls , is associated with a higher proton density originating from the presence of structural water molecules and resulting in twice as fast cp p signal buildup relative to crystalline ha . this observation is consistent with a ( simplified ) structural scenario where each apatite oh group is replaced by one h2o molecule ( see eq 8) . the local and intermediate - range structure of the amorphous cap clusters present in the pristine mbg is closely related to its biomimetic acp counterpart , as evidenced by their shared h and p nmr characteristics in terms of ( i ) chemical shifts , implying very similar local electron densities at the p sites ; ( ii ) nmr peak widths , which translates into comparable structural ( dis)order ; and ( iii ) very similar ( effective ) h p distances . these two amorphous phases mainly differ in their t1 relaxation properties , where acp manifests significantly faster h and p nmr signal damping , likely reflecting a higher mobility of the water molecules in its structure relative to those of the cap clusters . furthermore , while sharing essentially all of their structural features with bulk acp , the cap clusters also comprise small amounts of the ha - characteristic oh groups ( < 5% of their total proton population ) .

by exploiting 1h and 31p magic - angle spinning nuclear magnetic resonance ( nmr ) spectroscopy , we explore the proton and orthophosphate environments in biomimetic amorphous calcium phosphate ( acp ) and hydroxy - apatite ( ha ) , as grown in vitro at the surface of a 10cao85sio25p2o5 mesoporous bioactive glass ( mbg ) in either a simulated body fluid or buffered water . transmission electron microscopy confirmed the presence of a calcium phosphate layer comprising nanocrystalline ha . two - dimensional 1h31p heteronuclear correlation nmr established predominantly 1h2o31po43 and o1h31po43 contacts in the amorphous and crystalline component , respectively , of the mbg surface - layer ; these two pairs exhibit distinctly different 1h31p cross - polarization dynamics , revealing a twice as large squared effective 1h31p dipolar coupling constant in acp compared with ha . these respective observations are mirrored in synthetic ( well - crystalline ) ha , and the amorphous calcium orthophosphate ( cap ) clusters that are present in the pristine mbg pore walls : besides highlighting very similar local 1h and 31p environments in synthetic and biomimetic ha , our findings evidence closely related nmr characteristics , and thereby similar local structures , of the cap clusters in the pristine mbg relative to biomimetic acp .

Introduction Materials and Methods Results Discussion Conclusions

the organic matrix mainly involves fibrous collagen and to a lesser extent ( glyco)proteins and citrate , whereas the inorganic part constitutes ca - deficient hydroxy - carbonate apatite ( hca ; 39 wt % carbonate ) , substituted with additional ions , for example , na , mg , and f. repairing bone / tooth defects or fractures demands an implant with the minimum requirement of biocompatibility , that is , that it is nontoxic and will not be expelled by the human body . on contact with body fluids , they possess tissue - bonding abilities because of the formation of a biomimetic layer of amorphous calcium phosphate ( acp ) that subsequently converts into nanocrystalline hca . whereas the composition , structure , and evolution of the biomimetic phosphate layer generated in vitro at the surface of bioactive glasses ( bgs ) have been addressed by a diversity of characterization techniques , primarily powder x - ray diffraction , electron microscopy ( em ) coupled to energy - dispersive x - ray ( edx ) spectroscopy , and infrared and raman spectroscopy , very few studies employed solid - state nuclear magnetic resonance ( nmr ) . by utilizing si and p magic - angle spinning ( mas ) nmr , we recently reported a study(28 ) of the reactions leading to hca formation at the surface of an ordered mesoporous bioactive glass ( mbg ) of composition 10cao85sio25p2o5 in a simulated body fluid ( sbf). (43 ) here , we examine the characteristics of the biomimetically grown acp and hca constituents of the surface layer generated either in sbf or buffered water media from a very similar mbg specimen , by using transmission electron microscopy ( tem ) , and particularly p and h mas nmr . we contrast the hp cross - polarization ( cp ) dynamics observed from the amorphous and structurally ordered components of the phosphate layer with that of a synthetic crystalline hydroxyapatite sample ( labeled haref ) as well as with the amorphous calcium phosphate clusters ( henceforth referred to as cap ) initially present in the mbg pore wall . such experimentation provides qualitative information about the ( shortest ) h p internuclear distances in a structure , here revealing very similar h p spatial contacts in each pair of amorphous cap / acp and crystalline hca / haref phases : this underscores the close structural similarity between the orthophosphate component of the mbg structure and the biomimetic acp formed in vitro , on one hand , and the biomimetic apatite compared with the crystalline reference , on the other . tetraethyl orthosilicate ( teos ) , triethyl phosphate ( tep ) , and ca(no3)24h2o served as precursors for introducing each of the elements si , p , and ca , respectively . between 4096 and 14 336 transients were accumulated for variable contact intervals ( cp ) , using relaxation delays of 4.0 s. h p heteronuclear correlation ( hetcor ) 2d nmr spectra were acquired from the s85 , sbf16h , and tris16h specimens using hp cp for magnetization transfer ( cp = 1.5 ms ) and 800 accumulated signal transients / t1-value ( except for s85 : 1616 transients / t1 ) . cation compositions measured by edx over the displayed areas are reported in at % at the bottom left corner of the images a and d. representative tem images recorded from fragments of the sbf16h and tris16h specimens are displayed in figure 1b and 1c , respectively : they evidence a large number of 1050 nm sized elongated particles grown at the mbg surface . results from both powder x - ray diffraction and scanning electron microscopy ( sem ) coupled to edx further confirmed the presence of ha in the mbg surface layer. (29 ) onward , we will employ the label h(c)a when referring collectively to the biomimetic apatite phase forming in either sbf or tris solutions : note that acp crystallizes into ha in tris / hcl - buffered water , whereas the presence of carbonate ions in the sbf solution(43 ) leads to the formation of hca , analogously to bone and tooth formed in vivo . the simultaneous incorporation of na and co32 ions into the sbf - induced calcium phosphate layer at the s85 surface was recently evidenced by sem / edx,(29)na mas , and c cpmas nmr. figure 2a displays the results from the pristine s85 mbg sample and the crystalline hydroxyapatite reference specimen , hence , accompanying their common orthophosphate structural building blocks , both haref and the cap component of s85 produce a p nmr peak maximum around 3 ppm . however , the very different ordering of the two structures is reflected by their distinct peak shapes and full width at half - maximum ( fwhm ) height of the corresponding p nmr signals : the nmr response from the well - ordered haref structure features a narrow lorentzian peak ( 0.67 ppm ) , whereas that from the mbg is gaussian - shaped and an order of magnitude broader ( 6.3 ppm ) . as previously highlighted and explored further in this contribution , the cap clusters of s85 share many nmr and hence local structural features with the acp layer initially forming at the mbg surface on its contact with ( simulated ) body fluids . both reveal a significant signal - narrowing and the presence of two p sources in the specimens , reflecting one ordered phase ( i.e. the latter can not readily be distinguished by their 1d nmr - associated parameters ; however , as the surface - associated cap clusters tend to rapidly dissolve during the initial stages of the mbg soaking , their contributions to the p nmr spectra of the sbf16h and tris16h samples are expected to be minor . each amorphous ( acp / cap ) and crystalline ( h(c)a ) phosphate phase is primarily associated with proton environments stemming from water molecules and hydroxyl groups , respectively , which resonates at h 4 ppm ( broad peak ) and 0 ppm ( narrow signal ) , respectively . the dual presence of amorphous and crystalline phosphates in the sbf / tris - exposed samples is unambiguously evidenced from the 2d nmr spectra of figure 3b , c that primarily reveal two nmr correlations , centered at the pairs of shifts { h , p } around { 4 , 3 } ppm and { 0 , 3 } ppm : each respective slice along the p spectral dimension ( right panel ) comprises one broad and one narrow p nmr peak , as indeed suggested by the 1d p nmr spectra of figure 2 . given these observations , it is noteworthy that the broad p nmr signal from the cap clusters of the pristine s85 mbg correlates with those of both the h resonances from water molecules and the oh groups . this proves that whereas the structural properties of the cap clusters are predominantly shared by those of acp , they exhibit some features associated with ha , such as the presence of minor amounts of its characteristic hydroxy groups . previously , we suggested the presence of such oh moieties in the cap clusters of mbgs , observed tentatively from a 2d hetcor spectrum of an s85 sample associated with a composition close to the current one;(44 ) however , this oh nmr signal is sufficiently resolved in the present hetcor 2d spectrum to eliminate any ambiguity . whereas the h dimension of the hetcor spectrum from s85 is very broad , a weak nmr signal is discernible in the h region around 810 ppm , which may signify minor amounts of acidic p oh moieties associated with the cap clusters . however , in relatively dense structures , such as the calcium phosphates of the present work , a multitude of distinct through - space h whereas tcp is extractable as a phenomenological parameter by variable-cp cpmas experimentation , it may only be interpreted in terms of an effective dipolar coupling constant , beff , associated with an internuclear distance , reff . the most likely reason for this observation is enhanced h p contacts stemming from a larger total proton content in the acp / cap phases ( compared with that in the more ordered h(c)a structure ) , whereas the respective shortest internuclear distance is comparable within all structures . on the other hand , the absence of line shape alterations observed from the pristine s85 mbg throughout the entire range of cp values evidences uniform p h contacts in the cap clusters of its pore walls . to gain more quantitative insight into the cp dynamics and h p contacts in the various phosphorus - bearing phases grown from the s85-surface , we conducted a larger series of cpmas nmr experiments with variable cp values for the s85 , tris16h , and haref samples . the total signal buildup ( tot ; black symbols ) is shown together with its respective amorphous ( acp ; gray symbols ) and crystalline ( ha ; red ) components . ( b ) integrated signal intensities and best - fit curves obtained from the as - indicated samples of s85 , haref , and the tris16h(acp ) and tris16h(ha ) components displayed in panel a. for facilitating comparison , each signal buildup curve is normalized to the same maximum amplitude . before analyzing the hp cp dynamics in detail , we inspect the associated relaxation ( decay ) processes of the transverse h and p magnetization in the presence of an applied rf field ( t1 ) for each relevant h2o and oh resonance as well as that of each po43 site of the amorphous and crystalline phosphate constituents of the s85 , haref , and tris16h specimens . the rate of magnetization decay is dictated by t11 , where the time - constant t1 depends on both the external magnetic field and the mas frequency , but is primarily dictated by molecular motions over a time scale of the inverse rf nutation frequency ( nut1 ) that ( for instance ) may modulate heteronuclear dipolar interactions at the spin site to drive relaxation . except for the apatite oh groups present in the tris16h sample , whose signal is not discernable in directly excited h spectra ( see section ) , all proton relaxation time constants were obtained by direct integration of the well - resolved nmr peaks from oh groups in haref and from the h2o molecules in s85 and tris16h . transverse magnetization - decay of ( a ) h and ( b , c ) p due to relaxation during spin - locking by an rf field , plotted for variable spin - lock intervals ( sl ) and the integrated nmr signals of the following nuclei ( indicated by bold letters ) : ( a ) oh of haref and h2o in s85 and tris16h , respectively ; ( b ) po43 groups in the cap clusters of pristine s85 , and the acp phase of tris16h ; and ( c ) po43 of haref and the ha component of tris16h . the biexponential relaxation observed for the two s85-deriving specimens is not surprising , given their complex multicomponent character , whereas for the case of haref , they may stem from hydroxy and phosphate ions present at the surface and interior of the crystallites , respectively , as discussed by isobe et al. to determine both the underlying time constant ( tcp ) and damping parameters ( t1h , t1p ) of each cp buildup curve in figure 6 , we fitted the integrated signal intensity of each nmr peak , i(cp ) , to either of the following expressionsequations 46 are valid when ( i ) tcp(t1h , t1p ) and assume ( ii ) the absence of spin - diffusion among protons and that ( iii ) the h population grossly outnumbers that of p. the first two requirements are generally met under our experimental conditions ( a spinning rate of 9.0 khz and low h contents of the samples ) , but the third assumption is more questionable . furthermore , to improve the nmr spectral signal - to - noise and the cp - transfer stability , all of our cpmas experimentation involved ramping of the p rf field,(49 ) which decelerates the magnetization transfers relative to traditional constant amplitude we focus primarily on qualitatively comparing the cp kinetics and best - fit tcp , t1h , and t1p parameters among the three samples of the present study , which were all recorded under identical experimental conditions . the tcp values are indeed very similar for the two crystalline ha phases ( tcp 1.5 ms ) being roughly twice as large as those of the two amorphous cap / acp phosphates ( 0.69 ms to 0.80 ms ) . besides differences in the cp rate constants , the primary distinction between the crystalline and amorphous p - bearing phases is their nmr relaxation in the presence of rf fields : both the cap ( of pristine s85 ) and acp ( of tris16h ) phases display significantly faster nmr signal decays through their much smaller values of t1h and t1p . yet , its associated h and p nmr peak widths from the oh and po43 environments of the sbf / tris exposed samples are significantly larger than those of the well - ordered haref sample ( see table 1 ) : the p fwhm from the latter is 0.67 ppm , whereas the tris16h and sbf16h counterparts amount to 2.50 ppm and 2.81 ppm , respectively . their hetcor - observed h signals are also relatively broad , revealing fwhm values around 0.8 ppm ( tris16h ) and 1.2 ppm ( sbf16h ) , which may be contrasted with that of 0.5 ppm observed from haref . fwhm of the h and p signals , respectively ( separated by semicolon ) , obtained from the corresponding 2d slices in figure 3 . hence , whereas the signal narrowing in figure 2 is primarily attributed to a larger h(c)a contribution out of the total amount of p - bearing phases in the tris16h specimen relative to its sbf16h counterpart ( as discussed in detail elsewhere for a larger set of samples(29 ) ) , table 2 evidences an inherently narrower p signal from h(c)a grown in tris solution compared with sbf . this is attributed to an overall higher ordering of the former ha phase , stemming partially from its absence of co32 , na , and other substituted ions , as compared with the hca structure generated from sbf. concerns the present investigation involves to our knowledge the first detailed account of the h and p nmr relaxation properties and cp time constants associated with two such phases : biomimetically grown acp and the cap clusters of mbgs . our results confirm several observations from previous h and p mas nmr studies on synthetic acp , except for the presence of acidic protons in the structure , as either inferred from nmr or implied by generalized p contacts in synthetic and biomimetic hydroxyapatite as well as evidence closely related p nmr characteristics of the cap clusters in the s85 mbg relative to the acp surface layer formed in vitro . the roughly twice as large tcp values observed from the two ordered apatite structures ( ha and haref ) relative to the amorphous phosphates ( acp and cap ) reflect a higher proton density in the latter . equation 1 then implies the following relationship between the respective effective dipolar coupling constantsequation 7 is consistent with a multitude of distinct h however , h(c)a solely comprises oh groups , whereas water molecules constitute the proton source in the acp structure ( figure 3 ) ; then , one plausible and natural structural scenario consistent with eq 7 is the replacement of each oh group in the apatite structure by one h2o molecule ( accompanied by removal of one ca ion ) such that each shortest h this provides a doubled proton density in the latter structure and the following stoichiometric relationshipwhere the left - hand side is formulated by assuming the posner cluster as structural unit . furthermore , the markedly faster decays for both h and p of the amorphous phosphates , acp in tris16h and cap in s85 , are also mirrored in their significantly lower values of t1h and t1p , relative to haref and h(c)a ( table 2 ) . the main distinguishing property between the two amorphous components is the accelerated signal damping of the p sites in the acp phase of the tris16h sample , as compared with that of the cap clusters of the pristine mbg . this likely stems from a higher mobility of the water molecules of biomimetic acp relative to the cap clusters present in the mbg pore walls . yet , our tcp values for the ohpo43 and h2opo43 contacts in the crystalline and amorphous phases agree well with the corresponding results observed from natural bone specimens by maltsev et al.,(39 ) which were obtained at 12 khz mas . (36 ) interestingly , the amorphous ( and oh - poor ) cap clusters of s85 indeed display much slower spin lattice relaxation ( t1p = 128 s ; table 2 ) compared with haref ( t1p = 8.8 s ) : the latter value agrees well with previous studies . hence , these time constants fall in - between those observed from the crystalline ( haref ) and amorphous ( cap ) structures , which supports the proposition that the spin we have investigated structural features of the phosphate environments in amorphous ( acp ) and crystalline ( ha ) phases generated in vitro at the surface of an s85 tem / edx results obtained from s85 before and after its soaking in sbf or tris - solutions for 16 h revealed a biomimetically grown surface layer of calcium phosphate comprising domains of nanocrystalline h(c)a . h p 2d hetcor nmr spectra recorded from these sbf16h and tris16h specimens verified the coexistence of acp and h(c)a : each respective component phase displayed the pairs of correlations h2opo43(acp ) and ohpo43(apatite ) , meaning that the orthophosphate ions are in close spatial proximity solely to water molecules and hydroxyl groups in the acp and h(c)a phases , respectively . the structure of the two amorphous phases , biomimetic acp and the cap clusters of the mbg pore walls , is associated with a higher proton density originating from the presence of structural water molecules and resulting in twice as fast cp p signal buildup relative to crystalline ha . the local and intermediate - range structure of the amorphous cap clusters present in the pristine mbg is closely related to its biomimetic acp counterpart , as evidenced by their shared h and p nmr characteristics in terms of ( i ) chemical shifts , implying very similar local electron densities at the p sites ; ( ii ) nmr peak widths , which translates into comparable structural ( dis)order ; and ( iii ) very similar ( effective ) h p distances . these two amorphous phases mainly differ in their t1 relaxation properties , where acp manifests significantly faster h and p nmr signal damping , likely reflecting a higher mobility of the water molecules in its structure relative to those of the cap clusters .

construction of dsbd plasmids the plasmid pdzc1 was used to express isolated wild - type cdsbd bearing a c - terminal his6 tag and the plasmid pdzc5 a single cysteine variant ( c464a ) of cdsbd with a thrombin cleavage site preceding the c - terminal his6 tag . isolated ndsbd with the pelb signal peptide replacing the endogenous signal peptide and with a c - terminal streptavidin tag was expressed from the plasmid pdzn1 , and from that a single - cysteine variant ( c103a ) was produced . finally , the full - length construct in which ndsbd and cdsbd could be cleaved from tmdsbd ( thrombin - cleavable dsbd ) was expressed from pdsbd4 . details of the production of the above plasmids can be found in supplemental material . all experiments were performed in the e. coli strain bl21(de3 ) ( stratagene ) . all cells expressing unlabeled protein were grown at 37 c in 500-ml volumes of luria bertani ( lb ) broth in 2-liter flasks from overnight starter cultures ( grown at 37 c ) . sds - page analysis was carried out on 10 - 20% bistris6 nupage gels ( invitrogen ) with prestained protein markers ( seeblue plus 2 , invitrogen ) . bacteria transformed with pdsbd4 were grown with 100 g ml carbenicillin to an a600 of 0.7 before addition of 0.5 mm isopropyl -d - thiogalactopyranoside . after further incubation for 3 h the cells were harvested , and the crude membrane fraction was isolated using a french press . disruption of the cells was performed at 16,000 p.s.i . which was followed by centrifugation at 257,000 g for 1.5 h at 4 c . the membrane fraction was solubilized at a protein concentration of 5 mg ml for 1 h at 4 c under gentle agitation in 20 mm tris - hcl , 300 mm nacl , 20% v / v glycerol , 1% w / v n - dodecyl -d - maltoside ( ddm ) ( anatrace ) ( ph 7.5 ) . unsolubilized material was removed by centrifugation at 257,000 g for 45 min at 4 c . the supernatant was applied to 10 ml of fast flow chelating - sepharose ( amersham biosciences ) charged with ni . the column was washed with 20 mm tris - hcl , 150 mm nacl , 20 mm imidazole , 0.1% w / v ddm ( ph 7.5 ) , and the bound protein was eluted with 20 mm tris - hcl , 150 mm nacl , 200 mm imidazole , 0.1% w / v ddm ( ph 7.5 ) . a concentrated protein solution of 0.5 ml was applied to a superdex 200 ( hr ) size - exclusion column ( ge healthcare ) equilibrated in 20 mm tris - hcl , 150 mm nacl , 0.03% w / v ddm ( ph 7.5 ) . the purest fractions were pooled and concentrated to 0.5 ml . to prevent artifactual thiol - disulfide exchange reactions from occurring during the thrombin cleavage , free thiols were alkylated with n - ethylmaleimide ( sigma ) ( 16 ) . purified protein was incubated in the presence of 30 mm n - ethylmaleimide for 1 h at room temperature . after removal of the excess alkylating reagent , the protein was cleaved using the thrombin clean - cleave kit according to the manufacturer 's instructions ( sigma ) . the presence of the ndsbd - cdsbd mixed disulfide was confirmed by western blotting using two primary antibodies as follows : 1 ) penta - his hrp - conjugated monoclonal antibody ( qiagen ) , and 2 ) goat antiserum raised against the cdsbd sequence of e. coli dsbd and donkey anti - sheep alkaline phosphatase - conjugated antibody ( sigma ) , as primary and secondary antibodies , respectively . characterization of the ndsbd - cdsbd mixed disulfide formed in vivo the covalent complex between ndsbd and cdsbd was n - terminally sequenced from protein samples in sds - polyacrylamide gels that were electrophoretically transferred to a polyvinylidene difluoride membrane and stained with coomassie brilliant blue . the protein bands were excised and subjected to automated edman sequencing using an applied biosystems 494a procise protein sequencer . simultaneous overexpression of ndsbd and c464a - cdsbd bacteria transformed with pdzn1 and pdzc5 were grown with 100 g ml ampicillin and 20 g ml gentamycin to an a600 of 1.2 before addition of 1 mm isopropyl -d - thiogalactopyranoside . after further incubation for 4 h , the cells were harvested spheroplasted as described ( 17 ) omitting edta . western blotting of the extracted periplasm for the detection of the ndsbd - cdsbd mixed disulfide complex was done using two primary antibodies as follows : 1 ) strepmab - classic hrp - conjugated monoclonal antibody ( iba gmbh ) , and 2 ) penta - his hrp - conjugated monoclonal antibody ( qiagen ) . production and purification of the c103a - ndsbd - n - c464a - cdsbd complex for nmr studies the mixed disulfide complex was formed in vitro using c103a - ndsbd and uniformly n - labeled c464a - cdsbd purified separately from bl21(de3 ) cells ( stratagene ) . for the production of c103a - ndsbd , cells transformed with pdzn2 were grown at 30 c with 20 g ml gentamycin to an a600 of 1.5 before addition of 1 mm isopropyl -d - thiogalactopyranoside . after further incubation for 4 h at 37 c the cells were harvested and spheroplasted as described ( 17 ) omitting edta . the periplasmic fraction was applied to 5 ml of strep - tactin - sepharose ( iba gmbh ) equilibrated with 50 mm tris - hcl , 150 mm nacl ( ph 7.5 ) . the column was washed with 50 mm tris - hcl , 1 m nacl ( ph 7.5 ) , and the protein was eluted with 50 mm tris - hcl , 150 mm nacl , 2.5 mm desthiobiotin ( iba gmbh ) ( ph 7.5 ) according to the manufacturer 's instructions . production and purification of n - labeled c464a - cdsbd were done in the same way as for wild - type cdsbd , as described in previous work ( 15 ) . the c103a - ndsbd - n - c464a - cdsbd mixed disulfide complex was prepared using a reported method ( 12 ) . first , c103a - ndsbd was mixed with 10 mm dithiobisnitrobenzoic acid ( sigma ) at 25 c for 30 min . after removal of excess dithiobisnitrobenzoic acid , it was mixed with equimolar n - c464a - cdsbd and incubated as above . the reaction mixture was purified by two affinity chromatography steps , each specific to one of the domains . the mixture was applied to 3 ml of fast flow chelating - sepharose ( amersham biosciences ) charged with niand purified as described previously ( 15 ) . after removing the imidazole , the protein solution was applied to 3 ml of strep - tactin - sepharose ( iba gmbh ) and was purified as described above for c103a - ndsbd . the ndsbd - n - cdsbd mixed - disulfide sample was of high purity as assessed using sds - page ( supplemental fig . s1a ) , and its correct molecular weight was verified by electrospray ionization - mass spectrometry ( expected mass , 33,187 da ; observed mass , 33,185 da ) . production and purification of the n - labeled wild type and c464a - cdsbd n - labeled wild - type cdsbd and c464a - cdsbd were used as control proteins for the ph titration of the ndsbd - n - cdsbd mixed disulfide . ph titration of the ndsbd - n - cdsbd mixed disulfide using nmr spectroscopy the h and n resonances of c464a - cdsbd as a separate domain and in the ndsbd - n - cdsbd mixed disulfide were assigned using uniformly n - labeled samples of 0.8 mm protein for the isolated domains and 0.5 mm protein for the mixed disulfide , in 95% h2o , 5% d2o at ph 6.5 . assignments for c464a - cdsbd in the isolated domain and in the ndsbd - n - cdsbd complex were obtained by comparison of three - dimensional n - edited nuclear overhauser effect spectroscopy - hsqc and total correlation spectroscopy - hsqc spectra with the previously assigned spectra of reduced wild - type cdsbd ( 15 ) . nmr experiments for the determination of the pka values of asp in the active - site of cdsbd in the ndsbd - n - cdsbd mixed disulfide were performed using 0.5 mm of complex in 95% h2o , 5% d2o . determination of the pka values of asp in the active - site of n - labeled wild - type cdsbd and c464a - cdsbd was done in the same way as a control experiment . in the case of the n - labeled wild - type cdsbd , the sample was a 1:1 mixture of oxidized and reduced protein allowing simultaneous measurement of the pka value for the two oxidation states . the ph of solutions was adjusted by using small volumes of 0.1 - 1 m hcl or 0.1 - 1 m naoh . the ph of the samples was measured before and after each experiment , and the average of the two measurements was used for data analysis . two - dimensional h - n hsqc spectra were collected at 313 k on a home - built 750-mhz nmr spectrometer , which is controlled with ge / omega software and is equipped with a home - built triple - resonance pulsed - field gradient probe - head . sweep widths of 9345.79 hz and 2500 were used in f2 ( h ) and f1 ( n ) , respectively . 128 complex n increments of 8 , 12 , and 96 scans were collected with 1024 complex points in the acquisition dimension for n - labeled wild - type cdsbd , c464a - cdsbd , and the ndsbd - n - cdsbd mixed disulfide complex , respectively . nmr data were processed and visualized using nmrpipe , nmrdraw , and nmrview ( 18 , 19 ) . figure 2.western blots of purified full - length c464a thrombin - cleavable dsbd before and after thrombin cleavage . penta - his hrp - conjugated antibody was used in lanes 1 - 8 and goat antiserum raised against cdsbd of e. coli dsbd was used in lanes 9 - 16 . lanes 1 , 4 , 9 , and 12 show molecular mass markers ( from the top these are 49 , 38 , 28 , and 14 kda ) ; lanes 2 and 10 show the negative control ( ndsbd with no affinity tag ) ; lanes 3 and 11 show the positive control ( cdsbd bearing a c - terminal his6 tag ) . c464a thrombin - cleavable dsbd is shown in lanes 5 and 13 before alkylation of free thiols ( a ) and thrombin cleavage ( t ) , in lanes 6 and 14 without alkylation and after thrombin cleavage , in lanes 7 and 15 after alkylation and thrombin cleavage , and in lanes 8 and 16 after alkylation and thrombin cleavage in the presence of reductant ( r ) . the cdsbd band can be seen at 14 kda in lanes 3 and 11 , and in 6 - 8 and 14 - 16 in box iii ( this construct runs as a diffuse double band on sds - page because its pelb signal sequence , which targets the protein to the periplasm , is cleaved inefficiently by the signal peptidase , leaving a large fraction of the protein uncleaved ( confirmed by mass spectrometry and n - terminal sequencing ) ) . the uncleaved c464a thrombin - cleavable dsbd band can be seen at 49 kda ( the actual mass of the protein is 60.8 kda ; a difference between the two masses is common for membrane proteins ) in lanes 5 - 8 and 13 - 16 ( box i ) along with another band at 28 kda that is a contaminant commonly seen after the purification of this protein . the ndsbd - cdsbd mixed disulfide band can be seen at 32 kda in lanes 6 , 7 , 14 , and 15 ( box ii ) and it disappears in the presence of reductant in lanes 8 and 16 . 2 - 3 g of total protein were loaded in each lane . western blots of purified full - length c464a thrombin - cleavable dsbd before and after thrombin cleavage . penta - his hrp - conjugated antibody was used in lanes 1 - 8 and goat antiserum raised against cdsbd of e. coli dsbd was used in lanes 9 - 16 . lanes 1 , 4 , 9 , and 12 show molecular mass markers ( from the top these are 49 , 38 , 28 , and 14 kda ) ; lanes 2 and 10 show the negative control ( ndsbd with no affinity tag ) ; lanes 3 and 11 show the positive control ( cdsbd bearing a c - terminal his6 tag ) . c464a thrombin - cleavable dsbd is shown in lanes 5 and 13 before alkylation of free thiols ( a ) and thrombin cleavage ( t ) , in lanes 6 and 14 without alkylation and after thrombin cleavage , in lanes 7 and 15 after alkylation and thrombin cleavage , and in lanes 8 and 16 after alkylation and thrombin cleavage in the presence of reductant ( r ) . the cdsbd band can be seen at 14 kda in lanes 3 and 11 , and in 6 - 8 and 14 - 16 in box iii ( this construct runs as a diffuse double band on sds - page because its pelb signal sequence , which targets the protein to the periplasm , is cleaved inefficiently by the signal peptidase , leaving a large fraction of the protein uncleaved ( confirmed by mass spectrometry and n - terminal sequencing ) ) . the uncleaved c464a thrombin - cleavable dsbd band can be seen at 49 kda ( the actual mass of the protein is 60.8 kda ; a difference between the two masses is common for membrane proteins ) in lanes 5 - 8 and 13 - 16 ( box i ) along with another band at 28 kda that is a contaminant commonly seen after the purification of this protein . the ndsbd - cdsbd mixed disulfide band can be seen at 32 kda in lanes 6 , 7 , 14 , and 15 ( box ii ) and it disappears in the presence of reductant in lanes 8 and 16 . ph titration of [ 3-c]cysteine - labeled wild type and d455n - cdsbd h - c hsqc experiments for the determination of the pka value of cys at 313 k were performed on solutions containing 1.5 mm of [ 3-c]cysteine - labeled wild - type or d455n cdsbd in 99% d2o as described previously at 298 k ( 15 ) . pka values for asp were determined from h or n chemical shifts measured as a function of ph . pka values for cys were determined from h or c chemical shifts measured as a function of ph . the titration data were fitted to either one or two pka curves ( 20 ) using in - house software as described previously ( 15 ) . the pka value for asp was determined from the ph dependence of five chemical shift values ( asp / glu468 1h and n and gln488 15n ) . the pka value for cys was determined from the ph dependence of four to six chemical shift values ( cys / cysh and c ) . errors in the pka values were estimated as the standard deviation from the mean pka value . details of the parameters obtained in each fit are listed in supplemental tables s1 and s2 . in vivo studies of the formation of an ndsbd - cdsbd complex in full - length dsbd we have used a thrombin - cleavable variant of dsbd ( fig . 1a ) to attempt to identify a mixed - disulfide complex of ndsbd and cdsbd in vivo . this construct expresses full - length dsbd in which ndsbd and cdsbd can each be cleaved from the transmembrane domain by virtue of engineered thrombin cleavage sites . the c464a mutation in cdsbd was introduced in an attempt to trap the ndsbd - cdsbd mixed - disulfide complex because cys is believed to be responsible for driving the cleavage of the interdomain disulfide and the final transfer of reductant from cdsbd to ndsbd ( 12 ) . thrombin - cleavable dsbd was expressed , purified , incubated with n - ethylmaleimide to alkylate its free thiols , incubated with thrombin , and analyzed by sds - page and western blotting with antibodies against the his6 tag and cdsbd ; the results of the western blotting are shown in fig . 2 , lanes 6 , 7 , 14 , and 15 , that a band of the expected molecular weight for an ndsbd - cdsbd mixed disulfide ( 32 kda ) appeared after thrombin cleavage of thrombin - cleavable dsbd ( box ii ) . no band can be seen at this position before thrombin cleavage ( fig . 2 , lanes 5 and 13 ) . 2 , lanes 8 and 16 ) , confirming that it is a covalent complex containing a disulfide bond . the 32-kda covalent complex from the sds - page band was n - terminally sequenced , and both the ndsbd ( ( m)lfdapgrsq ) and cdsbd ( gsgqthlnft ) termini were detected . as seen in fig . 2 , lanes 6 and 7 or 14 and 15 , the band corresponding to the complex is present whether or not the free thiols of the protein were alkylated before thrombin cleavage . incubation with n - ethylmaleimide , therefore demonstrates that this complex was formed before cleavage of the full - length protein . a band for the full - length thrombin - cleavable dsbd can be seen ( at 49 kda ) even after thrombin cleavage ( fig . 2 , lanes 6 - 8 and 14 - 16 , box i ) ; this is attributed to inefficient thrombin cleavage because of the presence of the detergent ddm in the protein solutions . it is also likely that the presence of the protein in the detergent micelle could reduce the accessibility of the protease cleavage sites to the thrombin ( 21 ) . moreover , a c464a - cdsbd band can be seen ( at 14 kda ) after thrombin cleavage even in the absence of reductant ( fig . 2 , lanes 6 , 7 , 14 , and 15 , box iii ) . this is observed because there are molecules in the protein sample in which the ndsbd - cdsbd mixed disulfide had not formed and for which the thrombin cleavage led to release of the two separate periplasmic domains ( isolated ndsbd can not be seen in the western blot because it is not his6-tagged ) . figure 3.western blots of periplasmic extracts of e. coli expressing ndsbd with a streptavidin tag and c464a - cdsbd with a his6 tag . strepmab - classic hrp - conjugated antibody was used in lanes 1 - 4 and penta - his hrp - conjugated antibody was used in lanes 5 - 8 . lanes 1 and 5 show molecular mass markers ; lanes 2 and 6 show the negative control ( ndsbd with no affinity tag ) ; lanes 3 and 7 show positive controls ( full - length dsbd with a c - terminal streptavidin tag and cdsbd with a c - terminal his6 tag , respectively ) , and lanes 4 and 8 show the periplasmic extract . the full - length dsbd band can be seen at 49 kda in lane 3 along with a small amount of contaminant at 28 kda ( see explanation in fig . the ndsbd band can be seen at 17 kda in lane 4 and the cdsbd band can be seen at 14 kda ( the diffuse double band is explained in fig . 2 ) in lanes 7 and 8 along with the band of its homodimer at 33 kda . 15 - 20 g of total protein were loaded in each lane . western blots of periplasmic extracts of e. coli expressing ndsbd with a streptavidin tag and c464a - cdsbd with a his6 tag . strepmab - classic hrp - conjugated antibody was used in lanes 1 - 4 and penta - his hrp - conjugated antibody was used in lanes 5 - 8 . lanes 1 and 5 show molecular mass markers ; lanes 2 and 6 show the negative control ( ndsbd with no affinity tag ) ; lanes 3 and 7 show positive controls ( full - length dsbd with a c - terminal streptavidin tag and cdsbd with a c - terminal his6 tag , respectively ) , and lanes 4 and 8 show the periplasmic extract . the full - length dsbd band can be seen at 49 kda in lane 3 along with a small amount of contaminant at 28 kda ( see explanation in fig . the ndsbd band can be seen at 17 kda in lane 4 and the cdsbd band can be seen at 14 kda ( the diffuse double band is explained in fig . 2 ) in lanes 7 and 8 along with the band of its homodimer at 33 kda . 15 - 20 g of total protein were loaded in each lane . in vivo studies of complex formation between isolated ndsbd and cdsbd to assess the role of tmdsbd in mediating the interaction between ndsbd and cdsbd , the two periplasmic domains of dsbd , ndsbd bearing a c - terminal streptavidin tag and a c464a variant of cdsbd bearing a c - terminal his6 tag , were overexpressed simultaneously as soluble proteins exported to the periplasm . the periplasmic extract was analyzed by sds - page and western blotting with antibodies against the streptavidin tag , to detect ndsbd , and against a penta - histidine sequence , to detect cdsbd . the ndsbd - cdsbd mixed disulfide is expected to migrate on sds - page between the 28- and 38-kda molecular mass markers and to be detected by both antibodies ( fig . no band corresponding to the ndsbd - cdsbd mixed disulfide was observed in fig . 3 , lane 4 , only the ndsbd band at 17 kda is observed , and in lane 8 the c464a - cdsbd band at 14 kda and a band corresponding to its homodimer at 33 kda ( confirmed by western blotting , not shown ) are observed . a possible explanation for the failure of the ndsbd - cdsbd mixed - disulfide complex to form in vivo is that the proteins were present in the incorrect oxidation states for disulfide bond formation to occur ; interdomain disulfide bond formation requires the presence of oxidized ndsbd and reduced c464a - cdsbd . nmr studies of periplasmically overexpressed ndsbd show that it is present predominantly in the oxidized form . cdsbd can not form an intramolecular disulfide bond because it has a single cysteine and the homodimer is observed as only a minor species ( fig . 3 , lane 8) . for these reasons , attack of the cdsbd cysteine thiol on the disulfide of ndsbd , we therefore conclude that a complex between soluble ndsbd and cdsbd , co - expressed in the e. coli periplasm , does not form in the absence of the transmembrane domain that usually connects the two domains in the full - length protein . nmr analysis of the ndsbd - n - cdsbd mixed - disulfide complex a mixed - disulfide complex was produced using purified single - cysteine variants of ndsbd ( c103a ) and cdsbd ( c464a ) to ensure that the physiologically relevant disulfide was formed ( cys - ss - cys ) , to avoid any cleavage of this disulfide because of the presence of cys and to eliminate any complications during the pka determination of asp caused by the titration of the cys or cys thiols in the active site . nmr spectroscopy allows measurement of the pka value of asp in the active site of cdsbd when cdsbd is in close proximity to its periplasmic partner ndsbd . for this purpose we used uniformly n - labeled c464a - cdsbd covalently linked to unlabeled c103a - ndsbd , thus producing a c103a - ndsbd - n - c464a - cdsbd covalent complex ( subsequently referred to as ndsbd - n - cdsbd ) where only cdsbd gives nmr signals in the h - n hsqc spectrum but in which it is influenced by the presence of ndsbd . in this way we were able to simplify the nmr spectrum of a relatively large ( 32-kda ) protein complex and to focus on the domain of interest . previous studies of isolated cdsbd were carried out at 298 k. the h - n hsqc spectrum of the ndsbd - n - cdsbd complex at 298 k is significantly broader , because of the higher molecular weight of the complex , than that of isolated cdsbd , and many of the expected peaks are not observed ( supplemental fig . a significant improvement in the quality of the hsqc spectrum of the complex was observed at 313 k ( supplemental fig . both ndsbd and cdsbd have relatively high thermal stabilities and are completely folded and native at 313 k ( supplemental fig . the ph titration experiments described below were therefore performed at 313 k. pka determination for asp in the ndsbd- n - cdsbd complex we have monitored , between ph 4 and ph 10 , the n and h chemical shifts of the backbone amides of asp and glu and the side chain n of gln in hsqc spectra of the ndsbd - n - cdsbd mixed - disulfide complex ( fig . 4a ) . these shifts are all sensitive to titration of asp because of a hydrogen bond network involving asp , glu , and gln ( 10 , 11 ) . we have shown previously that cdsbd is stable between ph 4 and 12 ( 15 ) . h nmr spectra of the complex collected between ph 4 and 10 demonstrate that ndsbd in the complex is also stable over this ph range ( supplemental fig . analysis of the ph dependence of the n and h chemical shifts of asp and glu and of the side chain n of gln gave an average pka value of 8.5 0.1 for asp ( fig . this pka value is significantly higher than the values of 5.9 0.1 and 6.6 0.1 obtained previously for asp in reduced and oxidized cdsbd ( 15 ) . our previous study of cdsbd was carried out at 298 k , whereas the spectra of the complex were collected at 313 k. as a control , we have monitored the same chemical shifts in uniformly n - labeled wild - type cdsbd at 313 k. the experiment was performed , as before ( 15 ) , in a mixed oxidation state sample , and we obtained a pka value of 6.5 0.1 in the reduced state and of 7.0 0.1 for asp in the oxidized state ( fig . 5 , a - c ) . these values are 0.5 ph units higher than the values measured at 298 k but are nevertheless still significantly lower than the value of 8.5 measured for asp in the ndsbd - n - cdsbd complex . to ensure that the difference in the pka values of asp between complexed and uncomplexed cdsbd is not an effect of the c464a mutation , we have also performed the titration for uniformly n - labeled c464a - cdsbd at 313 k and obtained an average pka value of 7.4 0.1 for asp ( fig . therefore , it can be concluded that the pka value of asp is elevated by at least 1.1 ph units when cdsbd forms a complex with ndsbd . figure 4.overlay of two - dimensional h - n hsqc spectra of the ndsbd - n - c464a - cdsbd complex ( a ) and n - c464a - cdsbd collected at 750 mhz and 313 k for ph values ranging from 4 to 10 ( b ) . for the ndsbd - n - c464a - cdsbd complex , spectra collected at ph 4.635 ( light gray ) , 5.09 ( dark gray ) , 5.73 ( black ) , 6.57 ( red ) , 7.165 ( orange ) , 7.55 ( yellow ) , 8.00 ( green ) , 8.43 ( blue - green ) , 8.845 ( cyan ) , 9.32 ( blue ) , and 9.755 ( violet ) are shown . for n - c464a - cdsbd , spectra collected at ph 4.36 ( light gray ) , 4.895 ( dark gray ) , 5.53 ( black ) , 6.075 ( red ) , 6.61 ( orange ) , 7.21 ( yellow ) , 7.685 ( green ) , 8.155 ( blue - green ) , 8.61 ( cyan ) , 9.11 ( blue ) , and 9.86 ( violet ) are shown . the black lines drawn in a and b trace the ph - dependent chemical shift changes observed for asp . overlay of two - dimensional h - n hsqc spectra of the ndsbd - n - c464a - cdsbd complex ( a ) and n - c464a - cdsbd collected at 750 mhz and 313 k for ph values ranging from 4 to 10 ( b ) . for the ndsbd - n - c464a - cdsbd complex , spectra collected at ph 4.635 ( light gray ) , 5.09 ( dark gray ) , 5.73 ( black ) , 6.57 ( red ) , 7.165 ( orange ) , 7.55 ( yellow ) , 8.00 ( green ) , 8.43 ( blue - green ) , 8.845 ( cyan ) , 9.32 ( blue ) , and 9.755 ( violet ) are shown . for n - c464a - cdsbd , spectra collected at ph 4.36 ( light gray ) , 4.895 ( dark gray ) , 5.53 ( black ) , 6.075 ( red ) , 6.61 ( orange ) , 7.21 ( yellow ) , 7.685 ( green ) , 8.155 ( blue - green ) , 8.61 ( cyan ) , 9.11 ( blue ) , and 9.86 ( violet ) are shown . the black lines drawn in a and b trace the ph - dependent chemical shift changes observed for asp . figure 5.measurement of the pka value of the active - site residue asp from the ph dependence of the n chemical shift of asp ( a ) , the n chemical shift of glu ( b ) , and the n chemical shift of gln ( c ) . plots are shown for the ndsbd - c464a - cdsbd complex ( ) , isolated c464a - cdsbd ( ) , and for oxidized ( ) and reduced ( ) isolated wild - type cdsbd . the continuous lines show the best fit to a single pka value or to two pka values for each dataset . the fitting procedure has been described previously ( 15 ) ; see supplemental table s1 for a summary of the fitted parameters . measurement of the pka value of the active - site residue asp from the ph dependence of the n chemical shift of asp ( a ) , the n chemical shift of glu ( b ) , and the n chemical shift of gln ( c ) . plots are shown for the ndsbd - c464a - cdsbd complex ( ) , isolated c464a - cdsbd ( ) , and for oxidized ( ) and reduced ( ) isolated wild - type cdsbd . the continuous lines show the best fit to a single pka value or to two pka values for each dataset . the fitting procedure has been described previously ( 15 ) ; see supplemental table s1 for a summary of the fitted parameters . pka determination for cys at 313 k the pka value for cys was measured for wild - type cdsbd and for the d455n variant of cdsbd at 313 k for direct comparison with the pka values measured above for asp in the ndsbd - n - cdsbd complex at 313 k. analysis of the ph dependence of the c and h chemical shifts of cys and cys in cdsbd gives pka values of 10.4 0.1 and 9.2 0.2 for cys in wild - type and d455n cdsbd , respectively ( supplemental fig . s2 and supplemental table s2 ) . these values agree within experimental error with the values of 10.5 0.1 and 9.3 0.05 reported previously at 298 k. therefore , the pka value of cys does not show a temperature dependence . it has long been known that dsbd functions by interaction between the cysteine pairs in its three domains , although how these interactions occur within this protein , in which one of the three domains is membrane - embedded , has remained elusive . in this study we have used both in vivo and in vitro approaches to understand how the interaction and reaction between the two soluble domains are controlled . we have demonstrated using western blotting that a covalent ndsbd - cdsbd complex can be trapped in vivo when full - length dsbd is overexpressed . this result proves that the ndsbd - cdsbd interaction , which has previously only been probed in vitro ( 12 ) , is physiologically relevant . no such complex was detected when the individual domains , ndsbd and cdsbd , were co - expressed in the periplasm . the fact that the ndsbd - cdsbd complex was not detectable in the absence of the linking tmdsbd has important implications for the function of the membrane - embedded domain . the latter is known to interact with , and acquire reductant from , cytoplasmic thioredoxin and so effectively performs the transmembrane reductant transfer reaction ( 22 ) . in addition , it appears to have an important function in ensuring the proximity and correct orientation of the two periplasmic domains . it should be noted that the interaction interface identified in the ndsbd - cdsbd crystal structure is not very extensive and involves a relatively small number of interactions ( 12 ) . this limited interface may result in a relatively low affinity interaction between ndsbd and cdsbd . therefore , the transmembrane domain may be essential to ensure the high local concentration of the domains that would be required for the formation of a complex and subsequent electron transfer via the disulfide exchange reaction . in isolated cdsbd , cys , which catalyzes reduction of the cys - cys disulfide in ndsbd , has a pka value of 10.4 at 313 k ; this elevated value makes cys a poor nucleophile . the pka value of cys must therefore be lowered when it approaches ndsbd to make it more reactive . the high pka value for cys results from its close proximity to asp and glu in the active site . here we have used the covalent ndsbd - n - cdsbd complex to measure the pka value of asp in cdsbd when it is in close contact with ndsbd . we found that the pka value of asp is elevated by at least 1.1 ph units to a value of 8.5 in the complex at 313 k. at ph 7.0 , the ratio of deprotonated ( negatively charged ) to protonated ( uncharged ) asp in the complex will be 0.03:1 . this will have a similar effect as the d455n mutation that we have found previously leads to a 1.2 ph unit decrease in the pka value of cys ( from 10.4 to 9.2 at 313 k ) ( 15 ) . therefore , we conclude that when ndsbd and cdsbd form a complex , a reduction in the pka of cys by at least 1.2 ph units to a value of 9.2 will take place . the concentration of the thiolate form of cys will increase 16-fold at physiological ph making this cysteine a better nucleophile . this experimental result is broadly consistent with previous electrostatic calculations carried out using x - ray structures , which predicted pka values of 4.8 and 9.9 for asp in the isolated cdsbd and the ndsbd - cdsbd complex , respectively ( 10 ) . it should be noted that such calculations are extremely sensitive to small differences in x - ray structures ; the program propka gives predicted pka values for asp of 9.6 , 7.0 , and 7.7 using the three ndsbd - cdsbd molecules in the asymmetric unit ( 23 ) . a pka value of 5.4 is predicted by propka for asp in isolated reduced cdsbd . the increase in the predicted pka value for asp in the complex arises from an increase in the desolvation of the side chain of asp and from an increased charge - charge contribution from glu as this residue becomes more buried in the complex ( 23 ) . the side chains of cys and asp are in close proximity in the active site of cdsbd . these residues are homologous to cys and asp in thioredoxin ; microscopic pka values of 7.5 and 9.2 must be invoked to explain their titration behavior in thioredoxin ( 24 , 25 ) . if asp titrates first with a pka of 7.5 , then cys in the same molecule will have a pka value of 9.2 . conversely , if cys titrates with a pka of 7.5 , then asp will have an elevated pka of 9.2 . in isolated and reduced cdsbd microscopic pka values are not relevant because the measured pka values of 6.5 and 10.4 for asp and cys , respectively , at 313 k differ so significantly ( 15 ) . however , when cdsbd comes into contact with ndsbd , and the pka value of asp is raised to 8.5 , microscopic pka values may need to be considered . in this case , if asp titrates first with a pka of 8.5 , then cys in the same molecule will have a pka of 10.4 . however , if cys titrates first it will have a pka of 8.5 , and asp will have a pka of 10.4 . in the latter case , cys is significantly more reactive than in isolated cdsbd ( 80-fold increase in the concentration of the thiolate form of cys ) . this proposed lowering of the cys pka can not be confirmed experimentally by nmr because ndsbd and cdsbd do not form at stable high affinity complex in the absence of a mixed disulfide involving cys . the elevated pka value of 10.4 for cys in isolated cdsbd has the advantage of protecting cdsbd from nonspecific reoxidation , for example , by the powerful periplasmic oxidase dsba . when cdsbd and ndsbd interact , the pka value of cys is lowered leading to an increase in the nucleophilic character of this residue . asp is only able to exert this control over the reactivity of cys in cdsbd because it is buried and already has a relatively high pka value ( 6 ) in the isolated domain . thus , modulation of active - site pka values resulting from changes in the environment of the active site is key to the specificity of cdsbd . similar perturbations to pka values may occur in the active sites of other thioredoxin - like oxidoreductases . a general acid / base catalytic mechanism has been proposed for e. coli thioredoxin on the basis of pka values measured for the isolated protein ( 26 ) . in the final step of the mechanism , the deprotonated carboxyl group of asp ( which is the structural counterpart of asp in cdsbd ) is proposed to abstract a proton from the thiol of cys , either directly or via a bridging water molecule ( 27 ) , and the resulting thiolate attacks the mixed disulfide to form the reduced substrate and oxidized thioredoxin . a similar role for the analogous asp / glu is suggested in other thiol : disulfide oxidoreductases ( 26 ) . the pka value of 6.5 measured for asp in isolated cdsbd suggests it could play a similar role . however , the elevated pka value of 8.5 for asp in the ndsbd - cdsbd complex makes this role unlikely . other acidic groups in proximity to the thiol group of cys , such as glu , may play this role in cdsbd . an alternative mechanism for thioredoxin has been proposed in which the thiolate group in the substrate protein abstracts the proton from cys ( 28 , 29 ) ; this mechanism would also depend on the pka value of the substrate thiol in the complex . our results highlight the importance of considering the effects of protein - protein interactions in physiological complexes on the properties of active - site residues rather than simply considering protein domains in isolation .

the bacterial protein dsbd transfers reductant from the cytoplasm to the otherwise oxidizing environment of the periplasm . this reducing power is required for several essential pathways , including disulfide bond formation and cytochrome c maturation . dsbd includes a transmembrane domain ( tmdsbd ) flanked by two globular periplasmic domains ( ndsbd / cdsbd ) ; each contains a cysteine pair involved in electron transfer via a disulfide exchange cascade . the final step in the cascade involves reduction of the cys103-cys109 disulfide of ndsbd by cys461 of cdsbd . here we show that a complex between the globular periplasmic domains is trapped in vivo only when both are linked by tmdsbd . we have found previously ( mavridou , d. a. , stevens , j. m. , ferguson , s. j. , & redfield , c. ( 2007 ) j. mol . biol.370 , 643 -65817544440 ) that the attacking cysteine ( cys461 ) in isolated cdsbd has a high pka value ( 10.5 ) that makes this thiol relatively unreactive toward the target disulfide in ndsbd . here we show using nmr that active - site pka values change significantly when cdsbd forms a complex with ndsbd . this modulation of pka values is critical for the specificity and function of cdsbd . uncomplexed cdsbd is a poor nucleophile , allowing it to avoid nonspecific reoxidation ; however , in complex with ndsbd , the nucleophilicity of cdsbd increases permitting reductant transfer . the observation of significant changes in active - site pka values upon complex formation has wider implications for understanding reactivity in thiol : disulfide oxidoreductases .

EXPERIMENTAL PROCEDURES RESULTS DISCUSSION Supplementary Material

after removal of the excess alkylating reagent , the protein was cleaved using the thrombin clean - cleave kit according to the manufacturer 's instructions ( sigma ) . characterization of the ndsbd - cdsbd mixed disulfide formed in vivo the covalent complex between ndsbd and cdsbd was n - terminally sequenced from protein samples in sds - polyacrylamide gels that were electrophoretically transferred to a polyvinylidene difluoride membrane and stained with coomassie brilliant blue . ph titration of the ndsbd - n - cdsbd mixed disulfide using nmr spectroscopy the h and n resonances of c464a - cdsbd as a separate domain and in the ndsbd - n - cdsbd mixed disulfide were assigned using uniformly n - labeled samples of 0.8 mm protein for the isolated domains and 0.5 mm protein for the mixed disulfide , in 95% h2o , 5% d2o at ph 6.5 . nmr experiments for the determination of the pka values of asp in the active - site of cdsbd in the ndsbd - n - cdsbd mixed disulfide were performed using 0.5 mm of complex in 95% h2o , 5% d2o . determination of the pka values of asp in the active - site of n - labeled wild - type cdsbd and c464a - cdsbd was done in the same way as a control experiment . in the case of the n - labeled wild - type cdsbd , the sample was a 1:1 mixture of oxidized and reduced protein allowing simultaneous measurement of the pka value for the two oxidation states . lanes 1 , 4 , 9 , and 12 show molecular mass markers ( from the top these are 49 , 38 , 28 , and 14 kda ) ; lanes 2 and 10 show the negative control ( ndsbd with no affinity tag ) ; lanes 3 and 11 show the positive control ( cdsbd bearing a c - terminal his6 tag ) . the cdsbd band can be seen at 14 kda in lanes 3 and 11 , and in 6 - 8 and 14 - 16 in box iii ( this construct runs as a diffuse double band on sds - page because its pelb signal sequence , which targets the protein to the periplasm , is cleaved inefficiently by the signal peptidase , leaving a large fraction of the protein uncleaved ( confirmed by mass spectrometry and n - terminal sequencing ) ) . the uncleaved c464a thrombin - cleavable dsbd band can be seen at 49 kda ( the actual mass of the protein is 60.8 kda ; a difference between the two masses is common for membrane proteins ) in lanes 5 - 8 and 13 - 16 ( box i ) along with another band at 28 kda that is a contaminant commonly seen after the purification of this protein . lanes 1 , 4 , 9 , and 12 show molecular mass markers ( from the top these are 49 , 38 , 28 , and 14 kda ) ; lanes 2 and 10 show the negative control ( ndsbd with no affinity tag ) ; lanes 3 and 11 show the positive control ( cdsbd bearing a c - terminal his6 tag ) . c464a thrombin - cleavable dsbd is shown in lanes 5 and 13 before alkylation of free thiols ( a ) and thrombin cleavage ( t ) , in lanes 6 and 14 without alkylation and after thrombin cleavage , in lanes 7 and 15 after alkylation and thrombin cleavage , and in lanes 8 and 16 after alkylation and thrombin cleavage in the presence of reductant ( r ) . the cdsbd band can be seen at 14 kda in lanes 3 and 11 , and in 6 - 8 and 14 - 16 in box iii ( this construct runs as a diffuse double band on sds - page because its pelb signal sequence , which targets the protein to the periplasm , is cleaved inefficiently by the signal peptidase , leaving a large fraction of the protein uncleaved ( confirmed by mass spectrometry and n - terminal sequencing ) ) . the uncleaved c464a thrombin - cleavable dsbd band can be seen at 49 kda ( the actual mass of the protein is 60.8 kda ; a difference between the two masses is common for membrane proteins ) in lanes 5 - 8 and 13 - 16 ( box i ) along with another band at 28 kda that is a contaminant commonly seen after the purification of this protein . ph titration of [ 3-c]cysteine - labeled wild type and d455n - cdsbd h - c hsqc experiments for the determination of the pka value of cys at 313 k were performed on solutions containing 1.5 mm of [ 3-c]cysteine - labeled wild - type or d455n cdsbd in 99% d2o as described previously at 298 k ( 15 ) . errors in the pka values were estimated as the standard deviation from the mean pka value . in vivo studies of the formation of an ndsbd - cdsbd complex in full - length dsbd we have used a thrombin - cleavable variant of dsbd ( fig . the c464a mutation in cdsbd was introduced in an attempt to trap the ndsbd - cdsbd mixed - disulfide complex because cys is believed to be responsible for driving the cleavage of the interdomain disulfide and the final transfer of reductant from cdsbd to ndsbd ( 12 ) . 2 , lanes 8 and 16 ) , confirming that it is a covalent complex containing a disulfide bond . 2 , lanes 6 and 7 or 14 and 15 , the band corresponding to the complex is present whether or not the free thiols of the protein were alkylated before thrombin cleavage . 2 , lanes 6 - 8 and 14 - 16 , box i ) ; this is attributed to inefficient thrombin cleavage because of the presence of the detergent ddm in the protein solutions . it is also likely that the presence of the protein in the detergent micelle could reduce the accessibility of the protease cleavage sites to the thrombin ( 21 ) . this is observed because there are molecules in the protein sample in which the ndsbd - cdsbd mixed disulfide had not formed and for which the thrombin cleavage led to release of the two separate periplasmic domains ( isolated ndsbd can not be seen in the western blot because it is not his6-tagged ) . in vivo studies of complex formation between isolated ndsbd and cdsbd to assess the role of tmdsbd in mediating the interaction between ndsbd and cdsbd , the two periplasmic domains of dsbd , ndsbd bearing a c - terminal streptavidin tag and a c464a variant of cdsbd bearing a c - terminal his6 tag , were overexpressed simultaneously as soluble proteins exported to the periplasm . a possible explanation for the failure of the ndsbd - cdsbd mixed - disulfide complex to form in vivo is that the proteins were present in the incorrect oxidation states for disulfide bond formation to occur ; interdomain disulfide bond formation requires the presence of oxidized ndsbd and reduced c464a - cdsbd . for these reasons , attack of the cdsbd cysteine thiol on the disulfide of ndsbd , we therefore conclude that a complex between soluble ndsbd and cdsbd , co - expressed in the e. coli periplasm , does not form in the absence of the transmembrane domain that usually connects the two domains in the full - length protein . nmr analysis of the ndsbd - n - cdsbd mixed - disulfide complex a mixed - disulfide complex was produced using purified single - cysteine variants of ndsbd ( c103a ) and cdsbd ( c464a ) to ensure that the physiologically relevant disulfide was formed ( cys - ss - cys ) , to avoid any cleavage of this disulfide because of the presence of cys and to eliminate any complications during the pka determination of asp caused by the titration of the cys or cys thiols in the active site . nmr spectroscopy allows measurement of the pka value of asp in the active site of cdsbd when cdsbd is in close proximity to its periplasmic partner ndsbd . for this purpose we used uniformly n - labeled c464a - cdsbd covalently linked to unlabeled c103a - ndsbd , thus producing a c103a - ndsbd - n - c464a - cdsbd covalent complex ( subsequently referred to as ndsbd - n - cdsbd ) where only cdsbd gives nmr signals in the h - n hsqc spectrum but in which it is influenced by the presence of ndsbd . the ph titration experiments described below were therefore performed at 313 k. pka determination for asp in the ndsbd- n - cdsbd complex we have monitored , between ph 4 and ph 10 , the n and h chemical shifts of the backbone amides of asp and glu and the side chain n of gln in hsqc spectra of the ndsbd - n - cdsbd mixed - disulfide complex ( fig . our previous study of cdsbd was carried out at 298 k , whereas the spectra of the complex were collected at 313 k. as a control , we have monitored the same chemical shifts in uniformly n - labeled wild - type cdsbd at 313 k. the experiment was performed , as before ( 15 ) , in a mixed oxidation state sample , and we obtained a pka value of 6.5 0.1 in the reduced state and of 7.0 0.1 for asp in the oxidized state ( fig . to ensure that the difference in the pka values of asp between complexed and uncomplexed cdsbd is not an effect of the c464a mutation , we have also performed the titration for uniformly n - labeled c464a - cdsbd at 313 k and obtained an average pka value of 7.4 0.1 for asp ( fig . therefore , it can be concluded that the pka value of asp is elevated by at least 1.1 ph units when cdsbd forms a complex with ndsbd . figure 5.measurement of the pka value of the active - site residue asp from the ph dependence of the n chemical shift of asp ( a ) , the n chemical shift of glu ( b ) , and the n chemical shift of gln ( c ) . the fitting procedure has been described previously ( 15 ) ; see supplemental table s1 for a summary of the fitted parameters . measurement of the pka value of the active - site residue asp from the ph dependence of the n chemical shift of asp ( a ) , the n chemical shift of glu ( b ) , and the n chemical shift of gln ( c ) . the fitting procedure has been described previously ( 15 ) ; see supplemental table s1 for a summary of the fitted parameters . pka determination for cys at 313 k the pka value for cys was measured for wild - type cdsbd and for the d455n variant of cdsbd at 313 k for direct comparison with the pka values measured above for asp in the ndsbd - n - cdsbd complex at 313 k. analysis of the ph dependence of the c and h chemical shifts of cys and cys in cdsbd gives pka values of 10.4 0.1 and 9.2 0.2 for cys in wild - type and d455n cdsbd , respectively ( supplemental fig . these values agree within experimental error with the values of 10.5 0.1 and 9.3 0.05 reported previously at 298 k. therefore , the pka value of cys does not show a temperature dependence . it has long been known that dsbd functions by interaction between the cysteine pairs in its three domains , although how these interactions occur within this protein , in which one of the three domains is membrane - embedded , has remained elusive . in this study we have used both in vivo and in vitro approaches to understand how the interaction and reaction between the two soluble domains are controlled . we have demonstrated using western blotting that a covalent ndsbd - cdsbd complex can be trapped in vivo when full - length dsbd is overexpressed . the fact that the ndsbd - cdsbd complex was not detectable in the absence of the linking tmdsbd has important implications for the function of the membrane - embedded domain . therefore , the transmembrane domain may be essential to ensure the high local concentration of the domains that would be required for the formation of a complex and subsequent electron transfer via the disulfide exchange reaction . in isolated cdsbd , cys , which catalyzes reduction of the cys - cys disulfide in ndsbd , has a pka value of 10.4 at 313 k ; this elevated value makes cys a poor nucleophile . the high pka value for cys results from its close proximity to asp and glu in the active site . here we have used the covalent ndsbd - n - cdsbd complex to measure the pka value of asp in cdsbd when it is in close contact with ndsbd . we found that the pka value of asp is elevated by at least 1.1 ph units to a value of 8.5 in the complex at 313 k. at ph 7.0 , the ratio of deprotonated ( negatively charged ) to protonated ( uncharged ) asp in the complex will be 0.03:1 . this will have a similar effect as the d455n mutation that we have found previously leads to a 1.2 ph unit decrease in the pka value of cys ( from 10.4 to 9.2 at 313 k ) ( 15 ) . therefore , we conclude that when ndsbd and cdsbd form a complex , a reduction in the pka of cys by at least 1.2 ph units to a value of 9.2 will take place . this experimental result is broadly consistent with previous electrostatic calculations carried out using x - ray structures , which predicted pka values of 4.8 and 9.9 for asp in the isolated cdsbd and the ndsbd - cdsbd complex , respectively ( 10 ) . the increase in the predicted pka value for asp in the complex arises from an increase in the desolvation of the side chain of asp and from an increased charge - charge contribution from glu as this residue becomes more buried in the complex ( 23 ) . however , when cdsbd comes into contact with ndsbd , and the pka value of asp is raised to 8.5 , microscopic pka values may need to be considered . in the latter case , cys is significantly more reactive than in isolated cdsbd ( 80-fold increase in the concentration of the thiolate form of cys ) . the elevated pka value of 10.4 for cys in isolated cdsbd has the advantage of protecting cdsbd from nonspecific reoxidation , for example , by the powerful periplasmic oxidase dsba . when cdsbd and ndsbd interact , the pka value of cys is lowered leading to an increase in the nucleophilic character of this residue . asp is only able to exert this control over the reactivity of cys in cdsbd because it is buried and already has a relatively high pka value ( 6 ) in the isolated domain . thus , modulation of active - site pka values resulting from changes in the environment of the active site is key to the specificity of cdsbd . a general acid / base catalytic mechanism has been proposed for e. coli thioredoxin on the basis of pka values measured for the isolated protein ( 26 ) . in the final step of the mechanism , the deprotonated carboxyl group of asp ( which is the structural counterpart of asp in cdsbd ) is proposed to abstract a proton from the thiol of cys , either directly or via a bridging water molecule ( 27 ) , and the resulting thiolate attacks the mixed disulfide to form the reduced substrate and oxidized thioredoxin . a similar role for the analogous asp / glu is suggested in other thiol : disulfide oxidoreductases ( 26 ) . the pka value of 6.5 measured for asp in isolated cdsbd suggests it could play a similar role . however , the elevated pka value of 8.5 for asp in the ndsbd - cdsbd complex makes this role unlikely . an alternative mechanism for thioredoxin has been proposed in which the thiolate group in the substrate protein abstracts the proton from cys ( 28 , 29 ) ; this mechanism would also depend on the pka value of the substrate thiol in the complex . our results highlight the importance of considering the effects of protein - protein interactions in physiological complexes on the properties of active - site residues rather than simply considering protein domains in isolation .

abdominal aortic aneurysm ( aaa ) affects 25% of men and < 1% of women aged 6574 years . progressive dilatation of the aorta increases the risk of rupture and surgical intervention is indicated when the diameter reaches 5.5 cm . currently there are no recognized treatments to diminish aneurysm progression at an early stage and a pharmacological treatment that targeted the processes underlying aortic dilatation , as well as vascular disease in general would provide an attractive adjunct to the surveillance and surgery treatment paradigm . the major environmental exposures associated with aaa development include male sex , advancing age , cigarette smoking , dyslipidaemia , and hypertension . there is also a genetic component to the disease , with twin studies reporting heritability in the region of 70% . two recent genome - wide association studies ( gwas ) of aaa have reported robust associations with common variants in dab2ip and lrp1 , while the chromosome 9p21.3 locus associated with coronary heart disease ( chd ) also shows a strong association with aaa . these variants , however , explain only a small proportion of the observed heritability suggesting other contributory genetic mechanisms remain to be discovered . interleukin-6 ( il-6 ) signalling is initiated by binding of il-6 to its receptor ( il-6r ) , which forms a dimer with the ubiquitously expressed signal transducer glycoprotein-130 ( gp-130 ) . this , in turn leads to the activation of the intracellular receptor - associated kinases and downstream effects via the transcription factor stat3 . two forms of il-6 signalling have been described , namely classical and trans - signalling . in classical signalling , il-6 binds the membrane bound il-6r ( mil-6r ) , which is expressed in hepatocytes and cells of the innate immune system . in trans - signalling , il-6 binds to the circulating soluble il-6r ( sil-6r ) , and this complex is capable of binding to gp130 in a wide range of cell types . it has previously been shown that the expression of il-6 and downstream mediators of il-6 signalling , such as stat3 , are greater in aaa than in non - aneurysmal aortic tissue . a common non - synonymous sequence variant in il6r ( p.asp358ala , rs2228145 , also annotated as rs8192284 ) results in an increased proteolytic cleavage of the mil-6r . this variant is strongly associated with higher levels of circulating il-6 levels , but importantly , lower levels of downstream products of il-6 signalling , such as c - reactive protein and fibrinogen , suggesting that it acts by the attenuation of signalling via the il-6 receptor . furthermore , it was shown that this variant conferred protection against chd , prompting speculation that targeting the il-6r in chd is a potentially novel preventative strategy . whether or not this is the case for aaa has not yet been evaluated . in the present study , the primary hypothesis is that pro - inflammatory signalling via the il-6r plays a causal role in aaa development . first , a systematic review and meta - analysis of the published literature was performed in order to establish the relationship between circulating il-6 levels and aaa . secondly , novel analysis of the association between the functional asp358ala il6r variant and aaa was performed . finally , in vitro analyses were used to investigate the mechanism by which this variant could protect from cardiovascular disease . studies were identified using a two - stage search strategy following prisma guidelines ( supplementary material online ) . in the first stage , two electronic databases ( medline and embase ) were searched and in the second stage articles were identified by manually searching references of articles identified in the first stage and review articles . authors of large epidemiological studies of aaa were also approached to determine whether they had measures of il-6 in cases and controls . the medline database was searched from january 1966 to january 2012 , and the embase from 1980 to january 2012 . inclusion criteria were decided by consensus ( sch , rr , mvh , and seh ) , and the studies were screened and abstracted in duplicate by sch and rr . detailed descriptions of the study cohorts and demographic details are presented in the supplementary material online . briefly , for the genetic association analyses , we used data from five case control studies of aaa that have access to genetic information , and are part of an existing collaborative group investigating the genetics of aaa . all studies defined aaa as an infra - renal aortic diameter 3 cm by ultrasound or computed tomography imaging , or previous aaa rupture / repair . the aneurysm consortium ( ac ) genome wide association study of abdominal aortic aneurysm recruited 1596 cases of aaa from centres across the uk and western australia . control data were taken from the wellcome trust case control consortium ( n = 5855 ) . the new zealand study included 1373 individuals with aaa and 718 controls with no previous history of vascular disease . the secondary manifestations of arterial disease ( smart ) study included data from 631 cases of aaa and 6342 aaa free controls recruited from university medical center utrecht , the netherlands . the edinburgh artery study ( eas ) is a prospective population - based cohort that included data from 62 cases of aaa and 819 aaa free controls . in the utrectht study ( separate from the smart study ) , 862 individuals with aaa were recruited to the genetics aaa study and compared with controls from the ergo / rotterdam study ( n = 1866 ) . studies were identified as part of established collaborative efforts to understand the genetics of aaa . participants in the ac aaa gwas were genotyped using the illumina 660 k gene - chip , with previously described quality control filters . individuals in the smart study were genotyped using kaspar at kbiosciences , uk ( www.kbioscience.co.uk ) . for the new zealand study , samples were genotyped using the taqman allelic discrimination method ( using a pre - designed , functionally tested assay applied biosystems , assay c_26292282_10 ) . for the utrecht study , participants were genotyped using the illumina 610k chip . in the smart study these snps both tag the non - synonymous variant ( p.asp358ala , rs2228145 , r = 0.97 ) . linkage disequilibrium between snps was assessed using the snp annotation and proxy search resources ( www . epstein barr virus - transformed lymphoblastoid cell lines were used based on the genotype , confirmed using taqman allelic discrimination ( applied biosystems ) . after rna extraction , gene expression was analysed using taqman gene expression assays designed for targeting stat3 , myc , atf3 , icam1 , and bcl3 ( applied biosystems , carlsbad , usa ) using a 384-well plate format , following the standard protocol provided by applied biosystems . these target genes were chosen because they have been shown to be the major downstream target of il-6stat3 signalling . to estimate the association between circulating il-6 and aaa , we performed meta - analysis of summary statistics identified by our systematic review . for each study , we used mean concentrations of il-6 in cases and controls to determine a standardized mean difference ( smd ) and standard error . for studies that reported median concentration levels and inter - quartile ranges ( iqr ) , we took the median to be the mean value and divided the iqr by 1.35 in order to obtain the sds . mean differences in each study were pooled using inverse - weighted - fixed - effect meta - analysis . heterogeneity was assessed using the i statistic and cochrane 's q. owing to heterogeneity in units used to measure il-6 , and baseline levels , a smd was calculated preferentially to a weighted mean difference . snp - disease associations in each cohort were determined using logistic regression adjusted for age and gender , under an additive genetic model . meta - analysis was performed using fixed and random - effects modelling and we measured between study heterogeneity using the i statistic . gene expression was analysed using the rest expression software tool by mw pfaffl utilizing a pair - wise fixed reallocation randomization test . with 4523 case and 15 406 controls , there was over 80% to detect modest effects ( or > 1.12 ) on disease risk with a type 1 error rate of 1 in 1000 . studies were identified using a two - stage search strategy following prisma guidelines ( supplementary material online ) . in the first stage , two electronic databases ( medline and embase ) were searched and in the second stage articles were identified by manually searching references of articles identified in the first stage and review articles . authors of large epidemiological studies of aaa were also approached to determine whether they had measures of il-6 in cases and controls . the medline database was searched from january 1966 to january 2012 , and the embase from 1980 to january 2012 . inclusion criteria were decided by consensus ( sch , rr , mvh , and seh ) , and the studies were screened and abstracted in duplicate by sch and rr . detailed descriptions of the study cohorts and demographic details are presented in the supplementary material online . briefly , for the genetic association analyses , we used data from five case control studies of aaa that have access to genetic information , and are part of an existing collaborative group investigating the genetics of aaa . all studies defined aaa as an infra - renal aortic diameter 3 cm by ultrasound or computed tomography imaging , or previous aaa rupture / repair . the aneurysm consortium ( ac ) genome wide association study of abdominal aortic aneurysm recruited 1596 cases of aaa from centres across the uk and western australia . control data were taken from the wellcome trust case control consortium ( n = 5855 ) . the new zealand study included 1373 individuals with aaa and 718 controls with no previous history of vascular disease . the secondary manifestations of arterial disease ( smart ) study included data from 631 cases of aaa and 6342 aaa free controls recruited from university medical center utrecht , the netherlands . the edinburgh artery study ( eas ) is a prospective population - based cohort that included data from 62 cases of aaa and 819 aaa free controls . in the utrectht study ( separate from the smart study ) , 862 individuals with aaa were recruited to the genetics aaa study and compared with controls from the ergo / rotterdam study ( n = 1866 ) . studies were identified as part of established collaborative efforts to understand the genetics of aaa . participants in the ac aaa gwas were genotyped using the illumina 660 k gene - chip , with previously described quality control filters . individuals in the smart study were genotyped using kaspar at kbiosciences , uk ( www.kbioscience.co.uk ) . for the new zealand study , samples were genotyped using the taqman allelic discrimination method ( using a pre - designed , functionally tested assay applied biosystems , assay c_26292282_10 ) . for the utrecht study , participants were genotyped using the illumina 610k chip . in the smart study , rs7529229 was genotyped and in the new zealand study rs4129267 was genotyped . these snps both tag the non - synonymous variant ( p.asp358ala , rs2228145 , r = 0.97 ) . linkage disequilibrium between snps was assessed using the snp annotation and proxy search resources ( www . epstein barr virus - transformed lymphoblastoid cell lines were used based on the genotype , confirmed using taqman allelic discrimination ( applied biosystems ) . after rna extraction , gene expression was analysed using taqman gene expression assays designed for targeting stat3 , myc , atf3 , icam1 , and bcl3 ( applied biosystems , carlsbad , usa ) using a 384-well plate format , following the standard protocol provided by applied biosystems . these target genes were chosen because they have been shown to be the major downstream target of il-6stat3 signalling . to estimate the association between circulating il-6 and aaa , we performed meta - analysis of summary statistics identified by our systematic review . for each study , we used mean concentrations of il-6 in cases and controls to determine a standardized mean difference ( smd ) and standard error . for studies that reported median concentration levels and inter - quartile ranges ( iqr ) , we took the median to be the mean value and divided the iqr by 1.35 in order to obtain the sds . mean differences in each study were pooled using inverse - weighted - fixed - effect meta - analysis . heterogeneity was assessed using the i statistic and cochrane 's q. owing to heterogeneity in units used to measure il-6 , and baseline levels , a smd was calculated preferentially to a weighted mean difference . snp - disease associations in each cohort were determined using logistic regression adjusted for age and gender , under an additive genetic model . meta - analysis was performed using fixed and random - effects modelling and we measured between study heterogeneity using the i statistic . gene expression was analysed using the rest expression software tool by mw pfaffl utilizing a pair - wise fixed reallocation randomization test . with 4523 case and 15 406 controls , there was over 80% to detect modest effects ( or > 1.12 ) on disease risk with a type 1 error rate of 1 in 1000 . seven studies reporting circulating levels of plasma il-6 in aaa cases and controls were identified ( supplementary material online , figure s1 ) , including one previous unpublished study ( the nz study ) . two studies that reported higher il-6 in cases than controls , but did not report summary measures of il-6 ( mean and sd or median and iqr ) were not included in the meta - analysis . details of the studies included in the meta - analysis are provided in table 1 . all identified studies utilized a case control design and used recognized methods to diagnose and define the presence of aaa and to measure il-6 levels . all but one study selected controls that were matched for age and gender , but only one study matched for smoking status , but none specifically adjusted for these factors in the primary analyses . an assessment of the quality of the case control studies is provided in supplementary material online , table s1 . table 1characteristics of case control studies included in the meta - analysis of il-6 levels in abdominal aortic aneurysms cases and controlsauthoryearcountrystudy designcases / controlscase definition controlsdiagnosisil-6 cases , mean ( sd)il-6 controls , mean ( sd)treska2003czech republiccase control74/30small ( < 5 cm ) = 30 large ( 58 cm ) = 38 very large ( > 8 cm ) = 22 ruptured / symptomatic = 16**matched for age and gender no history of atherosclerosisuss59.3 ( 134.8)6.7 ( 5.1)fowkes et al.2006ukcase control89/98ap aortic diameter > 3 cm , size distribution nrnormal uss ( <3 cm ) matched for age and gender . control within the edinburgh artery studyuss2.8 ( 1.62)1.8 ( 1.04)dawson et al.2007ukcase - control27/15large aaa undergoing endovascular repairundergoing other vascular interventionct4.94 ( 2.49)2.65 ( 1.98)flondell - site2009swedencase control360/218small ( < 4.5 cm ) = 122 medium(4.55.5 cm ) = 108 large ( > 5.5 cm ) = 130age and sex matched no history of atherosclerosis / aaa undergoing routine preventative checksct / uss9.19 ( 31.91)2.08 ( 2.90)jones ( unpublished)2012new zealandcase control166/359all greater than 5 cm , awaiting repairmatched for age , free from atherosclerosis and aaa . recruited from screening programme in nz.ct/uss9.1 ( 6.60)6.3 ( 3.93)parry et al.2010ukcase control75/90all aaa < 5.5 cmmatched for age , gender , and race recruited from surgical clinicsuss3.13 ( 2.87)3.14 ( 2.32)wallinder et al.2009swedencase control case control78/41small ( < 5 cm ) = 38 awaiting elective repair ( > 5 cm ) = 40matched for age , gender , and smoking status volunteers with infra - renal aortic diameter <3 cmuss4.02 ( 3.79)2.3 ( 3.3)**values from ruptures not included in the meta - analysis . characteristics of case control studies included in the meta - analysis of il-6 levels in abdominal aortic aneurysms cases and controls * * values from ruptures not included in the meta - analysis . in meta - analysis , pooling data from 869 cases and 851 controls , individuals with aaa had higher circulating il-6 concentrations than controls ; smd = 0.46 , 95% ci : 0.250.66 , p random effects model = 1 . 1 10 , i = 70% . concentrations of il-6 were higher in cases than in controls in all but one of the studies in which no difference was observed . this study included only small aaa in the case group ( 35.5 cm ) , and reported different measurement units to all the other studies ( ng / ml vs. pg / ml ) . exclusion of this study reduced overall heterogeneity ( i = 56% ) and increased the statistical significance of the association ( p random effects = 2 10 ) ; however , this study did not overly influence the overall effect estimate greatly ( sensitivity analysis presented in the supplementary material online , figure s2 ) . for example , there is evidence that il-6 concentrations are correlated with the aaa size and subgroups analyses demonstrated that when the analysis was restricted to studies that compared il-6 in large aaa compared with controls , there was less heterogeneity ( i = 31.5% , figure 1 ) . furthermore , there was variation in how controls were selected , e.g. from distinct cohorts such as the general population or from surgical outpatients clinics , which is likely to contribute to heterogeneity . there was no evidence of publication bias using begg 's adjusted rank correlation test ( p = 0.7 ) . figure 1association between circulating il-6 levels and the presence of abdominal aortic aneurysms . in pooled analysis of seven studies reporting il-6 circulations in cases and controls ( 869/851 ) there was consistently higher concentrations in cases ( smd = 0.46 , 95% ci : 0.250.66 , p random effects model = 1 . 1 10 , i = 70% ) . subgroup analyses , comparing il-6 concentrations in large abdominal aortic aneurysms ( > 5 cm ) demonstrated less heterogeneity ( smd = 0.46 , 95% ci : 0.340.58 , p = 2.25 10 , i = 32% ) . in pooled analysis of seven studies reporting il-6 circulations in cases and controls ( 869/851 ) there was consistently higher concentrations in cases ( smd = 0.46 , 95% ci : 0.250.66 , p random effects model = 1 . 1 10 , i = 70% ) . subgroup analyses , comparing il-6 concentrations in large abdominal aortic aneurysms ( > 5 cm ) demonstrated less heterogeneity ( smd = 0.46 , 95% ci : 0.340.58 , p = 2.25 10 , i = 32% ) . demographic details of the five case controls studies are shown in table 2 and genotyping quality control measures are shown in table 3 . there was a consistent association between the rare allele of rs7529229 ( tagging ala358 ) and reduced risk of aaa in all cohorts . meta - analysis , pooling data from 4524 cases and 15 710 controls demonstrated a per - allele or of 0.84 ( 95% ci : 0.800.89 , p = 2.7 10 , i = 0% fixed - effect meta - analysis ) ( figure 2 ) . in time - to - event analyses of prospective data from the smart study , using cox proportional hazard models , adjusted for age and gender , the rare ala358 allele was also associated with a reduction in the risk of aaa clinical endpoints ( aaa rupture , repair , nevents = 223 ) ( figure 3 , per allele hr : 0.81 , 95% ci : 0.670.99 , p = 0.043 ) . table 2demographic details of cohorts used for gene association analysiscontrolscasessample setncontrols% femaleage ( sd)ncases% femaleage ( sd)ac585550na15964nasmart63523355 ( 12)6312265 ( 10)nz7182370 ( 6.6)13732074 ( 8.0)eas8194764 ( 5.6)6266 ( 5.6)74utrecht186656568621068.1 ( 8.3 ) table 3genotyping metrics from each of the studies ( hardy weinberg equilibrium)studygenotyping platformminor allele frequencycall rate ( % ) hwe , p - valueacillumina 670 k beadchip0.39>990.28new zealandabi taqman0.40>980.89smartkaspar0.39>970.77easabi taqman0.42>970.08utrechtillumina 610 k0.381000.97 figure 2association between rs7529229 and abdominal aortic aneurysms following fixed - effect meta - analysis of four case control studies ( 4524 cases/15 710 controls ) . per allele odds ratio = 0.84 ( 95% ci : 0.800.89 , i = 0 , p = 2.7 10 ) . figure 3time to event analysis curves showing the probability of abdominal aortic aneurysm - related endpoint ( rupture / endovascular repair / open repair ) by genotype at rs7529229 ( cc is the rare homozygote ) in 7139 individuals from the smart study , in who there were 223 events . the hazard ratio per c - allele is 0.81 , 95% ci = 0.670.99 , p = 0.043 . ) the p - value was calculated using cox regression , adjusting for age and gender . demographic details of cohorts used for gene association analysis genotyping metrics from each of the studies ( hardy weinberg equilibrium ) association between rs7529229 and abdominal aortic aneurysms following fixed - effect meta - analysis of four case control studies ( 4524 cases/15 710 controls ) . per allele odds ratio = 0.84 ( 95% ci : 0.800.89 , i = 0 , p = 2.7 10 ) . time to event analysis curves showing the probability of abdominal aortic aneurysm - related endpoint ( rupture / endovascular repair / open repair ) by genotype at rs7529229 ( cc is the rare homozygote ) in 7139 individuals from the smart study , in who there were 223 events . the hazard ratio per c - allele is 0.81 , 95% ci = 0.670.99 , p = 0.043 . ) the p - value was calculated using cox regression , adjusting for age and gender . the aim of these analyses was to determine the downstream effects of the asp358ala variant on the expression of il-6-mediated target genes identified in previous studies , namely stat3 , myc , bcl3 , icam1 , and atf3 . in lymphoblastoid cells , no difference in the expression of stat3 targets was observed at baseline ( figure 4a ) . after il-6 stimulation , there was a reduction in the expression of stat3 , myc , icam1 in ala358 homozygotes ( cc ) compared with asp358 homozygotes ( aa ) ( figure 4b ) . this genotype - dependent difference was abolished by the presence of excess sil-6r in the media ( figure 4c ) , suggesting that the effect observed with il-6 stimulation alone is via reducing signalling through the m - il6r is the presence of ala358 . figure 4gene expression in stat3 target genes in lymphoblast cell lines of different il6r rs2228145 ( aa = common homozygote ; cc , rare homozygote ) . ( a ) basal gene expression in lymphoblast cell lines shows no difference in gene expression between il6r genotypes . ( b ) gene expression following il-6 stimulation ; lower gene expression was observed in all stat3 target genes in lymphoblasts possessing the c allele . ( c ) gene expression following il-6 stimulation and addition of excess sil-6r . to examine if the effect seen in ( b ) was due to receptor shedding , excess soluble il-6r was added to the stimulated cells , suggesting that the mechanism is of genotype - specific reduction in stat3 targets is the result of increased shedding of the mil-6r . gene expression in stat3 target genes in lymphoblast cell lines of different il6r rs2228145 ( aa = common homozygote ; cc , rare homozygote ) . ( a ) basal gene expression in lymphoblast cell lines shows no difference in gene expression between il6r genotypes . ( b ) gene expression following il-6 stimulation ; lower gene expression was observed in all stat3 target genes in lymphoblasts possessing the c allele . ( c ) gene expression following il-6 stimulation and addition of excess sil-6r . to examine if the effect seen in ( b ) was due to receptor shedding , excess soluble il-6r was added to the stimulated cells , suggesting that the mechanism is of genotype - specific reduction in stat3 targets is the result of increased shedding of the mil-6r . seven studies reporting circulating levels of plasma il-6 in aaa cases and controls were identified ( supplementary material online , figure s1 ) , including one previous unpublished study ( the nz study ) . two studies that reported higher il-6 in cases than controls , but did not report summary measures of il-6 ( mean and sd or median and iqr ) were not included in the meta - analysis . details of the studies included in the meta - analysis are provided in table 1 . all identified studies utilized a case control design and used recognized methods to diagnose and define the presence of aaa and to measure il-6 levels . all but one study selected controls that were matched for age and gender , but only one study matched for smoking status , but none specifically adjusted for these factors in the primary analyses . an assessment of the quality of the case control studies is provided in supplementary material online , table s1 . table 1characteristics of case control studies included in the meta - analysis of il-6 levels in abdominal aortic aneurysms cases and controlsauthoryearcountrystudy designcases / controlscase definition controlsdiagnosisil-6 cases , mean ( sd)il-6 controls , mean ( sd)treska2003czech republiccase control74/30small ( < 5 cm ) = 30 large ( 58 cm ) = 38 very large ( > 8 cm ) = 22 ruptured / symptomatic = 16**matched for age and gender no history of atherosclerosisuss59.3 ( 134.8)6.7 ( 5.1)fowkes et al.2006ukcase control89/98ap aortic diameter > 3 cm , size distribution nrnormal uss ( <3 cm ) matched for age and gender . control within the edinburgh artery studyuss2.8 ( 1.62)1.8 ( 1.04)dawson et al.2007ukcase - control27/15large aaa undergoing endovascular repairundergoing other vascular interventionct4.94 ( 2.49)2.65 ( 1.98)flondell - site2009swedencase control360/218small ( < 4.5 cm ) = 122 medium(4.55.5 cm ) = 108 large ( > 5.5 cm ) = 130age and sex matched no history of atherosclerosis / aaa undergoing routine preventative checksct / uss9.19 ( 31.91)2.08 ( 2.90)jones ( unpublished)2012new zealandcase control166/359all greater than 5 cm , awaiting repairmatched for age , free from atherosclerosis and aaa . recruited from screening programme in nz.ct/uss9.1 ( 6.60)6.3 ( 3.93)parry et al.2010ukcase control75/90all aaa < 5.5 cmmatched for age , gender , and race recruited from surgical clinicsuss3.13 ( 2.87)3.14 ( 2.32)wallinder et al.2009swedencase control case control78/41small ( < 5 cm ) = 38 awaiting elective repair ( > 5 cm ) = 40matched for age , gender , and smoking status volunteers with infra - renal aortic diameter <3 cmuss4.02 ( 3.79)2.3 ( 3.3)**values from ruptures not included in the meta - analysis . characteristics of case control studies included in the meta - analysis of il-6 levels in abdominal aortic aneurysms cases and controls * * values from ruptures not included in the meta - analysis . in meta - analysis , pooling data from 869 cases and 851 controls , individuals with aaa had higher circulating il-6 concentrations than controls ; smd = 0.46 , 95% ci : 0.250.66 , p random effects model = 1 . 1 10 , i = 70% . concentrations of il-6 were higher in cases than in controls in all but one of the studies in which no difference was observed . this study included only small aaa in the case group ( 35.5 cm ) , and reported different measurement units to all the other studies ( ng / ml vs. pg / ml ) . exclusion of this study reduced overall heterogeneity ( i = 56% ) and increased the statistical significance of the association ( p random effects = 2 10 ) ; however , this study did not overly influence the overall effect estimate greatly ( sensitivity analysis presented in the supplementary material online , figure s2 ) . for example , there is evidence that il-6 concentrations are correlated with the aaa size and subgroups analyses demonstrated that when the analysis was restricted to studies that compared il-6 in large aaa compared with controls , there was less heterogeneity ( i = 31.5% , figure 1 ) . furthermore , there was variation in how controls were selected , e.g. from distinct cohorts such as the general population or from surgical outpatients clinics , which is likely to contribute to heterogeneity . there was no evidence of publication bias using begg 's adjusted rank correlation test ( p = 0.7 ) . figure 1association between circulating il-6 levels and the presence of abdominal aortic aneurysms . in pooled analysis of seven studies reporting il-6 circulations in cases and controls ( 869/851 ) there was consistently higher concentrations in cases ( smd = 0.46 , 95% ci : 0.250.66 , p random effects model = 1 . 1 10 , i = 70% ) . subgroup analyses , comparing il-6 concentrations in large abdominal aortic aneurysms ( > 5 cm ) demonstrated less heterogeneity ( smd = 0.46 , 95% ci : 0.340.58 , p = 2.25 10 , i = 32% ) . in pooled analysis of seven studies reporting il-6 circulations in cases and controls ( 869/851 ) there was consistently higher concentrations in cases ( smd = 0.46 , 95% ci : 0.250.66 , p random effects model = 1 . 1 10 , i = 70% ) . subgroup analyses , comparing il-6 concentrations in large abdominal aortic aneurysms ( > 5 cm ) demonstrated less heterogeneity ( smd = 0.46 , 95% ci : 0.340.58 , p = 2.25 10 , i = 32% ) . demographic details of the five case controls studies are shown in table 2 and genotyping quality control measures are shown in table 3 . there was a consistent association between the rare allele of rs7529229 ( tagging ala358 ) and reduced risk of aaa in all cohorts . meta - analysis , pooling data from 4524 cases and 15 710 controls demonstrated a per - allele or of 0.84 ( 95% ci : 0.800.89 , p = 2.7 10 , i = 0% fixed - effect meta - analysis ) ( figure 2 ) . in time - to - event analyses of prospective data from the smart study , using cox proportional hazard models , adjusted for age and gender , the rare ala358 allele was also associated with a reduction in the risk of aaa clinical endpoints ( aaa rupture , repair , nevents = 223 ) ( figure 3 , per allele hr : 0.81 , 95% ci : 0.670.99 , p = 0.043 ) . table 2demographic details of cohorts used for gene association analysiscontrolscasessample setncontrols% femaleage ( sd)ncases% femaleage ( sd)ac585550na15964nasmart63523355 ( 12)6312265 ( 10)nz7182370 ( 6.6)13732074 ( 8.0)eas8194764 ( 5.6)6266 ( 5.6)74utrecht186656568621068.1 ( 8.3 ) table 3genotyping metrics from each of the studies ( hardy weinberg equilibrium)studygenotyping platformminor allele frequencycall rate ( % ) hwe , p - valueacillumina 670 k beadchip0.39>990.28new zealandabi taqman0.40>980.89smartkaspar0.39>970.77easabi taqman0.42>970.08utrechtillumina 610 k0.381000.97 figure 2association between rs7529229 and abdominal aortic aneurysms following fixed - effect meta - analysis of four case control studies ( 4524 cases/15 710 controls ) . per allele odds ratio = 0.84 ( 95% ci : 0.800.89 , i = 0 , p = 2.7 10 ) . figure 3time to event analysis curves showing the probability of abdominal aortic aneurysm - related endpoint ( rupture / endovascular repair / open repair ) by genotype at rs7529229 ( cc is the rare homozygote ) in 7139 individuals from the smart study , in who there were 223 events . the hazard ratio per c - allele is 0.81 , 95% ci = 0.670.99 , p = 0.043 . ) the p - value was calculated using cox regression , adjusting for age and gender . demographic details of cohorts used for gene association analysis genotyping metrics from each of the studies ( hardy weinberg equilibrium ) association between rs7529229 and abdominal aortic aneurysms following fixed - effect meta - analysis of four case control studies ( 4524 cases/15 710 controls ) . per allele odds ratio = 0.84 ( 95% ci : 0.800.89 , i = 0 , p = 2.7 10 ) . time to event analysis curves showing the probability of abdominal aortic aneurysm - related endpoint ( rupture / endovascular repair / open repair ) by genotype at rs7529229 ( cc is the rare homozygote ) in 7139 individuals from the smart study , in who there were 223 events . the hazard ratio per c - allele is 0.81 , 95% ci = 0.670.99 , p = 0.043 . ) the p - value was calculated using cox regression , adjusting for age and gender . the aim of these analyses was to determine the downstream effects of the asp358ala variant on the expression of il-6-mediated target genes identified in previous studies , namely stat3 , myc , bcl3 , icam1 , and atf3 . in lymphoblastoid cells , no difference in the expression of stat3 targets was observed at baseline ( figure 4a ) . after il-6 stimulation , there was a reduction in the expression of stat3 , myc , icam1 in ala358 homozygotes ( cc ) compared with asp358 homozygotes ( aa ) ( figure 4b ) . this genotype - dependent difference was abolished by the presence of excess sil-6r in the media ( figure 4c ) , suggesting that the effect observed with il-6 stimulation alone is via reducing signalling through the m - il6r is the presence of ala358 . figure 4gene expression in stat3 target genes in lymphoblast cell lines of different il6r rs2228145 ( aa = common homozygote ; cc , rare homozygote ) . ( a ) basal gene expression in lymphoblast cell lines shows no difference in gene expression between il6r genotypes . ( b ) gene expression following il-6 stimulation ; lower gene expression was observed in all stat3 target genes in lymphoblasts possessing the c allele . ( c ) gene expression following il-6 stimulation and addition of excess sil-6r . to examine if the effect seen in ( b ) was due to receptor shedding , excess soluble il-6r was added to the stimulated cells , suggesting that the mechanism is of genotype - specific reduction in stat3 targets is the result of increased shedding of the mil-6r . gene expression in stat3 target genes in lymphoblast cell lines of different il6r rs2228145 ( aa = common homozygote ; cc , rare homozygote ) . ( a ) basal gene expression in lymphoblast cell lines shows no difference in gene expression between il6r genotypes . ( b ) gene expression following il-6 stimulation ; lower gene expression was observed in all stat3 target genes in lymphoblasts possessing the c allele . ( c ) gene expression following il-6 stimulation and addition of excess sil-6r . to examine if the effect seen in ( b ) was due to receptor shedding , excess soluble il-6r was added to the stimulated cells , suggesting that the mechanism is of genotype - specific reduction in stat3 targets is the result of increased shedding of the mil-6r . in this study , we used an extension of the mendelian randomisation ( mr ) paradigm to investigate the potential utility of targeting il-6 signalling in aaa . in the first stage , we reviewed the observational literature reporting associations between circulating il-6 and the presence of aaa . meta - analysis of case control studies demonstrated that il-6 was significantly higher in subjects with aaa compared with those without . these data support a hypothesis that il-6 and associated signalling pathways may play a role in the development of aaa , but it is not possible to directly infer a causal relationship , from observational data associations alone , owing to limitations inherent in such analyses . for example , many of the studies did not adjust for potential confounders such as smoking status , and previously published reports have found that aaas are a source of circulating il-6 , suggesting that the observed association could at least in part be the result of reverse causation . furthermore , none of the identified studies was prospective population cohorts , considered the gold standard in epidemiological studies and there was some heterogeneity in how cases and controls were selected in the papers identified by the literature search . to address this , we applied the principles of mr for drug target validation by studying the association between a functional non - synonymous variant in il6r and aaa . genotype is randomly allocated at conception and not subject to reverse causality and therefore represents a useful tool to examine causal relationships in complex diseases phenotypes . the asp358ala variant ( or close proxy ) was selected because it has previously been shown that it has concordant effects as tocilizumab on a range of inflammatory and cardiovascular biomarkers and , therefore , may be considered a useful genetic instrument to investigate the potential for targeting the il-6r pharmacologically . there was a consistent association between the ala358 variant and a reduced risk of aaa , a finding that suggests targeting the il-6r is a plausible strategy in aaa . this was a statistically robust association , surpassing threshold levels of significance commonly used in genome - wide studies . the il6r variants have not been previously identified in two separate gwas of aaa ; however , the largest of the discovery cohorts ( ncases = 1866 ) had only 20% power to detect a variant of this effect size at the significance threshold of p < 1 10 used to triage those snps to be submitted for analysis in replication studies . given the higher levels of circulating il-6 in patients with aaa , it may seem paradoxical that a variant associated with higher circulating il-6 is also associated with a lower risk of aaa . however , this variant has previously been associated with reduced concentrations of c - reactive protein and fibrinogen ( two downstream markers of il6r activation ) , a pattern consistent with a pharmacological blockade of the il-6 receptor with tocilizumab . furthermore , it is important to note that this study is not an mr study of circulating il-6 and aaa , but uses the mr concept to evaluate the potential utility of targeting the il-6r in aaa disease . the functional analysis suggests that the ala358 variant is associated with a reduction in the sensitivity of immune cells to il-6 in vitro . one possible interpretation of these data is that this variant reduces inflammation in the aortic wall in response to injury , via reduced signalling through the mil-6r in immune cells recruited to the injury , resulting in a lower risk of aaa development . although the lymphoblastoid cell line is useful to understand the functional consequences of this variant in vitro , further work on tissue specifically from aaa cases required to understand the in vivo mechanism of action , particularly in understating the balance of pro- and anti - inflammatory effects on remodelling of the arterial wall in response to damaging environmental exposures that promote vascular injury . these findings have potentially important translational implications as they support a hypothesis that targeting the il-6r pharmacologically is a strategy that merits evaluation in aaa . no randomized trials of tocilizumab ( a monoclonal antibody to the il6r used to treat rheumatoid arthritis ) have yet examined aaa as an outcome , although two recent open label studies have reported that in patients with rheumatoid arthritis , tocilizumab reduces arterial stiffness , which suggests that the il6r blockade has effects on the vasculature . indeed , a role for tocilizumab in the treatment of other forms of inflammatory vascular disease such as takayasu 's arteritis has been postulated , although randomized trials in these conditions have not yet been reported . furthermore , there is evidence that targeting inflammatory pathways can both prevent aneurysm formation and regress already established aneurysms in murine models of the disease and evidence that tocilizumab acts on this pathway . tocilizumab does , however , have a number of side effects ( such as hypersensitivity and respiratory tract infections ) and the potential for benefit in patients with aaa would of course need to be balanced against the potential risks . although the effect of the il6r asp358ala variant on aaa risk is modest , the potential for clinical benefit from pharmacological intervention at the il-6r should not be discounted . for example , snps in hmgcr ( the target for statins ) show only a modest ( but highly significant ) effect on chd risk , but the role of statins in the prevention of chd is well established . the fact that tocilizumab has been shown to be safe in large trials in humans adds further weight to the translational potential of the present findings . in common with two of the three previously identified well - validated loci associated with risk of aaa the il6r locus the magnitude of the effect identified in the present analysis ( or : 0.86 , 95% ci : 0.800.89 ) is , however , greater than that reported for the association with chd risk ( or : 0.95 ) . the association with chd does , however , strengthen the case for targeting the il-6r therapeutically , as the major non - aneurysm - related cause of death in patients with dilated aortae is cardiovascular disease . the number of cases and controls identified from the published literature for the case control analysis of il-6 levels and aaa is relatively small , but effects were consistent and the overall result statistically robust . the in vitro functional studies are in a lymphoblastoid cell - line , which may not mimic the in vivo immune response in the aortic wall . as there was limited phenotypic / risk factor data for some of the cohorts , we were unable to include the il6r variant in a multivariate regression model including other covariates . however , no convincing effect on phenotypes such as smoking , lipids , and blood pressure has been seen in large - scale association analysis suggesting that the effects are not primarily mediated through , or confounded by these intermediate phenotypes . finally , we would ideally have a large data set with validated measures of aortic expansion in the context of the il6r genotype and assess whether or not this could provide clinically useful information to guide surveillance programmes , but this is not available at present . this study has confirmed that aaa cases have higher concentrations of circulating il-6 than controls , and provided novel evidence that a common functional variant in il6r is associated with risk of aaa . this provides evidence that targeting the il-6r may be a useful and novel strategy in aaa , and supports development of clinical trials in this regard . the number of cases and controls identified from the published literature for the case control analysis of il-6 levels and aaa is relatively small , but effects were consistent and the overall result statistically robust . the in vitro functional studies are in a lymphoblastoid cell - line , which may not mimic the in vivo immune response in the aortic wall . as there was limited phenotypic / risk factor data for some of the cohorts , we were unable to include the il6r variant in a multivariate regression model including other covariates . however , no convincing effect on phenotypes such as smoking , lipids , and blood pressure has been seen in large - scale association analysis suggesting that the effects are not primarily mediated through , or confounded by these intermediate phenotypes . finally , we would ideally have a large data set with validated measures of aortic expansion in the context of the il6r genotype and assess whether or not this could provide clinically useful information to guide surveillance programmes , but this is not available at present . this study has confirmed that aaa cases have higher concentrations of circulating il-6 than controls , and provided novel evidence that a common functional variant in il6r is associated with risk of aaa . this provides evidence that targeting the il-6r may be a useful and novel strategy in aaa , and supports development of clinical trials in this regard . this study makes use of data generated by the wellcome trust case - control consortium . a full list of the investigators who contributed to the generation of the data is available from www.wtccc.org.uk . funding for the project was provided by the wellcome trust under award 076113 and 085475 . m.j.b is supported by a senior lectureship from the higher education funding council for england and the leicester nihr biomedical research unit in cardiovascular disease . the edinburgh artery study was supported by the british heart foundation and genotyping on this project was funded by a project grant from the chief scientist office for scotland . for the smart study , research was financially supported by bbmri - nl , a research infrastructure financed by the dutch government ( nwo 184.021.007 ) . folkert w. asselbergs is supported by a clinical fellowship from the netherlands organisation for health research and development ( zonmw grant 90700342 ) . a.f . b. was supported by a e. dekker grant from the netherlands heart foundation ( 2009t001 ) . the new zealand aaa data was provided by the vascular research consortium of new zealand , and was supported by programme grant funding from the health research council of new zealand . a.d.h is supported by the bhf as a senior research fellow ( fs 05/125 ) . the generation and management of gwas genotype data for the rotterdam study is supported by the netherlands organisation of scientific research nwo investments ( nr . this study is funded by the research institute for diseases in the elderly ( 014 - 93 - 015 ; ride2 ) , the netherlands genomics initiative ( ngi)/netherlands organisation for scientific research ( nwo ) project nr . we thank pascal arp , mila jhamai , marijn verkerk , lizbeth herrera and marjolein peters for their help in creating the gwas database , and karol estrada and maksim v. struchalin for their support in creation and analysis of imputed data . the rotterdam study is funded by erasmus medical center and erasmus university , rotterdam , netherlands organization for the health research and development ( zonmw ) , the research institute for diseases in the elderly ( ride ) , the ministry of education , culture and science , the ministry for health , welfare and sports , the european commission ( dg xii ) , and the municipality of rotterdam . the authors are grateful to the study participants , the staff from the rotterdam study and the participating general practitioners and pharmacists .

methodswe conducted a systematic review and meta - analysis of studies reporting circulating il-6 in aaa , and new investigations of the association between a common non - synonymous functional variant ( asp358ala ) in the il-6r gene ( il6r ) and aaa , followed the analysis of the variant both in vitro and in vivo.inflammation may play a role in the development of abdominal aortic aneurysms ( aaa ) . interleukin-6 ( il-6 ) signalling through its receptor ( il-6r ) is one pathway that could be exploited pharmacologically . we investigated this using a mendelian randomization approach.resultsup to october 2011 , we identified seven studies ( 869 cases , 851 controls ) . meta - analysis demonstrated that aaa cases had higher levels of il-6 than controls [ standardized mean difference ( smd ) = 0.46 sd , 95% ci = 0.250.66 , i2 = 70% , p = 1.1 105 random effects ] . meta - analysis of five studies ( 4524 cases/15 710 controls ) demonstrated that rs7529229 ( which tags the non - synonymous variant asp358ala , rs2228145 ) was associated with a lower risk of aaa , per ala358 allele odds ratio 0.84 , 95% ci : 0.800.89 , i2 = 0% , p = 2.7 1011 ) . in vitro analyses in lymphoblastoid cell lines demonstrated a reduction in the expression of downstream targets ( stat3 , myc and icam1 ) in response to il-6 stimulation in ala358 carriers.conclusionsa mendelian randomization approach provides robust evidence that signalling via the il-6r is likely to be a causal pathway in aaa . drugs that inhibit il-6r may play a role in aaa management .

Introduction Methods Observational association between interleukin-6 and abdominal aortic aneurysms Study populations Genotyping Functional analysis of the p.Asp358Ala (rs2228145) variant Statistical analysis and power Results Systematic review and meta-analysis of IL-6 and abdominal aortic aneurysms Association of Functional analyses of the Asp358Ala (rs2228145) Discussion Limitations Conclusion Supplementary material Funding Supplementary Material

interleukin-6 ( il-6 ) signalling is initiated by binding of il-6 to its receptor ( il-6r ) , which forms a dimer with the ubiquitously expressed signal transducer glycoprotein-130 ( gp-130 ) . it has previously been shown that the expression of il-6 and downstream mediators of il-6 signalling , such as stat3 , are greater in aaa than in non - aneurysmal aortic tissue . a common non - synonymous sequence variant in il6r ( p.asp358ala , rs2228145 , also annotated as rs8192284 ) results in an increased proteolytic cleavage of the mil-6r . this variant is strongly associated with higher levels of circulating il-6 levels , but importantly , lower levels of downstream products of il-6 signalling , such as c - reactive protein and fibrinogen , suggesting that it acts by the attenuation of signalling via the il-6 receptor . in the present study , the primary hypothesis is that pro - inflammatory signalling via the il-6r plays a causal role in aaa development . first , a systematic review and meta - analysis of the published literature was performed in order to establish the relationship between circulating il-6 levels and aaa . in the smart study these snps both tag the non - synonymous variant ( p.asp358ala , rs2228145 , r = 0.97 ) . to estimate the association between circulating il-6 and aaa , we performed meta - analysis of summary statistics identified by our systematic review . for each study , we used mean concentrations of il-6 in cases and controls to determine a standardized mean difference ( smd ) and standard error . these snps both tag the non - synonymous variant ( p.asp358ala , rs2228145 , r = 0.97 ) . to estimate the association between circulating il-6 and aaa , we performed meta - analysis of summary statistics identified by our systematic review . for each study , we used mean concentrations of il-6 in cases and controls to determine a standardized mean difference ( smd ) and standard error . seven studies reporting circulating levels of plasma il-6 in aaa cases and controls were identified ( supplementary material online , figure s1 ) , including one previous unpublished study ( the nz study ) . two studies that reported higher il-6 in cases than controls , but did not report summary measures of il-6 ( mean and sd or median and iqr ) were not included in the meta - analysis . table 1characteristics of case control studies included in the meta - analysis of il-6 levels in abdominal aortic aneurysms cases and controlsauthoryearcountrystudy designcases / controlscase definition controlsdiagnosisil-6 cases , mean ( sd)il-6 controls , mean ( sd)treska2003czech republiccase control74/30small ( < 5 cm ) = 30 large ( 58 cm ) = 38 very large ( > 8 cm ) = 22 ruptured / symptomatic = 16**matched for age and gender no history of atherosclerosisuss59.3 ( 134.8)6.7 ( 5.1)fowkes et al.2006ukcase control89/98ap aortic diameter > 3 cm , size distribution nrnormal uss ( <3 cm ) matched for age and gender . recruited from screening programme in nz.ct/uss9.1 ( 6.60)6.3 ( 3.93)parry et al.2010ukcase control75/90all aaa < 5.5 cmmatched for age , gender , and race recruited from surgical clinicsuss3.13 ( 2.87)3.14 ( 2.32)wallinder et al.2009swedencase control case control78/41small ( < 5 cm ) = 38 awaiting elective repair ( > 5 cm ) = 40matched for age , gender , and smoking status volunteers with infra - renal aortic diameter <3 cmuss4.02 ( 3.79)2.3 ( 3.3)**values from ruptures not included in the meta - analysis . characteristics of case control studies included in the meta - analysis of il-6 levels in abdominal aortic aneurysms cases and controls * * values from ruptures not included in the meta - analysis . in meta - analysis , pooling data from 869 cases and 851 controls , individuals with aaa had higher circulating il-6 concentrations than controls ; smd = 0.46 , 95% ci : 0.250.66 , p random effects model = 1 . exclusion of this study reduced overall heterogeneity ( i = 56% ) and increased the statistical significance of the association ( p random effects = 2 10 ) ; however , this study did not overly influence the overall effect estimate greatly ( sensitivity analysis presented in the supplementary material online , figure s2 ) . in pooled analysis of seven studies reporting il-6 circulations in cases and controls ( 869/851 ) there was consistently higher concentrations in cases ( smd = 0.46 , 95% ci : 0.250.66 , p random effects model = 1 . subgroup analyses , comparing il-6 concentrations in large abdominal aortic aneurysms ( > 5 cm ) demonstrated less heterogeneity ( smd = 0.46 , 95% ci : 0.340.58 , p = 2.25 10 , i = 32% ) . in pooled analysis of seven studies reporting il-6 circulations in cases and controls ( 869/851 ) there was consistently higher concentrations in cases ( smd = 0.46 , 95% ci : 0.250.66 , p random effects model = 1 . subgroup analyses , comparing il-6 concentrations in large abdominal aortic aneurysms ( > 5 cm ) demonstrated less heterogeneity ( smd = 0.46 , 95% ci : 0.340.58 , p = 2.25 10 , i = 32% ) . meta - analysis , pooling data from 4524 cases and 15 710 controls demonstrated a per - allele or of 0.84 ( 95% ci : 0.800.89 , p = 2.7 10 , i = 0% fixed - effect meta - analysis ) ( figure 2 ) . in time - to - event analyses of prospective data from the smart study , using cox proportional hazard models , adjusted for age and gender , the rare ala358 allele was also associated with a reduction in the risk of aaa clinical endpoints ( aaa rupture , repair , nevents = 223 ) ( figure 3 , per allele hr : 0.81 , 95% ci : 0.670.99 , p = 0.043 ) . table 2demographic details of cohorts used for gene association analysiscontrolscasessample setncontrols% femaleage ( sd)ncases% femaleage ( sd)ac585550na15964nasmart63523355 ( 12)6312265 ( 10)nz7182370 ( 6.6)13732074 ( 8.0)eas8194764 ( 5.6)6266 ( 5.6)74utrecht186656568621068.1 ( 8.3 ) table 3genotyping metrics from each of the studies ( hardy weinberg equilibrium)studygenotyping platformminor allele frequencycall rate ( % ) hwe , p - valueacillumina 670 k beadchip0.39>990.28new zealandabi taqman0.40>980.89smartkaspar0.39>970.77easabi taqman0.42>970.08utrechtillumina 610 k0.381000.97 figure 2association between rs7529229 and abdominal aortic aneurysms following fixed - effect meta - analysis of four case control studies ( 4524 cases/15 710 controls ) . per allele odds ratio = 0.84 ( 95% ci : 0.800.89 , i = 0 , p = 2.7 10 ) . the hazard ratio per c - allele is 0.81 , 95% ci = 0.670.99 , p = 0.043 . ) demographic details of cohorts used for gene association analysis genotyping metrics from each of the studies ( hardy weinberg equilibrium ) association between rs7529229 and abdominal aortic aneurysms following fixed - effect meta - analysis of four case control studies ( 4524 cases/15 710 controls ) . per allele odds ratio = 0.84 ( 95% ci : 0.800.89 , i = 0 , p = 2.7 10 ) . the hazard ratio per c - allele is 0.81 , 95% ci = 0.670.99 , p = 0.043 . ) the aim of these analyses was to determine the downstream effects of the asp358ala variant on the expression of il-6-mediated target genes identified in previous studies , namely stat3 , myc , bcl3 , icam1 , and atf3 . after il-6 stimulation , there was a reduction in the expression of stat3 , myc , icam1 in ala358 homozygotes ( cc ) compared with asp358 homozygotes ( aa ) ( figure 4b ) . seven studies reporting circulating levels of plasma il-6 in aaa cases and controls were identified ( supplementary material online , figure s1 ) , including one previous unpublished study ( the nz study ) . two studies that reported higher il-6 in cases than controls , but did not report summary measures of il-6 ( mean and sd or median and iqr ) were not included in the meta - analysis . table 1characteristics of case control studies included in the meta - analysis of il-6 levels in abdominal aortic aneurysms cases and controlsauthoryearcountrystudy designcases / controlscase definition controlsdiagnosisil-6 cases , mean ( sd)il-6 controls , mean ( sd)treska2003czech republiccase control74/30small ( < 5 cm ) = 30 large ( 58 cm ) = 38 very large ( > 8 cm ) = 22 ruptured / symptomatic = 16**matched for age and gender no history of atherosclerosisuss59.3 ( 134.8)6.7 ( 5.1)fowkes et al.2006ukcase control89/98ap aortic diameter > 3 cm , size distribution nrnormal uss ( <3 cm ) matched for age and gender . recruited from screening programme in nz.ct/uss9.1 ( 6.60)6.3 ( 3.93)parry et al.2010ukcase control75/90all aaa < 5.5 cmmatched for age , gender , and race recruited from surgical clinicsuss3.13 ( 2.87)3.14 ( 2.32)wallinder et al.2009swedencase control case control78/41small ( < 5 cm ) = 38 awaiting elective repair ( > 5 cm ) = 40matched for age , gender , and smoking status volunteers with infra - renal aortic diameter <3 cmuss4.02 ( 3.79)2.3 ( 3.3)**values from ruptures not included in the meta - analysis . characteristics of case control studies included in the meta - analysis of il-6 levels in abdominal aortic aneurysms cases and controls * * values from ruptures not included in the meta - analysis . in meta - analysis , pooling data from 869 cases and 851 controls , individuals with aaa had higher circulating il-6 concentrations than controls ; smd = 0.46 , 95% ci : 0.250.66 , p random effects model = 1 . exclusion of this study reduced overall heterogeneity ( i = 56% ) and increased the statistical significance of the association ( p random effects = 2 10 ) ; however , this study did not overly influence the overall effect estimate greatly ( sensitivity analysis presented in the supplementary material online , figure s2 ) . in pooled analysis of seven studies reporting il-6 circulations in cases and controls ( 869/851 ) there was consistently higher concentrations in cases ( smd = 0.46 , 95% ci : 0.250.66 , p random effects model = 1 . subgroup analyses , comparing il-6 concentrations in large abdominal aortic aneurysms ( > 5 cm ) demonstrated less heterogeneity ( smd = 0.46 , 95% ci : 0.340.58 , p = 2.25 10 , i = 32% ) . in pooled analysis of seven studies reporting il-6 circulations in cases and controls ( 869/851 ) there was consistently higher concentrations in cases ( smd = 0.46 , 95% ci : 0.250.66 , p random effects model = 1 . subgroup analyses , comparing il-6 concentrations in large abdominal aortic aneurysms ( > 5 cm ) demonstrated less heterogeneity ( smd = 0.46 , 95% ci : 0.340.58 , p = 2.25 10 , i = 32% ) . meta - analysis , pooling data from 4524 cases and 15 710 controls demonstrated a per - allele or of 0.84 ( 95% ci : 0.800.89 , p = 2.7 10 , i = 0% fixed - effect meta - analysis ) ( figure 2 ) . in time - to - event analyses of prospective data from the smart study , using cox proportional hazard models , adjusted for age and gender , the rare ala358 allele was also associated with a reduction in the risk of aaa clinical endpoints ( aaa rupture , repair , nevents = 223 ) ( figure 3 , per allele hr : 0.81 , 95% ci : 0.670.99 , p = 0.043 ) . table 2demographic details of cohorts used for gene association analysiscontrolscasessample setncontrols% femaleage ( sd)ncases% femaleage ( sd)ac585550na15964nasmart63523355 ( 12)6312265 ( 10)nz7182370 ( 6.6)13732074 ( 8.0)eas8194764 ( 5.6)6266 ( 5.6)74utrecht186656568621068.1 ( 8.3 ) table 3genotyping metrics from each of the studies ( hardy weinberg equilibrium)studygenotyping platformminor allele frequencycall rate ( % ) hwe , p - valueacillumina 670 k beadchip0.39>990.28new zealandabi taqman0.40>980.89smartkaspar0.39>970.77easabi taqman0.42>970.08utrechtillumina 610 k0.381000.97 figure 2association between rs7529229 and abdominal aortic aneurysms following fixed - effect meta - analysis of four case control studies ( 4524 cases/15 710 controls ) . per allele odds ratio = 0.84 ( 95% ci : 0.800.89 , i = 0 , p = 2.7 10 ) . the hazard ratio per c - allele is 0.81 , 95% ci = 0.670.99 , p = 0.043 . ) demographic details of cohorts used for gene association analysis genotyping metrics from each of the studies ( hardy weinberg equilibrium ) association between rs7529229 and abdominal aortic aneurysms following fixed - effect meta - analysis of four case control studies ( 4524 cases/15 710 controls ) . per allele odds ratio = 0.84 ( 95% ci : 0.800.89 , i = 0 , p = 2.7 10 ) . the hazard ratio per c - allele is 0.81 , 95% ci = 0.670.99 , p = 0.043 . ) the aim of these analyses was to determine the downstream effects of the asp358ala variant on the expression of il-6-mediated target genes identified in previous studies , namely stat3 , myc , bcl3 , icam1 , and atf3 . after il-6 stimulation , there was a reduction in the expression of stat3 , myc , icam1 in ala358 homozygotes ( cc ) compared with asp358 homozygotes ( aa ) ( figure 4b ) . these data support a hypothesis that il-6 and associated signalling pathways may play a role in the development of aaa , but it is not possible to directly infer a causal relationship , from observational data associations alone , owing to limitations inherent in such analyses . to address this , we applied the principles of mr for drug target validation by studying the association between a functional non - synonymous variant in il6r and aaa . there was a consistent association between the ala358 variant and a reduced risk of aaa , a finding that suggests targeting the il-6r is a plausible strategy in aaa . given the higher levels of circulating il-6 in patients with aaa , it may seem paradoxical that a variant associated with higher circulating il-6 is also associated with a lower risk of aaa . furthermore , it is important to note that this study is not an mr study of circulating il-6 and aaa , but uses the mr concept to evaluate the potential utility of targeting the il-6r in aaa disease . the functional analysis suggests that the ala358 variant is associated with a reduction in the sensitivity of immune cells to il-6 in vitro . one possible interpretation of these data is that this variant reduces inflammation in the aortic wall in response to injury , via reduced signalling through the mil-6r in immune cells recruited to the injury , resulting in a lower risk of aaa development . although the lymphoblastoid cell line is useful to understand the functional consequences of this variant in vitro , further work on tissue specifically from aaa cases required to understand the in vivo mechanism of action , particularly in understating the balance of pro- and anti - inflammatory effects on remodelling of the arterial wall in response to damaging environmental exposures that promote vascular injury . in common with two of the three previously identified well - validated loci associated with risk of aaa the il6r locus the magnitude of the effect identified in the present analysis ( or : 0.86 , 95% ci : 0.800.89 ) is , however , greater than that reported for the association with chd risk ( or : 0.95 ) . this study has confirmed that aaa cases have higher concentrations of circulating il-6 than controls , and provided novel evidence that a common functional variant in il6r is associated with risk of aaa . this provides evidence that targeting the il-6r may be a useful and novel strategy in aaa , and supports development of clinical trials in this regard . this study has confirmed that aaa cases have higher concentrations of circulating il-6 than controls , and provided novel evidence that a common functional variant in il6r is associated with risk of aaa . this provides evidence that targeting the il-6r may be a useful and novel strategy in aaa , and supports development of clinical trials in this regard .

micrornas ( mirnas ) are small ( 1924 nucleotides in length ) , noncoding rnas that bind both untranslated and coding regions of target mrnas , marking them for degradation or posttranscriptional modification . the biogenesis of mirnas begins in the nucleus where rna polymerase ii generates primary mirna ( pri - mirna ) transcripts . subsequently , pri - mirnas are processed by the rnase iii enzyme drosha , generating precursor mirnas ( pre - mirnas ) . nuclear pre - mirnas are then transported to the cytoplasm by exportin / ran - gtp where they are cleaved by the cytoplasmic rnase iii enzyme dicer , generating mature mirnas which are incorporated into the rna - induced silencing complex ( risc ) . this directs risc to the target mrna based on sequence complementarity , resulting in gene silencing [ 1 , 2 ] . mirnas are encoded by many different organisms and regulate a variety of cellular processes , including cell proliferation , apoptosis , differentiation , and development . viruses encode mirnas whose sequences and functions are unique from human mirnas and mirnas encoded by human herpesviruses have been increasingly well characterized . herpesviruses are enveloped , double - stranded dna viruses , and the human gamma - herpesviruses , epstein - barr virus ( ebv ) and kaposi 's sarcoma - associated herpesviruses ( kshv ) , are the etiologic agents of several forms of cancer . as with other herpesviruses , the kshv lifecycle involves two distinct phases : latent and lytic . during latency ( the predominant phase in the majority of infected cells ) only a limited number of viral genes provocation by a variety of stimuli induces lytic replication , resulting in virion assembly and release of infectious viral particles . existing data suggest that the oncogenic potential of kshv is largely dependent upon genes expressed during latency , although low level replication occurring in a small minority of cells is also critical for infection of nave cell targets , maintenance of the kshv reservoir , and tumor pathogenesis [ 68 ] . cancers caused by kshv , including multicentric castleman 's disease ( mcd ) , primary effusion lymphoma ( pel ) , and kaposi 's sarcoma ( ks ) , arise preferentially in the setting of immune suppression as seen with hiv infection and provision of immunosuppressive medications in the context of solid organ transplantation [ 911 ] . thus far , 12 kshv pre - mirnas , encoding 18 mature mirnas , have been identified [ 1214 ] . within the kshv genome , mirnas are located in the kshv latency - associated region ( klar ) . other proteins encoded within the klar are critical for maintenance of the viral episome and kshv oncogenesis , including the latency - associated nuclear antigen ( lana ) , virus - encoded cyclin ( vcyclin ) , viral flice inhibitory protein ( vflip ) , and kaposin ( k12 ) . 10 of 12 mirnas ( mir - k12 - 1~9 and 11 ) are located within the intron of k12 ; mir - k12 - 10 is located within the open reading frame of k12 a / c and the 3utr of k12 b , and mir - k12 - 12 is located within the 3utr of k12 [ 1214 ] . given their location within the klar , it follows that kshv mirnas facilitate maintenance of latent viral gene expression and the oncogenic potential of these genes . this paper will summarize recent findings regarding the expression of kshv mirnas and their regulatory functions and elaborate on emerging mechanistic concepts in this field . we will also review several recently published studies offering insight into the feasibility of targeting mirnas for therapeutic purposes . for an overview of kshv mirna targets and their putative functions , see table 1 . expression of kshv mirnas has been demonstrated within latently infected primary human cells and kshv - infected pel cells [ 1216 ] . pel cell lines exhibit significant conservation ( ~99.6% ) of kshv - mirna expression , although one group recently reported that mir - k12 - 9 may be mutationally inactivated in different pel lines . phylogenetic analyses of kshv mirna sequences from clinical samples of ks and mcd patients of divergent geographic backgrounds reveal the existence of 2 major sequence clusters , referred to as the major ( a / c ) and variant ( b / q ) clusters . further analyses of the pre - mirna sequences show that some kshv mirnas are highly conserved ( such as mir - k12 - 1 , 3 , 8 , 10 , 11 , and 12 ) , while others ( including mir - k12 - 2 , 4 , 5 , 6 , 7 , and 9 ) exhibit sequence alterations likely affecting their processing and function , although this hypothesis requires additional confirmation . in addition , one study found distinct polymorphisms within pri - mirnas , pre - mirnas , or mature mirnas encoded by circulating kshv in a european patient cohort , and some of these polymorphisms may affect mature mirna processing and associate with ks risk . collectively , these data indicate that individual kshv mirnas may regulate kshv pathogenesis in a disease - specific manner , and that they may exhibit cell type - specific functions within the tumor microenvironment . identification of viral and cellular factors governing these differences should illuminate additional mechanisms and help determine whether screening for mirna polymorphisms can be used to quantify one 's risk for developing kshv - associated tumors . we have reported that mir - k12 - 1 , 9 , and 11 increase macrophage and endothelial cell ( ec ) susceptibility to kshv entry and latent gene expression through upregulation of xct , an inducible amino acid exchanger and fusion - entry receptor for the virus . one mechanism for these observations involves upregulation of xct through mir - k12 - 11 repression of bach-1 , a negative transcription regulator of xct . these findings are consistent with earlier reports revealing direct targeting of bach-1 by mir - k12 - 11 [ 22 , 23 ] . mechanisms for regulation of xct expression by mir - k12 - 1 and 9 have not yet been elucidated . one related report noted involvement of kshv mirnas in endothelial cell reprogramming through repression of the cellular transcription factor maf ( cmaf ) . cmaf also serves as a negative transcription regulator for xct , so we have speculated that multiple kshv mirnas , through cooperative mechanisms , facilitate kshv entry . whether kshv mirnas regulate expression and/or function of other cellular receptors for kshv , including dc - sign and integrins [ 2529 ] , has not been established . increased cell permissiveness for kshv entry following initial infection and mirna expression may represent an evolutionary mechanism for kshv promotion of its own persistence . supporting this hypothesis , several reports have shown that a significant proportion of kshv - infected tumor cells contain multiple viral clones [ 3032 ] . moreover , downregulation of mhc class i ( mhc - i ) in kshv - infected cells is directly proportional to intracellular kshv episome copy number , implying that an increase in intracellular viral copies reduces kshv epitope presentation to cd8 t cells . for example , one group has demonstrated that several human mirnas regulate monocyte / macrophage susceptibility to hiv infection [ 34 , 35 ] . another group reported that mir-23b inhibits rhinoviruses 1b ( rv1b ) entry through targeting of the very low density lipoprotein receptor . therefore , it is plausible that kshv and human mirnas cooperatively regulate surface determinants of cell targeting by kshv and other viruses . furthermore , it is likely that kshv and other viral mirnas regulate secretion of microenvironmental factors by infected cells that influence susceptibility of neighboring cells to virus entry . we have shown that kshv mirna induce secretion of reactive nitrogen species ( rns ) , and that inhibition of the enzymatic generation of rns reduces cell susceptibility to kshv infection . given that both bach-1 and cmaf are negative transcription regulators for genes containing antioxidant response elements ( ares ) in their promoters , and since several genes involved in production of reactive nitrogen- and oxygen - based species ( rns and ros , resp . ) contain ares , we hypothesize that kshv mirna regulation of bach-1 and cmaf influences a more complex network of genes to generate tumor - promoting rns and ros while simultaneously protecting kshv - infected cells from oxidative damage inflicted by these species . these data have implications for development of therapeutic strategies to reduce kshv infection in the tumor microenvironment and , therefore , ks progression [ 68 ] . maintenance of latent kshv infection , coordinated with lytic reactivation within a small subset of infected cells , is critical for simultaneous promotion of kshv persistence and dissemination . studies published recently indicate a role for kshv mirnas in the regulation of this latent - lytic switch . mir - k12 - 7 and 9 bind and repress transcription of the kshv immediate - early gene orf50 which encodes the replication and transcription activator ( rta ) [ 37 , 38 ] . rta activation is critical for the initiation of lytic replication of the virus [ 37 , 38 ] . mir - k12 - 4 represses expression of the retinoblastoma ( rb)-like protein 2 ( rbl2 ) , a known repressor of dna methyl transferases ( dnmt)-1 , -3a and -3b . increased activity of these dnmts maintains methylation of the rta promoter and suppresses its expression . furthermore , mir - k12 - 1 targeting of ib , an inhibitor of nf-b complexes , promotes nf-b - dependent viral latency and cell survival . mir - k12 - 3 also promotes kshv latency through targeting of nuclear factor i / b , an activator of the rta promoter . conversely , mir - k12 - 5 and 9 repress the bcl-2-associated factor ( bclaf1 ) , resulting in an increase in lytic replication , albeit through mechanisms that have yet to be defined . one recent study indicates that mir - k12 - 11 targets and downregulates ikk , a signaling intermediate shown previously to facilitate lytic reactivation of kshv from latently infected cells . another report revealed upregulation of two mirnas , mir - k12 - 10 , and 12 , during chemical induction of kshv lytic reactivation , but whether these mirnas play an active role in regulation of the lytic switch for kshv remains to be determined . collectively , these data support the notion that kshv mirnas function primarily to maintain viral latency , congruous with their location within the klar . this is also supported by recent work revealing that cells infected with kshv deletion mutants lacking kshv mirnas exhibit increased expression of lytic viral genes , including orf50 [ 40 , 41 ] . several factors secreted by kshv - infected cells ( and other cells found within the tumor microenvironment ) , including vegf , il-8 , il-6 , il-10 , il-1 , and tnf- , support kshv - associated pathogenesis through complimentary mechanisms involving interference or augmentation of cellular functions relevant to cancer pathogenesis . more specifically , il-6 and il-10 collectively promote growth of kshv - infected tumor cells , angiogenesis and suppression of t - cell activation [ 5053 ] . we have demonstrated that kshv - mirnas induce il-6 and il-10 secretion by murine macrophages and human myelomonocytic cells , and that this is accomplished , in part , through mir - k12 - 3 and 7 repression of a dominant - negative isoform of c / ebp which serves as a transcriptional repressor of il-6 and il-10 . however , it remains unclear whether this effect results from direct targeting of c / ebp by these mirnas or an indirect effect within a more complex regulatory network . furthermore , whether these events occur following de novo infection of human primary cells is unknown . a more recent study reported that mir - k12 - 11 induces splenic b - cell expansion and kshv - associated lymphomagenesis through direct targeting of c / ebp . it is plausible that lymphomagenesis in this model is dependent on mirna regulation of cytokine responses through targeting of c / ebp , congruous with existing clinical data suggesting a role for cytokines in pel pathogenesis . another study reveals that kshv - encoded orf57 competes with human mirnas for binding of transcripts for both human il-6 ( hil-6 ) and the kshv - encoded viral homolog for il-6 ( vil-6 ) . in doing so , orf57 impairs hil-6 and vil-6 rna association with human mirna - specified riscs , thereby stabilizing il-6 rna . existing data further indicate that kshv mirnas may facilitate conditional suppression of cytokine responses and immune recognition . mir - k12 - 10 repression of the tumor necrosis factor - like weak inducer of apoptosis receptor ( tweakr ) in primary human ecs results in decreased expression of il-8 and monocyte chemoattractant protein 1 ( mcp-1 ) which are normally induced following tweakr interactions with its cognate ligand , tweak . in addition , one group has found that kshv mirnas repress expression of the stress - induced natural killer ( nk ) cell ligand , micb , thereby permitting escape of kshv - infected cells from nk cell recognition and killing . it seems likely that kshv mirna regulation of cytokine responses and immune evasion is a finely coordinated effort hinging on intracellular and/or exogenous microenvironmental signals that are cell type - specific . maintenance of viability for kshv - infected cells depends , in part , on kshv regulation of cellular pathways promoting cell survival and antiapoptotic signaling . microarray analyses using cells stably expressing kshv - encoded mirnas revealed that 3utrs of select cell proliferation / apoptosis - associated genes , including spp1 , s100a2 , and prg1 , are likely targeted by multiple kshv mirnas . however to our knowledge , specific target sequences within the 3utrs for these genes have not yet been validated , and functional correlates for kshv mirnas targeting these genes have not been determined . as mentioned previously , mir - k12 - 10 represses tweakr , and cells transfected with mir - k12 - 10 are more resistant another group showed that expression of the cellular cyclin - dependent kinase inhibitor p21 , a key inducer of cell cycle arrest , is repressed through its direct targeting by mir - k12 - 1 . ectopically expressed mir - k12 - 1 strongly attenuated cell cycle arrest induced during p53 activation through repression of endogenous p21 . in summary , kshv mirna support of anti - apoptotic signaling , coupled with their regulation of cytokine responses and their putative role in suppression of immune recognition , suggests that kshv mirnas invoke cooperative mechanisms critical for survival of kshv - infected cells . online mirna databases ( http://www.mirbase.org/ ) and bioinformatics programs have been developed to predict virus - encoded mirna targets [ 22 , 5759 ] . several groups have utilized these programs for identifying putative targets of kshv mirna [ 14 , 20 , 22 , 24 , 40 , 44 ] , although the use of seed sequence matching as the principal predictive tool for these programs is too stringent given that many valid targets of mirnas will not meet predetermined sequence matching criteria . this has led to interest in developing screening tools involving more direct assessment of viral mirna regulation of potential targets . one group published their use of a tandem array - based screening approach : first , they quantified expression of host genes under conditions of either kshv mirna overexpression or inhibition of single kshv mirnas in latently infected cells ; second , they identified targets using stringent criteria including seed sequence complementarity at positions 28 which , although not required for targeting , has been associated with more reliable prediction of target downregulation . through this effort , they identified one gene targeted by mir - k12 - 5 ( bclaf1 ) . as noted by the authors , limitations for this approach are its labor - intensive nature and lack of all - inclusiveness in target identification . another group performed immunoprecipitation of riscs followed by microarray analysis of the risc - bound mirna targets ( rip - chip ) of kshv mirnas , ebv - mirnas , and human mirnas using latently infected or stably transduced human b - cell lines . two targets were validated for ebv mirnas , and transcript half - life of human and viral mirna targets correlated inversely with recruitment to risc complexes , indicating that rip - chip may offer a quantitative estimate of viral mirna function . furthermore , two putative targets exhibited mirna binding sites within their coding sequences , not within 3utrs . additional studies should clarify whether these and other methods are ultimately cost - effective and yield more reliable identification of viral mirna targets relative to bioinformatics screens . although the majority of published work has thus far focused on defining kshv mirna targets and functional correlations , data published more recently also suggest that kshv - encoded proteins regulate cellular machinery by virtue of their regulation or interference with cellular mirna functioning . in ks and pel tumors , tumor - suppressor mirnas , including mir-221 , mir-222 , and let-7 family members , are underrepresented . furthermore , pre - mirna signatures may define the stages of ec transformation following kshv infection . more specifically , the loss of mir-221 expression marks the transition from immortalization to tumorigenicity for these cells . since the publication of these studies , several groups have identified specific mechanisms for kshv regulation of cellular mirnas . kshv - encoded vflip represses expression of the chemokine receptor cxcr4 through nf-b - mediated upregulation of mir-146a . since kshv encodes redundant mechanisms for nf-b upregulation , and since multiple cellular mirnas have nf-b binding sites within their promoters , this study illuminates an important mechanism for kshv regulation of the cellular mirna machinery . another elegant study recently confirmed that kshv induces ec migration through regulation of cellular transcription factors , and the authors identified two complimentary mechanisms for this effect . first , they found that the transcription factors ets2 and ets1 are downstream targets of cellular mir-221 and mir-222 , respectively . they confirmed that two kshv - encoded latent proteins , lana , and kaposin b , downregulate the mir-221/mir-222 cluster through direct interactions with the mir-221/mir-222 promoter resulting in upregulation of ets1/2-induced ec mobility . second , they found that kshv upregulates ec expression of mir-31 , thereby repressing expression of the tumor suppressor fat4 . they confirmed the presence of mir-31 binding sites within the coding region of fat4 , and that kshv / mir-31-induced suppression of fat4 results in enhanced ec mobility . the same group published additional data suggesting that the minor variant of kshv - encoded k15 induces cell migration and invasion through induction of mir-31 . kshv infection induces expression of mir-132 , thereby reducing expression of interferon ( ifn)-stimulated genes and facilitating viral replication in ec . and as previously mentioned , kshv - encoded orf57 competes with cellular mirnas for binding of transcripts for il-6 , thereby stabilizing il-6 rna . these studies further underscore the complex regulatory network of viral and human mirnas that contribute to tumor pathogenesis , and future studies will confirm whether inhibition of kshv regulation of human mirnas offers a viable therapeutic strategy for kshv - associated diseases . less well understood are mechanisms for transcriptional and posttranscriptional regulation of mirnas themselves , including viral mirnas , although burgeoning data suggest that these processes are important for cancer pathogenesis [ 68 , 69 ] . mirnas are under the control of a wide range of transcription factors , including some tumor suppressors and oncogenes [ 7072 ] , and recent reports reveal that certain environmental conditions like hypoxic stress influence mirna expression . mir-210 is induced by hypoxia - inducible factor-1 alpha ( hif-1 ) to promote cell survival and adaptation to hypoxic environmental conditions , and hif-1 alters mir-101 expression in a prostate cancer model . interestingly , hif-1 is highly expressed in hiv - associated ks lesions , and kshv - encoded ifn regulatory factor 3 ( virf3 ) stabilizes hif-1 , thereby inducing vascular endothelial growth factor ( vegf ) expression . kshv - encoded lana also functions both as an inhibitor of a hif-1 suppressor , the von hippel - lindau protein , and as an inducer of nuclear accumulation of hif-1 during latent kshv infection [ 77 , 78 ] . these data would support additional work to determine whether kshv regulation of hif-1 dysregulates human mirna expression and tumor pathogenesis . other factors regulate mirna expression at the posttranscriptional level , including drosha and its interactional protein dgcr8 [ 7981 ] . one study also noted that a single nucleotide polymorphism within the mir - k12 - 5 precursor stem - loop reduces drosha processing and inhibits mature mir - k12 - 5 expression in bcbl-1 cells . this implies that mutations within mirna genes themselves may arise during the transformation of an infected cell and differential expression of kshv mirnas which favor specific pathogenic events . dna methylation and histone deacetylation also contribute to regulation of mirna transcription [ 8385 ] . a report referenced previously found that genomic dna from cells infected with a kshv deletion mutant lacking 10 of the 12 mature kshv mirnas exhibited a striking loss of methylation , but whether mirna expression is regulated through epigenetic mechanisms , possibly involving mirnas themselves , has not been elucidated . despite challenges in achieving efficient and selective approaches for suppressing mirna functions in vivo , the concept of targeting mirnas for therapeutic benefit has gained considerable attention with the publication of elegant studies revealing effective methods for suppressing mirna - associated tumor progression in animal models . one of the first examples of chemical modification of oligonucleotides for mirna inhibition was the development of antagomirs , small ribonucleotide chains whose 2-hydroxyl on the ribose is replaced by a 2-o - methyl group for stability . commercially available antagomirs have additional modifications that stabilize mirna - antagomir binding and prevent recognition of cognate mrnas by mirnas . antagomirs have demonstrated utility for inhibiting kshv mirna - induced pathogenesis in kshv - infected cells in vitro [ 20 , 44 , 56 ] . intravenous delivery of antagomirs has also demonstrated utility in vivo [ 8688 ] , but off - target effects and excessive doses required to suppress mirna expression have raised concerns about the utility of this approach . examples of other chemical modifications of oligonucleotides for clinical applications include morpholinos and locked nucleic acids ( lnas ) . morpholinos contain six - member morpholine rings rather than five - member ribose rings , conferring resistance to nucleases . morpholinos may be further engineered to bind and protect mrna target sequences from mirna to confer superior target specificity . morpholinos conjugated to peptides to enhance cell penetration have demonstrated utility in animal models , and a modified drug based on this technology , delivered by intramuscular injection , is undergoing evaluation in one clinical trial . lnas contain a biochemical modification where the 2-oxygen and 4-carbon atoms of the ribose rings are chemically bridged . an lna - based mir-122 inhibitor is under evaluation for the treatment of hepatitis c . in fact , lnas have demonstrated utility in a number of animal model systems [ 9599 ] . as noted previously , mir - k12 - 11 , a kshv - encoded ortholog of cellular mir-155 , targets c / ebp , , and one study indicates that silencing of mir-155 in mice using lnas leads to derepression of c / ebp . it is interesting to speculate whether lnas could be used to suppress kshv - associated lymphoma progression in vivo using this approach to target mir - k12 - 11 . as we discussed previously , kshv itself suppresses expression of human mirnas serving as tumor suppressors , including mir-221 and mir-222 . this raises the question of whether delivery of selected mirnas would interfere with kshv pathogenesis in vivo . the utility of mirna delivery for cancer therapeutics has been supported recently through studies indicating successful suppression of tumors in vivo using liposomal nanoparticles containing mirna or lipid - based delivery reagents which are commercially available [ 100 , 101 ] . in one study , systemic delivery of mir-34a inhibited prostate cancer metastasis and extended survival of tumor - bearing mice , in part through targeting of cd44 . cd44 is one of two well - characterized receptors for hyaluronic acid ( ha ) , and we have recently reported effective sensitization of human kshv - infected lymphoma cells to chemotherapy using various approaches for interfering with ha - receptor interactions . whether kshv regulation of human mirnas initiates upregulation of ha receptors and drug resistance for kshv - infected cells remains unknown . regardless , this reinforces the complex interplay between viral and human mirna and the redundancy of viral mirna regulatory mechanisms , and implies that viral cancer treatment approaches targeting a single mirna would likely be limited in their clinical efficacy . combining mirna targeting with existing therapies for viral tumors may be a more tractable approach . of note , in vivo effects of targeting multiple mirnas simultaneously have not been defined , although simultaneous use of multiple antagomirs targeting kshv mirnas demonstrates additive or synergistic suppression of kshv pathogenesis in vitro [ 20 , 44 , 56 ] . as the etiologic agent of diverse forms of human cancer and by virtue of its tropism for a variety of human cell types , kshv represents a model pathogen for the study of viral mirna expression and function . elegant studies performed recently underscore the importance of kshv mirnas and their interactions with human mirna for cancer pathogenesis , including viral biology and gene expression , cytokine responses , immune evasion , and anti - apoptotic signaling . the plasticity of these interactions and challenges inherent to mirna targeting in vivo incur substantial obstacles for development of mirna - based therapies , but recent advances hold considerable promise for eventual clinical application of therapeutic approaches targeting viral mirnas in the treatment of viral malignancies .

the human genome contains micrornas ( mirnas ) , small noncoding rnas that orchestrate a number of physiologic processes through regulation of gene expression . burgeoning evidence suggests that dysregulation of mirnas may promote disease progression and cancer pathogenesis . virus - encoded mirnas , exhibiting unique molecular signatures and functions , have been increasingly recognized as contributors to viral cancer pathogenesis . a large segment of the existing knowledge in this area has been generated through characterization of mirnas encoded by the human gamma - herpesviruses , including the kaposi 's sarcoma - associated herpesvirus ( kshv ) . recent studies focusing on kshv mirnas have led to a better understanding of viral mirna expression in human tumors , the identification of novel pathologic check points regulated by viral mirnas , and new insights for viral mirna interactions with cellular ( human ) mirnas . elucidating the functional effects of inhibiting kshv mirnas has also provided a foundation for further translational efforts and consideration of clinical applications . this paper summarizes recent literature outlining mechanisms for kshv mirna regulation of cellular function and cancer - associated pathogenesis , as well as implications for interactions between kshv and human mirnas that may facilitate cancer progression . finally , insights are offered for the clinical feasibility of targeting mirnas as a therapeutic approach for viral cancers .

1. Introduction 2. Expression Patterns for KSHV miRNAs 3. KSHV miRNA Regulation of Virus Entry 4. KSHV miRNA Regulation of Viral Gene Expression 5. KSHV miRNA Regulation of Cytokine Responses, Immune Recognition, and Cell Survival 6. Future Directions 7. Conclusion

micrornas ( mirnas ) are small ( 1924 nucleotides in length ) , noncoding rnas that bind both untranslated and coding regions of target mrnas , marking them for degradation or posttranscriptional modification . subsequently , pri - mirnas are processed by the rnase iii enzyme drosha , generating precursor mirnas ( pre - mirnas ) . nuclear pre - mirnas are then transported to the cytoplasm by exportin / ran - gtp where they are cleaved by the cytoplasmic rnase iii enzyme dicer , generating mature mirnas which are incorporated into the rna - induced silencing complex ( risc ) . mirnas are encoded by many different organisms and regulate a variety of cellular processes , including cell proliferation , apoptosis , differentiation , and development . viruses encode mirnas whose sequences and functions are unique from human mirnas and mirnas encoded by human herpesviruses have been increasingly well characterized . herpesviruses are enveloped , double - stranded dna viruses , and the human gamma - herpesviruses , epstein - barr virus ( ebv ) and kaposi 's sarcoma - associated herpesviruses ( kshv ) , are the etiologic agents of several forms of cancer . as with other herpesviruses , the kshv lifecycle involves two distinct phases : latent and lytic . during latency ( the predominant phase in the majority of infected cells ) only a limited number of viral genes provocation by a variety of stimuli induces lytic replication , resulting in virion assembly and release of infectious viral particles . existing data suggest that the oncogenic potential of kshv is largely dependent upon genes expressed during latency , although low level replication occurring in a small minority of cells is also critical for infection of nave cell targets , maintenance of the kshv reservoir , and tumor pathogenesis [ 68 ] . cancers caused by kshv , including multicentric castleman 's disease ( mcd ) , primary effusion lymphoma ( pel ) , and kaposi 's sarcoma ( ks ) , arise preferentially in the setting of immune suppression as seen with hiv infection and provision of immunosuppressive medications in the context of solid organ transplantation [ 911 ] . thus far , 12 kshv pre - mirnas , encoding 18 mature mirnas , have been identified [ 1214 ] . other proteins encoded within the klar are critical for maintenance of the viral episome and kshv oncogenesis , including the latency - associated nuclear antigen ( lana ) , virus - encoded cyclin ( vcyclin ) , viral flice inhibitory protein ( vflip ) , and kaposin ( k12 ) . given their location within the klar , it follows that kshv mirnas facilitate maintenance of latent viral gene expression and the oncogenic potential of these genes . this paper will summarize recent findings regarding the expression of kshv mirnas and their regulatory functions and elaborate on emerging mechanistic concepts in this field . we will also review several recently published studies offering insight into the feasibility of targeting mirnas for therapeutic purposes . for an overview of kshv mirna targets and their putative functions , see table 1 . expression of kshv mirnas has been demonstrated within latently infected primary human cells and kshv - infected pel cells [ 1216 ] . further analyses of the pre - mirna sequences show that some kshv mirnas are highly conserved ( such as mir - k12 - 1 , 3 , 8 , 10 , 11 , and 12 ) , while others ( including mir - k12 - 2 , 4 , 5 , 6 , 7 , and 9 ) exhibit sequence alterations likely affecting their processing and function , although this hypothesis requires additional confirmation . in addition , one study found distinct polymorphisms within pri - mirnas , pre - mirnas , or mature mirnas encoded by circulating kshv in a european patient cohort , and some of these polymorphisms may affect mature mirna processing and associate with ks risk . collectively , these data indicate that individual kshv mirnas may regulate kshv pathogenesis in a disease - specific manner , and that they may exhibit cell type - specific functions within the tumor microenvironment . identification of viral and cellular factors governing these differences should illuminate additional mechanisms and help determine whether screening for mirna polymorphisms can be used to quantify one 's risk for developing kshv - associated tumors . we have reported that mir - k12 - 1 , 9 , and 11 increase macrophage and endothelial cell ( ec ) susceptibility to kshv entry and latent gene expression through upregulation of xct , an inducible amino acid exchanger and fusion - entry receptor for the virus . mechanisms for regulation of xct expression by mir - k12 - 1 and 9 have not yet been elucidated . one related report noted involvement of kshv mirnas in endothelial cell reprogramming through repression of the cellular transcription factor maf ( cmaf ) . cmaf also serves as a negative transcription regulator for xct , so we have speculated that multiple kshv mirnas , through cooperative mechanisms , facilitate kshv entry . whether kshv mirnas regulate expression and/or function of other cellular receptors for kshv , including dc - sign and integrins [ 2529 ] , has not been established . increased cell permissiveness for kshv entry following initial infection and mirna expression may represent an evolutionary mechanism for kshv promotion of its own persistence . for example , one group has demonstrated that several human mirnas regulate monocyte / macrophage susceptibility to hiv infection [ 34 , 35 ] . therefore , it is plausible that kshv and human mirnas cooperatively regulate surface determinants of cell targeting by kshv and other viruses . furthermore , it is likely that kshv and other viral mirnas regulate secretion of microenvironmental factors by infected cells that influence susceptibility of neighboring cells to virus entry . we have shown that kshv mirna induce secretion of reactive nitrogen species ( rns ) , and that inhibition of the enzymatic generation of rns reduces cell susceptibility to kshv infection . given that both bach-1 and cmaf are negative transcription regulators for genes containing antioxidant response elements ( ares ) in their promoters , and since several genes involved in production of reactive nitrogen- and oxygen - based species ( rns and ros , resp . ) contain ares , we hypothesize that kshv mirna regulation of bach-1 and cmaf influences a more complex network of genes to generate tumor - promoting rns and ros while simultaneously protecting kshv - infected cells from oxidative damage inflicted by these species . studies published recently indicate a role for kshv mirnas in the regulation of this latent - lytic switch . rta activation is critical for the initiation of lytic replication of the virus [ 37 , 38 ] . mir - k12 - 4 represses expression of the retinoblastoma ( rb)-like protein 2 ( rbl2 ) , a known repressor of dna methyl transferases ( dnmt)-1 , -3a and -3b . another report revealed upregulation of two mirnas , mir - k12 - 10 , and 12 , during chemical induction of kshv lytic reactivation , but whether these mirnas play an active role in regulation of the lytic switch for kshv remains to be determined . this is also supported by recent work revealing that cells infected with kshv deletion mutants lacking kshv mirnas exhibit increased expression of lytic viral genes , including orf50 [ 40 , 41 ] . several factors secreted by kshv - infected cells ( and other cells found within the tumor microenvironment ) , including vegf , il-8 , il-6 , il-10 , il-1 , and tnf- , support kshv - associated pathogenesis through complimentary mechanisms involving interference or augmentation of cellular functions relevant to cancer pathogenesis . we have demonstrated that kshv - mirnas induce il-6 and il-10 secretion by murine macrophages and human myelomonocytic cells , and that this is accomplished , in part , through mir - k12 - 3 and 7 repression of a dominant - negative isoform of c / ebp which serves as a transcriptional repressor of il-6 and il-10 . it is plausible that lymphomagenesis in this model is dependent on mirna regulation of cytokine responses through targeting of c / ebp , congruous with existing clinical data suggesting a role for cytokines in pel pathogenesis . another study reveals that kshv - encoded orf57 competes with human mirnas for binding of transcripts for both human il-6 ( hil-6 ) and the kshv - encoded viral homolog for il-6 ( vil-6 ) . existing data further indicate that kshv mirnas may facilitate conditional suppression of cytokine responses and immune recognition . mir - k12 - 10 repression of the tumor necrosis factor - like weak inducer of apoptosis receptor ( tweakr ) in primary human ecs results in decreased expression of il-8 and monocyte chemoattractant protein 1 ( mcp-1 ) which are normally induced following tweakr interactions with its cognate ligand , tweak . in addition , one group has found that kshv mirnas repress expression of the stress - induced natural killer ( nk ) cell ligand , micb , thereby permitting escape of kshv - infected cells from nk cell recognition and killing . it seems likely that kshv mirna regulation of cytokine responses and immune evasion is a finely coordinated effort hinging on intracellular and/or exogenous microenvironmental signals that are cell type - specific . maintenance of viability for kshv - infected cells depends , in part , on kshv regulation of cellular pathways promoting cell survival and antiapoptotic signaling . microarray analyses using cells stably expressing kshv - encoded mirnas revealed that 3utrs of select cell proliferation / apoptosis - associated genes , including spp1 , s100a2 , and prg1 , are likely targeted by multiple kshv mirnas . however to our knowledge , specific target sequences within the 3utrs for these genes have not yet been validated , and functional correlates for kshv mirnas targeting these genes have not been determined . as mentioned previously , mir - k12 - 10 represses tweakr , and cells transfected with mir - k12 - 10 are more resistant another group showed that expression of the cellular cyclin - dependent kinase inhibitor p21 , a key inducer of cell cycle arrest , is repressed through its direct targeting by mir - k12 - 1 . in summary , kshv mirna support of anti - apoptotic signaling , coupled with their regulation of cytokine responses and their putative role in suppression of immune recognition , suggests that kshv mirnas invoke cooperative mechanisms critical for survival of kshv - infected cells . online mirna databases ( http://www.mirbase.org/ ) and bioinformatics programs have been developed to predict virus - encoded mirna targets [ 22 , 5759 ] . several groups have utilized these programs for identifying putative targets of kshv mirna [ 14 , 20 , 22 , 24 , 40 , 44 ] , although the use of seed sequence matching as the principal predictive tool for these programs is too stringent given that many valid targets of mirnas will not meet predetermined sequence matching criteria . this has led to interest in developing screening tools involving more direct assessment of viral mirna regulation of potential targets . one group published their use of a tandem array - based screening approach : first , they quantified expression of host genes under conditions of either kshv mirna overexpression or inhibition of single kshv mirnas in latently infected cells ; second , they identified targets using stringent criteria including seed sequence complementarity at positions 28 which , although not required for targeting , has been associated with more reliable prediction of target downregulation . another group performed immunoprecipitation of riscs followed by microarray analysis of the risc - bound mirna targets ( rip - chip ) of kshv mirnas , ebv - mirnas , and human mirnas using latently infected or stably transduced human b - cell lines . two targets were validated for ebv mirnas , and transcript half - life of human and viral mirna targets correlated inversely with recruitment to risc complexes , indicating that rip - chip may offer a quantitative estimate of viral mirna function . additional studies should clarify whether these and other methods are ultimately cost - effective and yield more reliable identification of viral mirna targets relative to bioinformatics screens . although the majority of published work has thus far focused on defining kshv mirna targets and functional correlations , data published more recently also suggest that kshv - encoded proteins regulate cellular machinery by virtue of their regulation or interference with cellular mirna functioning . in ks and pel tumors , tumor - suppressor mirnas , including mir-221 , mir-222 , and let-7 family members , are underrepresented . since the publication of these studies , several groups have identified specific mechanisms for kshv regulation of cellular mirnas . kshv - encoded vflip represses expression of the chemokine receptor cxcr4 through nf-b - mediated upregulation of mir-146a . since kshv encodes redundant mechanisms for nf-b upregulation , and since multiple cellular mirnas have nf-b binding sites within their promoters , this study illuminates an important mechanism for kshv regulation of the cellular mirna machinery . another elegant study recently confirmed that kshv induces ec migration through regulation of cellular transcription factors , and the authors identified two complimentary mechanisms for this effect . they confirmed that two kshv - encoded latent proteins , lana , and kaposin b , downregulate the mir-221/mir-222 cluster through direct interactions with the mir-221/mir-222 promoter resulting in upregulation of ets1/2-induced ec mobility . they confirmed the presence of mir-31 binding sites within the coding region of fat4 , and that kshv / mir-31-induced suppression of fat4 results in enhanced ec mobility . and as previously mentioned , kshv - encoded orf57 competes with cellular mirnas for binding of transcripts for il-6 , thereby stabilizing il-6 rna . these studies further underscore the complex regulatory network of viral and human mirnas that contribute to tumor pathogenesis , and future studies will confirm whether inhibition of kshv regulation of human mirnas offers a viable therapeutic strategy for kshv - associated diseases . less well understood are mechanisms for transcriptional and posttranscriptional regulation of mirnas themselves , including viral mirnas , although burgeoning data suggest that these processes are important for cancer pathogenesis [ 68 , 69 ] . mirnas are under the control of a wide range of transcription factors , including some tumor suppressors and oncogenes [ 7072 ] , and recent reports reveal that certain environmental conditions like hypoxic stress influence mirna expression . mir-210 is induced by hypoxia - inducible factor-1 alpha ( hif-1 ) to promote cell survival and adaptation to hypoxic environmental conditions , and hif-1 alters mir-101 expression in a prostate cancer model . interestingly , hif-1 is highly expressed in hiv - associated ks lesions , and kshv - encoded ifn regulatory factor 3 ( virf3 ) stabilizes hif-1 , thereby inducing vascular endothelial growth factor ( vegf ) expression . kshv - encoded lana also functions both as an inhibitor of a hif-1 suppressor , the von hippel - lindau protein , and as an inducer of nuclear accumulation of hif-1 during latent kshv infection [ 77 , 78 ] . these data would support additional work to determine whether kshv regulation of hif-1 dysregulates human mirna expression and tumor pathogenesis . other factors regulate mirna expression at the posttranscriptional level , including drosha and its interactional protein dgcr8 [ 7981 ] . this implies that mutations within mirna genes themselves may arise during the transformation of an infected cell and differential expression of kshv mirnas which favor specific pathogenic events . a report referenced previously found that genomic dna from cells infected with a kshv deletion mutant lacking 10 of the 12 mature kshv mirnas exhibited a striking loss of methylation , but whether mirna expression is regulated through epigenetic mechanisms , possibly involving mirnas themselves , has not been elucidated . despite challenges in achieving efficient and selective approaches for suppressing mirna functions in vivo , the concept of targeting mirnas for therapeutic benefit has gained considerable attention with the publication of elegant studies revealing effective methods for suppressing mirna - associated tumor progression in animal models . one of the first examples of chemical modification of oligonucleotides for mirna inhibition was the development of antagomirs , small ribonucleotide chains whose 2-hydroxyl on the ribose is replaced by a 2-o - methyl group for stability . antagomirs have demonstrated utility for inhibiting kshv mirna - induced pathogenesis in kshv - infected cells in vitro [ 20 , 44 , 56 ] . intravenous delivery of antagomirs has also demonstrated utility in vivo [ 8688 ] , but off - target effects and excessive doses required to suppress mirna expression have raised concerns about the utility of this approach . in fact , lnas have demonstrated utility in a number of animal model systems [ 9599 ] . as noted previously , mir - k12 - 11 , a kshv - encoded ortholog of cellular mir-155 , targets c / ebp , , and one study indicates that silencing of mir-155 in mice using lnas leads to derepression of c / ebp . as we discussed previously , kshv itself suppresses expression of human mirnas serving as tumor suppressors , including mir-221 and mir-222 . cd44 is one of two well - characterized receptors for hyaluronic acid ( ha ) , and we have recently reported effective sensitization of human kshv - infected lymphoma cells to chemotherapy using various approaches for interfering with ha - receptor interactions . whether kshv regulation of human mirnas initiates upregulation of ha receptors and drug resistance for kshv - infected cells remains unknown . regardless , this reinforces the complex interplay between viral and human mirna and the redundancy of viral mirna regulatory mechanisms , and implies that viral cancer treatment approaches targeting a single mirna would likely be limited in their clinical efficacy . of note , in vivo effects of targeting multiple mirnas simultaneously have not been defined , although simultaneous use of multiple antagomirs targeting kshv mirnas demonstrates additive or synergistic suppression of kshv pathogenesis in vitro [ 20 , 44 , 56 ] . as the etiologic agent of diverse forms of human cancer and by virtue of its tropism for a variety of human cell types , kshv represents a model pathogen for the study of viral mirna expression and function . elegant studies performed recently underscore the importance of kshv mirnas and their interactions with human mirna for cancer pathogenesis , including viral biology and gene expression , cytokine responses , immune evasion , and anti - apoptotic signaling . the plasticity of these interactions and challenges inherent to mirna targeting in vivo incur substantial obstacles for development of mirna - based therapies , but recent advances hold considerable promise for eventual clinical application of therapeutic approaches targeting viral mirnas in the treatment of viral malignancies .

probes capable of recognizing specific mixed - sequence double - stranded dna ( dsdna ) regions have been long - sought - after as they can be developed into tools that enable modulation of gene expression at the transcriptional level , gene editing , and detection of specific genetic signatures . early examples of dsdna - targeting probes include triplex - forming oligonucleotides ( tfos ) and peptide nucleic acids ( pnas ) , as well as minor groove binding polyamides . unfortunately , only a subset of the possible target regions is available for recognition by these probes due to requirements for exclusive purine content ( tfos / pnas ) or short target regions ( polyamides ) . consequentially , significant efforts have been devoted to develop alternative approaches , which has resulted in tfos and pnas with reduced target site limitations . more recently , engineered proteins such as zinc finger nucleases , transcription activator - like effector nucleases ( talens ) and in particular crispr / cas9 systems , have gained a tremendous amount of attention , despite mounting concerns regarding recognition specificity and cellular delivery . another class of compounds that has emerged from these efforts are the so - called pseudocomplementary dna and pna ( pcdna / pcpna ) , in which a short dna or pna duplex is modified to contain pseudocomplementary base pairs between 2-thiothymine and 2,6-diaminopurine ( figure 1a ) . the steric clash between the 2-thio and the 2-amino group perturbs and weakens the hydrogen bonding between these moieties , resulting in a destabilized probe duplex . in contrast , 2-thiothymine and 2,6-diaminopurine form stable base pairs with canonical adenine and thymine , respectively . the difference in thermodynamic stability between probe duplexes and duplexes between individual probe strands and complementary dna ( cdna ) allows for double - duplex invasion of dsdna target regions under certain conditions ( figure 1b ) . thus , pcdna can recognize terminal target regions , while pcpna also recognize internal target regions of dna duplexes , albeit at low ionic strengths . however , a recent study has suggested that recognition of mixed - sequence dsdna regions by pcpna may be possible under the highly viscous conditions found in the nucleus . we , and later others , have pursued an alternative strategy for the construction of energetically activated double - stranded probes for recognition of mixed - sequence dsdna regions , which is based on forced intercalation of aromatic moieties . our invader probes are short dna duplexes containing + 1 interstrand zipper arrangements of intercalator - functionalized nucleotides ( figure 1c ; for a definition of the zipper nomenclature , see experimental section ) . this motif , which we denote as an energetic hotspot , forces two intercalators into the same region of the duplex , resulting in unwinding and destabilization as the nearest neighbor exclusion principle is violated . according to this principle , the space between the two nearest base pairs on either side of a bound intercalator , will not be bound by a second intercalator due to limitations in local helix expandability ( every intercalation event unwinds the duplex by 3.4 ) , and/or to avoid disruption of highly stable stacking interactions between the first bound intercalator and neighboring nucleobases . on the other hand , each of the two strands comprising an invader probe display very high affinity toward cdna since the intercalators stack strongly with the neighboring base pairs , acting as molecular glue ( figure 1c ) . we have previously demonstrated that the differences in thermostability between invader probes and probe - cdna duplexes can drive mixed - sequence recognition of isosequential dna duplexes , dna hairpins , and chromosomal dna targets . initially , 2-n-(pyren-1-yl)-2-amino--l - lna ( locked nucleic acid ) monomers were used to construct the energetic hotspots of invader probes . however , the challenging synthesis of these building blocks , prompted us to conduct a search for more feasible structural and functional mimics , which , among others , resulted in the identification of 2-n-(pyren-1-yl)methyl-2-n - methyl-2-aminouridine monomer x as a next - generation invader modification ( figure 1a ) . access to building blocks that are based on simpler sugar scaffolds has facilitated extensive structure property relationship studies aiming at improving the recognition efficiency of invader probes . in the present study , we set out to study if the dsdna - recognition potential of invader probes can be increased further through incorporation of pseudocomplementary base pairs . we hypothesized that these chimeric pseudocomplementary invader probes will be more energetically labile , yet form even more stable duplexes with cdna than either canonical invader or pcdna probes , leading to more favorable energetics for dsdna - recognition ( figure 1b ) . toward this end , two different approaches were followed . in the first , we wanted to integrate the intercalator as part of a pseudocomplementary nucleotide , which led to the identification of 2-n-(pyren-1-yl)methyl-2-n - methyl-2-amino-2-thiouridine monomer y as a target ( figure 1a ) . incorporation of this monomer in a + 1 interstrand zipper arrangement opposite of conventional 2,6-diaminopurine dna d monomers would furnish a double - stranded probe with a pseudocomplementary energetic hotspot ( dy probes ) ( figure 1b ) . in the second approach , we separated the two key structural features by using conventional 2-thiothymine dna monomer s and 2,6-diaminopurine dna monomer d , alongside energetic hotspots comprised of the conventional invader monomer x ( dsx probes ) ( figure 1b ) . we demonstrate that the latter type of probes is particularly interesting for dsdna - recognition applications . ( b ) representation of energy levels of different dsdna - targeting probes and the corresponding duplexes with cdna ( only one probe - target duplex is shown ) . note , the large difference in energy between pc - invader : cdna duplexes and pc - invader probe duplexes . the key 2-n-(pyren-1-yl)methyl-2-n - methyl-2-amino-2-thiouridine phosphoramidite 6 was obtained from known nucleoside 1 , following a similar general strategy as was used for the synthesis of 2-o-[2-(methoxy)ethyl]-2-thiothymidine ( scheme 1 ) . nucleoside 1obtained in 61% yield over six steps from uridine was first subjected to a sequence of protecting group manipulations , i.e. , 3-o - acetylation , 5-o - detritylation and 5-o - methanesulfonylation , to afford nucleoside 2 in 55% yield over three steps . prolonged refluxing in anhydrous ethanol in the presence of sodium bicarbonate , results in the formation of 2-o - ethyluridine derivative 3 in 52% yield , presumably via a nucleophilic opening of an o2,o5-anhydrouridine intermediate . subsequent o5-dimethoxytritylation of 3 using standard conditions affords nucleoside 4 in 88% yield , which upon treatment with h2s - saturated pyridine in the presence of 1,1,3,3-tetramethylguanidine provides 2-thiouridine derivative 5 in 82% yield . treatment of nucleoside 5 with 2-cyanoethyl - n , n - diisopropylchlorophosphoramidite and n , n - diisopropylethylamine affords target phosphoramidite 6 in 81% yield , corresponding to an overall yield of 17% from nucleoside 1 . dmtr = 4,4-dimethoxytrityl ; py = pyren-1-yl ; ms = methanesulfonyl ; tmg = 1,1,3,3-tetramethylguanidine ; pcl reagent = phosphoramidite 6 was used to incorporate monomer y into oligodeoxyribonucleotides ( ons ) via automated solid - phase dna synthesis . extended hand - coupling ( 15 min ) and the use of 4,5-dicyanoimidazole as an activator resulted in stepwise coupling yields of 95% . ons modified with monomer x were synthesized as previously described ( 15 min coupling , 5-[3,5-bis(trifluoromethyl)phenyl]-1h - tetrazole as an activator , 99% coupling yield ) . the corresponding phosphoramidites of monomers s and d were obtained from commercial sources and incorporated into ons using the conditions for incorporation of monomer y ( stepwise coupling yields > 95% ) . to prevent desulfurization in ons modified with y or s monomers , nucleotide phosphite to phosphate oxidation was performed using tert - butylhydroperoxide / ch3cn / h2o ( 10 min ) rather than the standard aqueous iodine solution . the identity and purity of the modified ons were established through maldi - tof ( table s1 ) and ion - pair reverse - phase hplc ( > 90% purity unless otherwise noted ) , respectively . pyrene - functionalized 2-thiouracil monomer y was incorporated into the same 9-mer mixed - sequence ons that was previously used for evaluation of invader monomer x. thermal denaturation temperatures ( tm s ) of duplexes between y - modified ons and cdna or crna were determined in a medium salt phosphate buffer ( [ na ] = 110 mm ) and compared relative to unmodified and corresponding x - modified duplexes . the resulting denaturation curves display the expected monophasic sigmoidal transitions ( figure s1 ) . duplexes between y1y4 and cdna are significantly more stable than unmodified reference duplexes ( tm between + 2.5 and + 11.5 c , table 1 ) , whereas heteroduplexes with crna are much less stable ( tm between 6.5 to + 2.5 c , table s2 ) . ons in which y monomers are flanked by 3-purines result in greater duplex stabilization than ons with 3-flanking pyrimidines ( e.g. , compare tm s for y1 and y3 , table 1 ) . this sequence dependence , along with the prominent dna selectivity ( table s3 ) , is typical for ons modified with intercalating pyrene moieties . surprisingly , y - modified ons form slightly less stable duplexes with cdna than their x - modified counterparts ( compare tm s of x1x4 and y1y4 , table 1 ) , which suggests that the binding modes responsible for the stabilizing contributions of the pyrene and 2-thiouracil moieties are not fully compatible . to generate invader probes with pseudocomplementary energetic hotspots , we synthesized ons in which 2-amino-2-deoxyadenosine monomers flank monomer y ( i.e. , the dy series ) . replacing regular 2-deoxyadenosines with d monomers increases the cdna / crna affinity of y - modified ons by 15 c , presumably due to stabilization from the extra hydrogen bond in d : t base pairs relative to normal a : t pairs ( e.g. , compare tm for y1 and dy1 , table 1 and table s2 ) . similar relative increases in cdna affinity are observed when 2-deoxyadenosines are replaced with d monomers in otherwise unmodified ons ( see tm for d1d4 vs cdna , table 1 ) , which is in agreement with previous studies . tm = change in tm relative to reference duplex dna1:dna2 ( tm 29.5 c ) , where dna1 : 5-gtg ata tgc and dna2 : 3-cac tat acg . tm s are determined as the maximum of the first derivative of melting curves ( a260 vs t ) recorded in medium salt phosphate buffer ( [ na ] = 110 mm , [ cl ] = 100 mm , ph 7.0 ( nah2po4/na2hpo4 ) ) , using 1.0 m of each strand . reported tm s are averages of at least two measurements within 1.0 c ; a = adenin-9-yl dna monomer , c = cytosin-1-yl dna monomer , g = guanin-9-yl dna monomer , and t = thymin-1-yl dna monomer . for structures of monomers next , we set out to study dna duplexes with different interstrand zipper arrangements of y monomers and dy segments to identify probe architectures that are strongly activated for dsdna - recognition . as expected from our previous studies on invader probes , y1:y3 , which features a + 1 interstrand zipper of y monomers , is much more thermolabile than y1:y2 featuring a 1 interstrand zipper ( table 2 ) , or duplexes between y - modified ons and cdna ( table 1 ) . the destabilization is likely a consequence of the nearest neighbor exclusion principle being violated . in line with our hypothesis , introduction of a 2,6-diaminopurine d monomer opposite of the pyrene - functionalized 2-thiouracil y monomer , decreases the tm s of the duplexes ( e.g. , compare tm of dy2:dy3 and y1:y2 , table 2 ) . interestingly , the destabilizing effect of the pseudocomplementary base pairs is more pronounced when the y monomers are positioned in a 1 zipper orientation ( drop in tm of 9.5 c from y1:y2 to dy2:dy3 , relative to drop of 2.0 c from y1:y3 to dy1:dy4 , table 2 ) . this indicates that the pseudocomplementary energetic hotspot architecture of dy1:dy4 does not fully harness the activating effects from both structural elements . the tm - based conclusions were largely corroborated by the gibbs free energies associated with duplex formation , which were derived from denaturation curves via line fitting ( table 2 ) . thus : ( i ) duplexes between y - modified ons and cdna are much more stable than unmodified reference duplexes ( g between 16 and 7 kj / mol , first and second g columns , table 2 ) due to more favorable enthalpy ( h between 76 and 44 kj / mol , table s7 ) . ( ii ) in comparison , the corresponding x - modified duplexes are slightly more stable , while the d - modified duplexes are far less stable ( e.g. , compare g for y1:cdna , x1:cdna and d1:cdna , table 2 ) . ( iii ) duplexes between ons with dy motifs and cdna are generally less stable than the corresponding y - modified duplexes ( e.g. , compare g for y1:cdna and dy2:cdna , table 2 ) . this contrasts the tm - based conclusions ( table 1 ) , but likely reflects the different entropies of these duplexes ( table s8 ) , leading to different temperature dependencies of the gibbs free energies . ( iv ) formation of y1:y3 ( + 1 zipper ) is less thermodynamically favorable than y1:y2 ( 1 zipper ) ( third g column , table 2 ) . ( v ) duplexes with pseudocomplementary energetic hotspots are slightly less stable than the corresponding duplexes featuring only regular energetic hotspots ( e.g. , compare g for dy1:dy4 and y1:y3 , table 2 ) . the energetic nature of dy1:dy4 is the result of particularly unfavorable enthalpy ( h = + 95 kj / mol , table s7 ) , likely reflecting a violation of the nearest neighbor exclusion principle and concomitant formation of destabilizing pseudocomplementary base pairs . ( vi ) pc - dna without any energetic hotspots range between being slightly more stable to slightly less stable than unmodified duplexes ( e.g. , see g for d1:y2 and y1:d3 , table 2 ) . consequentially , + 1 zipper duplex y1:y3 is much more energetically activated than 1 zipper duplex y1:y2 , as gauged by the free energy available for recognition of isosequential dsdna targets grec293 ( ona : onb ) = g ( ona : cdna ) + g ( cdna : onb ) g ( ona : onb ) g ( dsdna ) , where ona : onb denotes a double - stranded probe and dsdna is the isosequential dsdna target for ona : onb ( compare grec293 for y1:y2 and y1:y3 , table 2 ) . dy1:dy4 , featuring a pseudocomplementary energetic hotspot , is slightly more energetically activated for dsdna - recognition than y1:y3 ( compare grec293 for dy1:dy4 and y1:y3 , table 2 ) . as expected , dy2:dy3 , which also features two pseudocomplementary base pairs but has an 1 interstrand zipper arrangement of y monomers , is far less activated for dsdna - recognition ( compare grec293 for dy2:dy3 and dy1:dy4 , table 2 ) . pc - dna without energetic hotspots are also far less activated for dsdna - recognition ( e.g. , compare grec293 of y1:d3 and dy1:dy4 , table 2 ) . unexpectedly , dy1:dy4 has lower thermodynamic dsdna - recognition potential than conventional invader probe x1:x3 ( compare grec293 for dy1:dy4 and x1:x3 , table 2 ) , which likely is due to two factors : ( i ) intercalation of the pyrene moieties perturbs the local duplex geometry , which weakens the normally stabilizing base pairs between 2,6-diaminopurine : thymine ( d : t ) and 2-thiouracil : adenine ( y : a ) , leading to lower - than - expected cdna - affinity of dy - modified ons ( e.g. , compare g for x1:cdna , y1:cdna and dy1:cdna , table 2 ) , and ( ii ) + 1 interstrand zipper arrangements of nucleotide monomers with intercalating pyrene moieties perturb local duplex geometries , which , in turn , are likely to reduce the steric clash between 2,6-diaminopurine and 2-thiouracil normally occurring in pseudocomplementary base pairs , resulting in less pronounced probe destabilization . it is also possible that the pseudocomplementary base pairs of dy1:dy4 interfere with the forced and destabilizing intercalation of the two pyrene moieties ; this is supported by the uv vis absorption characteristics of dy1:dy4 . normally , dna duplexes with + 1 interstrand zipper motifs of intercalating pyrene - functionalized monomers exhibit markedly blue - shifted pyrene absorption relative to dna duplexes with other interstrand zipper motifs , which is indicative of reduced pyrene - nucleobase interactions due to a locally perturbed duplex geometry ( e.g. , compare max for x1:x3 relative to x1:x2 , table 2 ) . the same trend is observed for y1:y3 ( compare max for y1:y3 relative to y1:y2 ) , but the trend is less pronounced for dy1:dy4 ( compare max for dy1:dy4 relative to dy2:dy3 ) , indicating that dy1:dy4 is not strongly perturbed . zp = zipper . for conditions of thermal denaturation , see table 1 . grec293 ( ona : onb ) = g ( ona : cdna ) + g ( cdna : onb ) g ( ona : onb ) g ( dsdna ) . absorption spectra were recorded at t = 10 c using each strand at 1.0 m concentration in tm buffer . data for x1:x2 and x1:x3 are from ref ( 32 ) and are included to facilitate comparison . on the basis of the observed grec293 values , we decided to determine the dsdna - recognition properties of y1:y3 and dy1:dy4 relative to benchmark invader x1:x3 using an electrophoretic mobility shift assay from our earlier studies . thus , a digoxigenin ( dig ) labeled dna hairpin ( dh)comprised of a 9-mer double - stranded mixed - sequence stem , which is linked by a t10 loop was used as a model dsdna target ( figure 2a and 2b ) . room temperature incubation of dh1 with y1:y3 , dy1:dy4 , or x1:x3 in a hepes buffer of considerable ionic strength , results in dose - dependent formation of a ternary recognition complex as evidenced by the emergence of a slower migrating band on nondenaturing page gels ( figure 2c ) . response curves reveal that x1:x3 , y1:y3 , and dy1:dy4 have c50 values of 0.8 m , 2.8 m , and 1.5 m , respectively ( figure 2d ) , which mirrors the trend in grec293 values ( table 2 ) . the binding specificities of y1:y3 , dy1:dy4 , and x1:x3 were examined by incubating a 200-fold molar excess of the probes with dna hairpins dh2dh7 , which deviate in the nucleotide sequence at one position relative to the invader probes ( figure 2b ) . no recognition was observed , demonstrating that invader - mediated dsdna - recognition proceeds with excellent binding specificity ( figure 2e ) . ( a ) illustration of recognition process ; ( b ) sequences and thermal denaturation temperatures of dna hairpins with isosequential ( dh1 ) or nonisosequential stems ( dh2dh7 ) ; underlined nucleotides indicate sequence deviations relative to probes ; ( c ) representative electrophoretograms of recognition of dh1 using 1- to 500-fold molar excess of y1:y3 or dy1:dy4 ; ( d ) dose response curves ( average of at least three independent experiments ; error bars represent standard deviation ) ; ( e ) electrophoretograms illustrating incubation of dh1dh7 with 200-fold molar excess of x1:x3 , y1:y3 , or dy1:dy4 . experimental conditions for electrophoretic mobility shift assay : separately preannealed targets ( 34.4 nm ) and probes ( variable concentrations ) were incubated for 1216 h at ambient temperature in 1x hepes buffer ( 50 mm hepes , 100 mm nacl , 5 mm mgcl2 , 10% sucrose , 1.4 mm spermine tetrahydrochloride , ph 7.2 ) and then resolved on 16% nondenaturing page ( 70 v , 2.5 h , 4 c ) using 0.5 tbe as a running buffer ( 45 mm tris , 45 mm boric acid , 1 mm edta ) ; dig : digoxigenin . the above results suggest that incorporation of pseudocomplementary energetic hotspots i.e . , 5-yd-3:3-dy-5 cassettes provide less of a benefit for dsdna - recognition than originally anticipated . at this stage , we hypothesized that the two structural elements that activate invader and pc - dna probes for dsdna - recognition the intercalator - functionalized nucleotides forming the energetic hotspots , and the pseudocomplementary base pairs between 2,6-diaminopurine and 2-thiouracil need to be spatially separated in order to harness their full benefits . accordingly , a series of 13-mer dsx - modified invader probes were designed , i.e. , double - stranded probes featuring energetic hotspots comprised of conventional invader building block x , along with pseudocomplementary base pairs between regular 2,6-diaminopurine d and 2-thiothymine s monomers . the following probes were synthesized : ( i ) two different dsx - modified invader probes , in which the energetic hotspot either is next to or one nucleotide away from two pseudocomplementary base pairs ( dsx1:dsx2 and dsx3:dsx4 ) , ( ii ) an invader probe comprised of regular x monomers ( x5:x6 ) , ( iii ) an invader probe with a single pseudocomplementary energetic hotspot ( dy5:dy6 ) , and ( iv ) three pcdna probes , each containing two differentially spaced , regular pseudocomplementary base pairs ( sd1:sd2 , sd3:sd4 and sd5:sd6 ) ( table 3 ) . first , tm s for duplexes between individual probe strands and cdna ( table 3 , first two tm columns ) or crna ( table s9 ) were determined . in line with observations from the 9-mer series , x - modified ons form highly thermostable duplexes with cdna ( tm = 1112 c ) and less thermostable duplexes with crna ( tm = 3 c ) . in comparison , ons with dy motifs display slightly higher crna affinity ( tm for dy5/dy6 = 4.55.0 c , table s9 ) and slightly lower cdna affinity ( tm = 10 c , table 3 ) , again suggesting that the normally stabilizing base - pairs between 2,6-diaminoadenosine : thymine and 2-thiouracil : adenine are weakened by proximal intercalation of the pyrene moieties . encouragingly , dsx - modified ons display significantly increased cdna and crna affinity relative to x - modified ons ( tm , dna = 1415 c , table 3 ; tm , rna = 7.08.5 c , table s9 ) , suggesting that separation of the intercalators and the modified nucleobases is beneficial for binding affinity . in comparison , regular pcdna strands form far less stable duplexes with cdna ( tm , dna = 2.54.0 c , table 3 ) . benchmark invader probe x5:x6 and dy5:dy6 are both relatively thermolabile ( tm = 0.5 c and 1.5 c , respectively , table 3 ) . dsx probes are slightly less stable ( tm = 2.0 c and 3.0 c for dsx1:dsx2 and dsx3:dsx4 , respectively , table 3 ) . however , comparison with the corresponding pcdna probes suggests that the full destabilizing effect of the pseudocomplementary base pairs still is not fully realized with these probe architectures ( e.g. , compare tm of 8.0 , 0.5 , and 3.0 for sd5:sd6 , x5:x6 and dsx3:dsx4 , respectively , table 3 ) . the above tm - based conclusions are substantiated by the gibbs free energies for formation of duplexes ( table 3 ) . as a result of these stability trends , dsx probes are significantly more thermodynamically activated for dsdna - recognition than x5:x6 , dy5:dy6 , or any of the regular pcdna ( trend in grec293 values : dsx3:dsx4 < dsx1:dsx2 x5:x6 < dy5:dy6 sd5:sd6 < sd3:sd4 < sd1:sd2 , table 3 ) . tm = change in tm relative to reference duplexes dna3:dna4 ( tm 37.5 c ) , where dna3 : 5-ggta tatata ggc , dna4 : 3-ccat atatat ccg . x , y , d and s , see figure 1 . on the basis of the observed grec293 values , we decided to evaluate the dsdna - targeting efficiency of dsx1:dsx2 , dsx3:dsx4 , x5:x6 , and dy5:dy6 using a similar electrophoretic mobility shift assay as used in the 9-mer series ( figure 3a ) . thus , a dig - labeled dna hairpin ( dh8)comprised of a 13-mer double - stranded mixed - sequence stem that is linked by a t10 loop was used as a model dsdna target . incubation of dh8 with the various 13-mer invader probes results in dose - dependent formation of a slower moving band on nondenaturing page gels ( figure 3b ) . as expected from the initial 9-mer studies , the parent invader x5:x6 recognizes dh8 more effectively at low probe concentrations than dy5:dy6 , which has a pseudocomplementary energetic hotspot ( figure 3c ) . gratifyingly , invader probes with separated pseudocomplementary base pairs and energetic hotspots display improved recognition efficiency ( see curves for dsx1:dsx2 and dsx3:dsx4 , respectively , figure 3c ) , which follows the observed trend in grec293 values . it is particularly noteworthy that the use of as little as 1.0 mol equiv of dsx3:dsx4 results in 20% recognition of dh8 , especially when considering that optimal invader design normally calls for incorporation of multiple energetic hotspots . this suggests that spatial separation of pseudocomplementary base pairs and energetic hotspots is a promising principle for the design of efficient dsdna - targeting invader probes . in the present study , although there is an absence of suitable pseudocomplementary base pairs for cg - steps , we have previously shown that energetic hotspots can be comprised of intercalator - functionalized monomers that are based on any of the natural nucleobases ( although there is a preference for pyrimidine monomers ) . it is , therefore , likely possible to design dsx - like probes against dna targets with a higher gc - content than studied herein . recognition of dsdna model target dh8 using different invader probes . ( a ) illustration of recognition process ; ( b ) representative electrophoretograms for recognition of dh8 using 1- to 500-fold excess of x5:x6 , dy5:dy6 dsx1:dsx2 , or dsx3:dsx4 ; ( c ) dose response curves ( average of at least three independent experiments , error bars represent standard deviation ) . the sequence of dna hairpin dh8 is shown in figure 4 . for experimental conditions , lastly , binding specificity was studied by incubating the invader probes with dna hairpins dh9dh14 , which differ in the nucleotide sequence at one position in the stem region relative to the probes ( figure 4a ) . excellent discrimination of the nontarget dna hairpins is observed , even when using a 200-fold molar excess of x5:x6 , dy5:dy6 or dsx1:dsx2 ( figure 4b ) . similarly , high - affinity invader probe dsx3:dsx4 only results in trace recognition of dh10 and dh14 . to gain further insight into the underlying reasons for the excellent binding fidelity , we evaluated individual probe strands against singly mismatched single - stranded dna targets ( tables s10s12 ) . excellent mismatch discrimination is generally observed . in agreement with the observations from the dsdna - recognition experiments , the discrimination is least efficient when dsx3 or dsx4 are hybridized with single - stranded targets that have a sequence corresponding to the target region in dh10 and dh14 ( tm s only reduced by 57 c , relative to matched duplexes , table s12 ) . the binding of invader probes to noncomplementary dsdna targets would accordingly entail formation of two mismatched and destabilized probe - target duplexes , which is energetically prohibitive in most cases . moreover , like other structured probes , the double - stranded invader probes are also likely to exhibit improved binding specificity due to a thus , pseudocomplementary invader probes allow for strong and highly specific binding to mixed - sequence dsdna target regions at ionic conditions . ( a ) sequences and thermal denaturation temperatures of dna hairpins with isosequential ( dh8 ) or nonisosequential stems ( dh9dh14 ) ; underlined nucleotides denote sequence deviations relative to invader probes . ( b ) representative gel electrophoretograms illustrating incubation of dh8dh14 with 200-fold molar excess of x5:x6 , dy5:dy6 , dsx1:dsx2 , or dsx3:dsx4 . for experimental conditions , see figure 2 . a synthetic route to 2-n-(pyren-1-yl)methyl-2-amino-2-deoxy-2-n - methyl-2-thiouridine phosphoramidite 6 has been developed ( 10% overall yield over 13 steps from uridine ) . ons modified with the corresponding y monomer display high affinity toward cdna ( tm up to + 11.5 c ) . incorporation of 2-aminoadenosine monomer d next to the y monomer further increases cdna affinity . i.e. , 5-yd-3:3-dy-5 cassettes ) are thermolabile and activated for recognition of dsdna targets , but less so than regular invader probes with hotspots comprised of 2-n-(pyren-1-yl)methyl-2-n - methyl-2-aminouridine monomer x. in other words , close proximity of the two structural elements that normally activate pseudocomplementary dna and invader probes for dsdna - recognition i.e . , the weak base pairs between 2,6-diaminopurine and 2-thiouracil , and the destabilizing + 1 interstrand zipper arrangements of intercalating pyrene moieties does not provide a clear design benefit . in contrast , dsx probes , in which the two destabilizing structural motifs are separated , are strongly activated for recognition of mixed - sequence dsdna targets . this was confirmed in studies with model dsdna targets as efficient recognition was demonstrated to occur with excellent specificity . thus , the use of chimeric pseudocomplementary invader probes presents itself as a promising strategy for mixed - sequence recognition of dsdna for applications in molecular biology and nucleic acid diagnostics , especially since we have recently demonstrated that conventional invader probes can recognize target regions in chromosomal dna . nucleoside 1(32 ) ( 3.40 g , 4.39 mmol ) was coevaporated with anhydrous 1,2-dichloroethane ( 2 30 ml ) , redissolved in anhydrous pyridine ( 55 ml ) and cooled to 0 c . to this was added 4-dimethylaminopyridine ( dmap , 55 mg , 0.44 mmol ) and acetic anhydride ( 1.25 ml , 13.18 mmol ) . after stirring at ambient temperature for 12 h , the reaction mixture was diluted with etoac ( 150 ml ) and washed with h2o ( 80 ml ) and saturated aqueous nahco3 ( 80 ml ) . the organic layer was evaporated to near dryness and coevaporated with absolute etoh : toluene ( 2:1 v / v , 3 30 ml ) . the resulting residue ( 3.5 g ) was suspended in acoh : h2o ( 4:1 v / v , 55 ml ) and the reaction mixture was stirred overnight at room temperature . solvents were evaporated off and the resulting residue was purified by silica gel column chromatography ( 05% meoh / ch2cl2 , v / v ) to afford o5-hydroxy derivative ( 1.8 g ) as a white solid material . this material was coevaporated with anhydrous pyridine : ch2cl2 ( 1:1 v / v , 2 30 ml ) , redissolved in anhydrous pyridine : ch2cl2 ( 1:1 v / v , 44 ml ) and cooled to 20 c ( ice / salt ) . methanesulfonyl chloride ( 0.75 ml , 9.62 mmol ) was added dropwise over 30 min and the reaction mixture was stirred at 20 c for 2 h more . the reaction mixture was diluted with ch2cl2 ( 80 ml ) and washed with saturated aqueous nahco3 ( 50 ml ) . the aqueous layer was back - extracted with ch2cl2 ( 3 15 ml ) and the organic layers were combined and evaporated to dryness , followed by coevaporation with absolute etoh : toluene ( 2:1 v / v , 3 30 ml ) . the resulting residue was purified by silica gel column chromatography ( 03% meoh / ch2cl2 , v / v ) to afford nucleoside 2 ( 1.43 g , 55% ) as a white foam . rf = 0.4 ( 5% meoh in ch2cl2 , v / v ) ; maldi - hrms m / z 614.1600 ( [ m + na ] , c30h29n3o8sna , calc . 614.1568 ) ; h nmr ( 500 mhz , dmso - d6 ) 11.47 ( d , 1h , ex , j = 2.2 hz , nh ) , 8.338.36 ( d , 1h , j = 9.0 hz , py ) , 8.258.29 ( ap t , 2h , j = 7.6 hz , py ) , 8.21 ( d , 1h , j = 7.5 hz , py ) , 8.15 ( br s , 2h , py ) , 8.108.12 ( d , 1h , j = 9.0 hz , py ) , 8.048.08 ( t , 1h , j = 7.6 hz , py ) , 7.92 ( d , 1h , j = 7.5 hz , py ) , 7.73 ( d , 1h , j = 8.2 hz , h6 ) , 6.40 ( d , 1h , j = 7.4 hz , h1 ) , 5.67 ( dd , 1h , j = 8.2 hz , 2.2 hz , h5 ) , 5.39 ( dd , 1h , j = 6.6 hz , 3.6 hz , h3 ) , 4.444.48 ( m , 2h , h5 ) , 4.344.43 ( m , 3h , ch2py , h4 ) , 3.84 ( ap t , 1h , j 7.5 hz , h2 ) , 3.20 ( s , 3h , ch3so2 ) , 2.34 ( s , 3h , ch3n ) , 2.13 ( s , 3h , ch3co ) ; c nmr ( 125 mhz , dmso - d6 ) 169.7 , 162.7 , 150.5 , 140.6 ( c6 ) , 131.9 , 130.7 , 130.3 , 130.2 , 129.1 , 127.7 ( py ) , 127.3 ( py ) , 127.0 ( py ) , 126.8 ( py ) , 126.1 ( py ) , 125.1 ( py ) , 124.4 ( py ) , 124.2 , 123.8 , 123.7 ( py ) , 102.6 ( c5 ) , 83.7 ( c1 ) , 80.0 ( c4 ) , 71.7 ( c3 ) , 69.1 ( c5 ) , 64.9 ( c2 ) , 57.5 ( ch2py ) , 37.7 ( ch3n ) , 36.8 ( ch3so2 ) , 20.9 ( ch3co ) . nucleoside 2 ( 0.77 g , 1.30 mmol ) was dried through coevaporation with anhydrous 1,2-dichloroethane ( 3 6 ml ) and suspended in anhydrous etoh ( 25 ml ) . to this was added nahco3 ( 275 mg , 3.25 mmol ) and the reaction mixture was refluxed for 4 days . ch2cl2 ( 100 ml ) was added and the precipitate was filtered off and washed with ch2cl2 . the combined organic layers were evaporated to dryness and the resulting residue was purified via silica gel column chromatography ( 07% meoh in ch2cl2 , v / v ) to afford nucleoside 3 ( 0.34 g , 52% ) as a white foam . rf = 0.3 ( 10% meoh in ch2cl2 , v / v ) ; maldi - hrms m / z 522.1985 ( [ m + na ] , c29h29n3o5na , calc . 522.1999 ) ; h nmr ( 500 mhz , dmso - d6 ) 8.398.42 ( d , 1h , j = 9.3 hz , py ) , 8.248.28 ( m , 2h , py ) , 8.198.22 ( d , 1h , j = 8.0 hz , py ) , 8.14 ( br s , 2h , py ) , 8.038.10 ( d+t , 2h , py ) , 7.967.99 ( d , 1h , j = 8.0 hz , py ) , 7.93 ( d , 1h , j = 7.7 hz , h6 ) , 6.35 ( d , 1h , j = 8.5 hz , h1 ) , 5.83 ( d , 1h , j = 7.7 hz , h5 ) , 5.53 ( d , 1h , ex , j = 4.9 hz , 3oh ) , 5.12 ( t , 1h , ex , j = 5.2 hz , 5oh ) , 4.434.51 ( m , 3h , ch2py , h3 ) , 4.124.27 ( m , 2h , och2ch3 ) , 3.994.01 ( m , 1h , h4 ) , 3.583.62 ( m , 2h , h5 ) , 3.47 ( dd , 1h , j = 8.5 hz , 5.2 hz , h2 ) , 2.36 ( s , 3h , ch3n ) , 1.06 ( t , 3h , j = 7.1 hz , ch3ch2o ) ; c nmr ( 125 mhz , dmso - d6 ) 169.3 , 155.0 , 138.1 ( c6 ) , 132.7 , 130.7 , 130.3 , 130.1 , 129.0 , 127.5 ( py ) , 127.3 ( py ) , 126.9 ( py ) , 126.8 ( py ) , 126.1 ( py ) , 125.02 ( py ) , 124.99 ( py ) , 124.4 ( py ) , 124.1 , 123.8 , 123.6 ( py ) , 108.4 ( c5 ) , 87.6 ( c4 ) , 85.0 ( c1 ) , 71.2 ( c3 ) , 68.8 ( c2 ) , 64.2 ( och2ch3 ) , 61.7 ( c5 ) , 57.1 ( ch2py ) , 39.0 ( ch3n overlap with dmso - d6 signal ) , 13.6 ( ch3ch2o ) . nucleoside 3 ( 0.32 g , 0.63 mmol ) was coevaporated with anhydrous 1,2-dichloroethane ( 2 5 ml ) and redissolved in anhydrous pyridine ( 6 ml ) . to this was added dmtrcl ( 0.26 g , 0.76 mmol ) and 4-dimethylaminopyridine ( dmap , 8 mg , 0.06 mmol ) and the reaction mixture was stirred at ambient temperature for 14 h at which point it was diluted with chcl3 ( 80 ml ) and washed with saturated aqueous nahco3 ( 30 ml ) and h2o ( 30 ml ) . the aqueous layer was back - extracted with chcl3 ( 3 15 ml ) and the combined organic layers were dried ( na2so4 ) and evaporated to dryness . the resulting residue was purified by silica gel column chromatography ( 02.5% meoh in chcl3 , v / v ) to afford nucleoside 4 ( 0.45 g , 88% ) as a pale orange foam . rf = 0.7 ( 10% meoh in ch2cl2 , v / v ) ; maldi - hrms m / z 824.3319 ( [ m + na ] , c50h47n3o7na , calc . 824.3306 ) ; h nmr ( 500 mhz , dmso - d6 ) 8.388.41 ( d , 1h , j = 9.3 hz , py ) , 8.258.29 ( m , 2h , py ) , 8.178.19 ( d , 1h , j = 7.8 hz , py ) , 8.118.15 ( 2d , 2h , j = 9.1 hz , 9.1 hz , py ) , 8.048.09 ( m , 2h , py ) , 7.988.00 ( d , j = 7.8 hz , py ) , 7.69 ( d , 1h , j = 7.8 hz , h6 ) , 7.197.38 ( m , 9h , dmtr ) , 6.836.88 ( m , 4h , dmtr ) , 6.32 ( d , 1h , j = 7.9 hz , h1 ) , 5.605.64 ( m , 2d , 1 ex , j = 7.8 hz , 5.0 hz , h5 , 3oh ) , 4.484.54 ( m , 2h , ch2py ) , 4.444.48 ( m , 1h , h3 ) , 4.104.24 ( m , 3h , h4 , och2ch3 ) , 3.71 ( s , 3h , ch3o ) , 3.70 ( s , 3h , ch3o ) , 3.523.58 ( dd , 1h , j = 7.9 hz , 5.8 hz , h2 ) , 3.313.35 ( dd , 1h , j = 10.5 hz , 5.0 hz , h5 - partial overlap with h2o ) , 3.153.20 ( dd , 1h , j = 10.5 hz , 3.5 hz , h5 ) , 2.43 ( s , 3h , ch3n ) , 1.05 ( t , 3h , j = 7.0 hz , ch3ch2o ) ; c nmr ( 125 mhz , dmso - d6 ) 169.2 , 158.09 , 158.08 , 154.9 , 144.5 , 138.1 ( c6 ) , 135.3 , 135.0 , 132.6 , 130.7 , 130.2 , 130.1 , 129.7 ( dmtr ) , 129.6 ( dmtr ) , 129.0 , 127.8 ( dmtr ) , 127.6 ( dmtr ) , 127.5 ( py ) , 127.3 ( py ) , 126.91 ( py ) , 126.86 ( py ) , 126.7 ( dmtr ) , 126.1 ( py ) , 125.0 ( py ) , 124.4 ( py ) , 124.1 , 123.9 , 123.6 ( py ) , 113.20 ( dmtr ) , 113.17 ( dmtr ) , 108.1 ( c5 ) , 85.9 , 85.7 ( c4 ) , 85.3 ( c1 ) , 71.0 ( c3 ) , 68.1 ( c2 ) , 64.2 ( och2ch3 ) , 64.0 ( c5 ) , 57.1 ( ch2py ) , 55.0 ( ch3o ) , 38.8 ( ch3n ) , 13.6 ( ch3ch2o ) . a trace impurity of chcl3 was identified in the c nmr at 79.1 ppm . an ice - cold solution of anhydrous 1,1,3,3-tetramethylguanidine ( tmg , 0.68 ml , 5.42 mmol ) in anhydrous pyridine ( 10 ml ) was saturated with hydrogen sulfide gas for 1 h while maintaining the temperature at 0 c . the solution was transferred , using an argon - flushed syringe , to a precooled flask containing nucleoside 4 ( 0.44 g , 0.54 mmol ) and the reaction mixture was allowed to reach room temperature . after stirring under an argon atmosphere for 72 h , etoac ( 100 ml ) was added and the organic layer was washed with saturated aqueous nahco3 ( 50 ml ) and h2o ( 50 ml ) . the aqueous layer was back - extracted with ch2cl2 ( 3 20 ml ) and the combined organic layers were evaporated to dryness and coevaporated with absolute etoh : toluene ( 2:1 v / v , 3 15 ml ) . the resulting residue was purified by silica gel column chromatography ( 070% etoac in petroleum ether , v / v ) to afford nucleoside 5 ( 0.35 g , 82% ) as a white foam . rf = 0.8 ( 80% etoac in petroleum ether , v / v ) ; maldi - hrms m / z 812.2765 ( [ m + na ] , c48h43n3o6sna , calc . 812.2797 ) ; h nmr ( 500 mhz , dmso - d6 ) 12.77 ( br s , 1h , ex , nh ) , 8.498.52 ( d , 1h , j = 9.3 hz , py ) , 8.248.29 ( m , 2h , py ) , 8.178.20 ( d , 1h , j = 8.0 hz , py ) , 8.128.16 ( 2d , 2h , j = 9.1 hz , 9.1 hz , py ) , 8.028.11 ( m , 3h , py ) , 7.75 ( d , 1h , j = 8.2 hz , h6 ) , 7.207.39 ( m , 10h , dmtr , h1 ) , 6.846.89 ( m , 4h , dmtr ) , 5.62 ( dd , 1h , j = 8.2 hz , 1.7 hz , h5 ) , 5.55 ( d , 1h , ex , j = 4.9 hz , 3oh ) , 4.534.57 ( d , 1h , j = 13.0 hz , ch2py ) , 4.434.51 ( m , 2h , ch2py , h3 ) , 4.084.12 ( m , 1h , h4 ) , 3.71 ( s , 3h , ch3o ) , 3.70 ( s , 3h , ch3o ) , 3.483.52 ( m , 1h , h2 ) , 3.333.37 ( dd , 1h , j = 10.5 hz , 5.0 hz , h5 ) , 3.183.22 ( dd , 1h , j = 10.5 hz , 3.0 hz , h5 ) , 2.44 ( s , 3h , ch3n ) ; c nmr ( 125 mhz , dmso - d6 ) 176.6 , 159.0 , 158.11 , 158.10 , 144.5 , 140.8 ( c6 ) , 135.3 , 135.0 , 132.6 , 130.7 , 130.3 , 130.2 , 129.73 ( dmtr ) , 129.68 ( dmtr ) , 129.2 , 128.0 ( py ) , 127.9 ( dmtr ) , 127.6 ( dmtr ) , 127.3 ( py ) , 127.0 ( py ) , 126.8 ( py ) , 126.7 ( dmtr ) , 126.1 ( py ) , 125.02 ( py ) , 125.00 ( py ) , 124.4 ( py ) , 124.1 , 124.0 ( py ) , 123.9 , 113.3 ( dmtr ) , 113.2 ( dmtr ) , 106.9 ( c5 ) , 88.0 ( c1 ) , 86.0 , 85.4 ( c4 ) , 71.0 ( c3 ) , 68.6 ( c2 ) , 63.9 ( c5 ) , 57.6 ( ch2py ) , 55.0 ( ch3o ) , 39.2 ( ch3n ; overlap with dmso - d6 ) . to a flame - dried round - bottomed flask containing nucleoside 5 ( 150 mg , 0.19 mmol ) was added anhydrous ch2cl2 ( 2 ml ) , anhydrous n , n - diisopropylethylamine ( dipea , 165 l , 0.95 mmol ) and 2-cyanoethyl - n , n - diisopropylchlorophosphoramidite ( pcl reagent , 85 l , 0.38 mmol ) . the reaction mixture was stirred at room temperature for 3.5 h at which point ice - cold etoh ( 1.0 ml ) was added . the reaction mixture was evaporated to dryness and the resulting residue was purified by silica gel column chromatography ( 055% etoac in petroleum ether , v / v ) followed by precipitation from cold petroleum ether to afford nucleoside 6 ( 153 mg , 81% ) as a white foam . rf = 0.6 ( 50% etoac in petroleum ether , v / v ) ; maldi - hrms m / z 1012.3843 ( [ m + na ] , c57h60n5o7psna , calc . 1012.3887 ) ; p nmr ( 121 mhz , cdcl3 ) 150.9 , 149.6 . modified ons were synthesized on a 0.2 mol scale using a dna synthesizer , succinyl - linked lcaa - cpg ( long chain alkyl amine controlled pore glass ) columns with a pore size of 500 , and standard protocols for incorporation of a , c , g and t dna phosphoramidites . the following hand - coupling conditions were used for incorporation of the corresponding phosphoramidites of monomers x , y , s ( n3/o4-toluoyl protected ) and d ( bis(diisobutylaminomethylidene)-protected ) ( coupling time ; activator ; coupling yield ) : x ( 15 min ; 5-[3,5-bis(trifluoromethyl)phenyl]-1h - tetrazole ; 99% ) , y ( 15 min ; 4,5-dicyanoimidazole ; 95% ) and s / d ( 15 min ; 4,5-dicyanoimidazole ; 99% ) . modified phosphoramidites were used at 50-fold molar excess and 0.05 m concentration in ch3cn . extended oxidation ( 45 s ) with standard 0.05 m aqueous iodine was used for d1d4 and x1x6 . extended oxidation ( 2 5 min oxidation with an acetonitrile wash between oxidations ) using a tert - butylhydroperoxide / ch3cn / h2o solution ( 10/87/3 , v / v / v ) was used for all ons containing s and y modifications to prevent desulfurization . cleavage from solid support and removal of protecting groups was accomplished by treatment with 32% aq . ons were purified in the dmt - on mode via ion - pair reverse phase hplc ( c18 column ) using a 0.05 m triethylammonium acetate . acoh ) and precipitation ( naoac / naclo4/acetone , 18 c for 1216 h ) . the identity of synthesized ons was established through maldi - ms analysis ( table s1 ) recorded in positive ions mode on a quadrupole time - of - flight tandem mass spectrometer equipped with a maldi source using anthranilic acid , 3-hydroxypicolinic acid ( 3-hpa ) or 2,4,6-trihydroxyacetophenone ( thap ) as matrices . purity was verified by ion - pair reverse phase hplc running in analytical mode ( > 90% unless otherwise mentioned ) . on concentrations were estimated using the following extinction coefficients for dna ( od/mol ) : g ( 12.01 ) , a ( 15.20 ) , t ( 8.40 ) , c ( 7.05 ) ; rna ( od/mol ) : g ( 13.70 ) , a ( 15.40 ) , u ( 10.00 ) , c ( 9.00 ) ; pyrene ( 22.4),d ( 8.5 ) , s ( 10.0 ) and y ( 32.4 ) . strands were thoroughly mixed and denatured by heating to 7085 c , followed by cooling to the starting temperature of the experiment . thermal denaturation temperatures ( tm s ) of duplexes ( 1.0 m final concentration of each strand ) were measured using a uv / vis spectrophotometer equipped with a 12-cell peltier temperature controller and determined as the maximum of the first derivative of thermal denaturation curves ( a260 vs t ) recorded in medium salt phosphate buffer ( tm buffer : 100 mm nacl , 0.1 mm edta and ph 7.0 adjusted with 10 mm na2hpo4 and 5 mm na2hpo4 ) . the temperature of the denaturation experiments ranged from at least 15 c below tm to 20 c above tm ( although not below 3 c ) . thermodynamic parameters for duplex formation were determined through fitting of baselines of denaturation curves ( vant hoff analysis ) using software provided with the uv / vis spectrometer . bimolecular reactions , two - state melting behavior , and a heat capacity change of cp = 0 upon hybridization were assumed . a minimum of two experimental denaturation curves were each analyzed at least three times to minimize errors arising from baseline choice . uv vis absorption spectra ( range 200600 nm ) were recorded at 10 c using the same samples and instrumentation as in the thermal denaturation experiments . steady - state fluorescence emission spectra of y- or dy - modified ons and the corresponding duplexes with complementary dna / rna targets , were recorded in nondeoxygenated thermal denaturation buffer ( each strand at 1.0 m concentration ) and obtained as an average of five scans using an excitation wavelength of ex = 350 nm . excitation and emission slits of 5.0 and 2.5 nm , respectively , were used along with a scan speed of 600 nm / min . experiments were determined at 5 c ( to ascertain maximal hybridization of probes to dna / rna targets ) under n2 flow ( to prevent condensation ) . unmodified dna hairpins dh1dh14 were obtained from commercial sources and used without further purification . the dna hairpins were 3-dig - labeled using the second generation dig gel shift kit ( roche applied bioscience ) following the manufacturer s recommendation . dig - labeled ons obtained in this manner were diluted and used without further purification in the recognition experiments . preannealed probes ( 85 c for 10 min , cooled to room temperature over 15 min ) and dig - labeled dna hairpins ( 34.4 nm ) were mixed and incubated in hepes buffer ( 50 mm hepes , 100 mm nacl , 5 mm mgcl2 , 10% sucrose , 1.44 mm spermine tetrahydrochloride , ph 7.2 ) for the specified time at ambient temperature ( 21 3 c ) . the reaction mixtures were then diluted with 6x dna loading dye ( fermentas ) and loaded onto a 16% nondenaturing polyacrylamide gel . electrophoresis was performed using a constant voltage of 70 v for 2.5 h at 4 c using 0.5 tbe as a running buffer ( 45 mm tris , 45 mm boric acid , 1 mm edta ) . gels were blotted onto positively charged nylon membranes ( roche applied bioscience ) using constant voltage with external cooling ( 100 v , 4 c ) . the membranes were exposed to antidigoxigenin - ap fab fragments as recommended by the manufacturer of the dig gel shift kit , transferred to a hybridization jacket , and incubated with the substrate ( cspd ) in detection buffer for 10 min at 37 c . the chemiluminescence of the formed product was captured on x - ray film , which was developed using an x - omatic 1000a x - ray film developer ( kodak ) . the efficiency of dna recognition was determined as the intensity ratio between the recognition complex band and the total lane . nonlinear regression was used to fit data points from dose response experiments , using a script written for the solver module in microsoft office excel . the following nomenclature describes the relative arrangement between two pyrene - functionalized monomers positioned on opposing strands in a duplex : the number n describes the distance measured in number of base pairs and has a positive value if a monomer is shifted toward the 5-side of its own strand relative to a second reference monomer on the other strand . conversely , n has a negative value if a monomer is shifted toward the 3-side of its own strand relative to a second reference monomer on the other strand .

the development of molecular strategies that enable recognition of specific double - stranded dna ( dsdna ) regions has been a longstanding goal as evidenced by the emergence of triplex - forming oligonucleotides , peptide nucleic acids ( pnas ) , minor groove binding polyamides , and more recently engineered proteins such as crispr / cas9 . despite this progress , an unmet need remains for simple hybridization - based probes that recognize specific mixed - sequence dsdna regions under physiological conditions . herein , we introduce pseudocomplementary invader probes as a step in this direction . these double - stranded probes are chimeras between pseudocomplementary dna ( pcdna ) and invader probes , which are activated for mixed - sequence dsdna - recognition through the introduction of pseudocomplementary base pairs comprised of 2-thiothymine and 2,6-diaminopurine , and + 1 interstrand zipper arrangements of intercalator - functionalized nucleotides , respectively . we demonstrate that certain pseudocomplementary invader probe designs result in very efficient and specific recognition of model dsdna targets in buffers of high ionic strength . these chimeric probes , therefore , present themselves as a promising strategy for mixed - sequence recognition of dsdna targets for applications in molecular biology and nucleic acid diagnostics .

Introduction Results and Discussion Conclusion Experimental Section

probes capable of recognizing specific mixed - sequence double - stranded dna ( dsdna ) regions have been long - sought - after as they can be developed into tools that enable modulation of gene expression at the transcriptional level , gene editing , and detection of specific genetic signatures . early examples of dsdna - targeting probes include triplex - forming oligonucleotides ( tfos ) and peptide nucleic acids ( pnas ) , as well as minor groove binding polyamides . more recently , engineered proteins such as zinc finger nucleases , transcription activator - like effector nucleases ( talens ) and in particular crispr / cas9 systems , have gained a tremendous amount of attention , despite mounting concerns regarding recognition specificity and cellular delivery . another class of compounds that has emerged from these efforts are the so - called pseudocomplementary dna and pna ( pcdna / pcpna ) , in which a short dna or pna duplex is modified to contain pseudocomplementary base pairs between 2-thiothymine and 2,6-diaminopurine ( figure 1a ) . in contrast , 2-thiothymine and 2,6-diaminopurine form stable base pairs with canonical adenine and thymine , respectively . the difference in thermodynamic stability between probe duplexes and duplexes between individual probe strands and complementary dna ( cdna ) allows for double - duplex invasion of dsdna target regions under certain conditions ( figure 1b ) . however , a recent study has suggested that recognition of mixed - sequence dsdna regions by pcpna may be possible under the highly viscous conditions found in the nucleus . we , and later others , have pursued an alternative strategy for the construction of energetically activated double - stranded probes for recognition of mixed - sequence dsdna regions , which is based on forced intercalation of aromatic moieties . our invader probes are short dna duplexes containing + 1 interstrand zipper arrangements of intercalator - functionalized nucleotides ( figure 1c ; for a definition of the zipper nomenclature , see experimental section ) . on the other hand , each of the two strands comprising an invader probe display very high affinity toward cdna since the intercalators stack strongly with the neighboring base pairs , acting as molecular glue ( figure 1c ) . we have previously demonstrated that the differences in thermostability between invader probes and probe - cdna duplexes can drive mixed - sequence recognition of isosequential dna duplexes , dna hairpins , and chromosomal dna targets . however , the challenging synthesis of these building blocks , prompted us to conduct a search for more feasible structural and functional mimics , which , among others , resulted in the identification of 2-n-(pyren-1-yl)methyl-2-n - methyl-2-aminouridine monomer x as a next - generation invader modification ( figure 1a ) . in the present study , we set out to study if the dsdna - recognition potential of invader probes can be increased further through incorporation of pseudocomplementary base pairs . we hypothesized that these chimeric pseudocomplementary invader probes will be more energetically labile , yet form even more stable duplexes with cdna than either canonical invader or pcdna probes , leading to more favorable energetics for dsdna - recognition ( figure 1b ) . in the first , we wanted to integrate the intercalator as part of a pseudocomplementary nucleotide , which led to the identification of 2-n-(pyren-1-yl)methyl-2-n - methyl-2-amino-2-thiouridine monomer y as a target ( figure 1a ) . incorporation of this monomer in a + 1 interstrand zipper arrangement opposite of conventional 2,6-diaminopurine dna d monomers would furnish a double - stranded probe with a pseudocomplementary energetic hotspot ( dy probes ) ( figure 1b ) . in the second approach , we separated the two key structural features by using conventional 2-thiothymine dna monomer s and 2,6-diaminopurine dna monomer d , alongside energetic hotspots comprised of the conventional invader monomer x ( dsx probes ) ( figure 1b ) . we demonstrate that the latter type of probes is particularly interesting for dsdna - recognition applications . the identity and purity of the modified ons were established through maldi - tof ( table s1 ) and ion - pair reverse - phase hplc ( > 90% purity unless otherwise noted ) , respectively . pyrene - functionalized 2-thiouracil monomer y was incorporated into the same 9-mer mixed - sequence ons that was previously used for evaluation of invader monomer x. thermal denaturation temperatures ( tm s ) of duplexes between y - modified ons and cdna or crna were determined in a medium salt phosphate buffer ( [ na ] = 110 mm ) and compared relative to unmodified and corresponding x - modified duplexes . duplexes between y1y4 and cdna are significantly more stable than unmodified reference duplexes ( tm between + 2.5 and + 11.5 c , table 1 ) , whereas heteroduplexes with crna are much less stable ( tm between 6.5 to + 2.5 c , table s2 ) . for structures of monomers next , we set out to study dna duplexes with different interstrand zipper arrangements of y monomers and dy segments to identify probe architectures that are strongly activated for dsdna - recognition . as expected from our previous studies on invader probes , y1:y3 , which features a + 1 interstrand zipper of y monomers , is much more thermolabile than y1:y2 featuring a 1 interstrand zipper ( table 2 ) , or duplexes between y - modified ons and cdna ( table 1 ) . in line with our hypothesis , introduction of a 2,6-diaminopurine d monomer opposite of the pyrene - functionalized 2-thiouracil y monomer , decreases the tm s of the duplexes ( e.g. the tm - based conclusions were largely corroborated by the gibbs free energies associated with duplex formation , which were derived from denaturation curves via line fitting ( table 2 ) . the energetic nature of dy1:dy4 is the result of particularly unfavorable enthalpy ( h = + 95 kj / mol , table s7 ) , likely reflecting a violation of the nearest neighbor exclusion principle and concomitant formation of destabilizing pseudocomplementary base pairs . consequentially , + 1 zipper duplex y1:y3 is much more energetically activated than 1 zipper duplex y1:y2 , as gauged by the free energy available for recognition of isosequential dsdna targets grec293 ( ona : onb ) = g ( ona : cdna ) + g ( cdna : onb ) g ( ona : onb ) g ( dsdna ) , where ona : onb denotes a double - stranded probe and dsdna is the isosequential dsdna target for ona : onb ( compare grec293 for y1:y2 and y1:y3 , table 2 ) . dy1:dy4 , featuring a pseudocomplementary energetic hotspot , is slightly more energetically activated for dsdna - recognition than y1:y3 ( compare grec293 for dy1:dy4 and y1:y3 , table 2 ) . as expected , dy2:dy3 , which also features two pseudocomplementary base pairs but has an 1 interstrand zipper arrangement of y monomers , is far less activated for dsdna - recognition ( compare grec293 for dy2:dy3 and dy1:dy4 , table 2 ) . pc - dna without energetic hotspots are also far less activated for dsdna - recognition ( e.g. unexpectedly , dy1:dy4 has lower thermodynamic dsdna - recognition potential than conventional invader probe x1:x3 ( compare grec293 for dy1:dy4 and x1:x3 , table 2 ) , which likely is due to two factors : ( i ) intercalation of the pyrene moieties perturbs the local duplex geometry , which weakens the normally stabilizing base pairs between 2,6-diaminopurine : thymine ( d : t ) and 2-thiouracil : adenine ( y : a ) , leading to lower - than - expected cdna - affinity of dy - modified ons ( e.g. , compare g for x1:cdna , y1:cdna and dy1:cdna , table 2 ) , and ( ii ) + 1 interstrand zipper arrangements of nucleotide monomers with intercalating pyrene moieties perturb local duplex geometries , which , in turn , are likely to reduce the steric clash between 2,6-diaminopurine and 2-thiouracil normally occurring in pseudocomplementary base pairs , resulting in less pronounced probe destabilization . it is also possible that the pseudocomplementary base pairs of dy1:dy4 interfere with the forced and destabilizing intercalation of the two pyrene moieties ; this is supported by the uv vis absorption characteristics of dy1:dy4 . normally , dna duplexes with + 1 interstrand zipper motifs of intercalating pyrene - functionalized monomers exhibit markedly blue - shifted pyrene absorption relative to dna duplexes with other interstrand zipper motifs , which is indicative of reduced pyrene - nucleobase interactions due to a locally perturbed duplex geometry ( e.g. on the basis of the observed grec293 values , we decided to determine the dsdna - recognition properties of y1:y3 and dy1:dy4 relative to benchmark invader x1:x3 using an electrophoretic mobility shift assay from our earlier studies . thus , a digoxigenin ( dig ) labeled dna hairpin ( dh)comprised of a 9-mer double - stranded mixed - sequence stem , which is linked by a t10 loop was used as a model dsdna target ( figure 2a and 2b ) . room temperature incubation of dh1 with y1:y3 , dy1:dy4 , or x1:x3 in a hepes buffer of considerable ionic strength , results in dose - dependent formation of a ternary recognition complex as evidenced by the emergence of a slower migrating band on nondenaturing page gels ( figure 2c ) . response curves reveal that x1:x3 , y1:y3 , and dy1:dy4 have c50 values of 0.8 m , 2.8 m , and 1.5 m , respectively ( figure 2d ) , which mirrors the trend in grec293 values ( table 2 ) . the binding specificities of y1:y3 , dy1:dy4 , and x1:x3 were examined by incubating a 200-fold molar excess of the probes with dna hairpins dh2dh7 , which deviate in the nucleotide sequence at one position relative to the invader probes ( figure 2b ) . , 5-yd-3:3-dy-5 cassettes provide less of a benefit for dsdna - recognition than originally anticipated . at this stage , we hypothesized that the two structural elements that activate invader and pc - dna probes for dsdna - recognition the intercalator - functionalized nucleotides forming the energetic hotspots , and the pseudocomplementary base pairs between 2,6-diaminopurine and 2-thiouracil need to be spatially separated in order to harness their full benefits . , double - stranded probes featuring energetic hotspots comprised of conventional invader building block x , along with pseudocomplementary base pairs between regular 2,6-diaminopurine d and 2-thiothymine s monomers . the following probes were synthesized : ( i ) two different dsx - modified invader probes , in which the energetic hotspot either is next to or one nucleotide away from two pseudocomplementary base pairs ( dsx1:dsx2 and dsx3:dsx4 ) , ( ii ) an invader probe comprised of regular x monomers ( x5:x6 ) , ( iii ) an invader probe with a single pseudocomplementary energetic hotspot ( dy5:dy6 ) , and ( iv ) three pcdna probes , each containing two differentially spaced , regular pseudocomplementary base pairs ( sd1:sd2 , sd3:sd4 and sd5:sd6 ) ( table 3 ) . as a result of these stability trends , dsx probes are significantly more thermodynamically activated for dsdna - recognition than x5:x6 , dy5:dy6 , or any of the regular pcdna ( trend in grec293 values : dsx3:dsx4 < dsx1:dsx2 x5:x6 < dy5:dy6 sd5:sd6 < sd3:sd4 < sd1:sd2 , table 3 ) . on the basis of the observed grec293 values , we decided to evaluate the dsdna - targeting efficiency of dsx1:dsx2 , dsx3:dsx4 , x5:x6 , and dy5:dy6 using a similar electrophoretic mobility shift assay as used in the 9-mer series ( figure 3a ) . thus , a dig - labeled dna hairpin ( dh8)comprised of a 13-mer double - stranded mixed - sequence stem that is linked by a t10 loop was used as a model dsdna target . gratifyingly , invader probes with separated pseudocomplementary base pairs and energetic hotspots display improved recognition efficiency ( see curves for dsx1:dsx2 and dsx3:dsx4 , respectively , figure 3c ) , which follows the observed trend in grec293 values . this suggests that spatial separation of pseudocomplementary base pairs and energetic hotspots is a promising principle for the design of efficient dsdna - targeting invader probes . in the present study , although there is an absence of suitable pseudocomplementary base pairs for cg - steps , we have previously shown that energetic hotspots can be comprised of intercalator - functionalized monomers that are based on any of the natural nucleobases ( although there is a preference for pyrimidine monomers ) . recognition of dsdna model target dh8 using different invader probes . to gain further insight into the underlying reasons for the excellent binding fidelity , we evaluated individual probe strands against singly mismatched single - stranded dna targets ( tables s10s12 ) . in agreement with the observations from the dsdna - recognition experiments , the discrimination is least efficient when dsx3 or dsx4 are hybridized with single - stranded targets that have a sequence corresponding to the target region in dh10 and dh14 ( tm s only reduced by 57 c , relative to matched duplexes , table s12 ) . the binding of invader probes to noncomplementary dsdna targets would accordingly entail formation of two mismatched and destabilized probe - target duplexes , which is energetically prohibitive in most cases . moreover , like other structured probes , the double - stranded invader probes are also likely to exhibit improved binding specificity due to a thus , pseudocomplementary invader probes allow for strong and highly specific binding to mixed - sequence dsdna target regions at ionic conditions . , 5-yd-3:3-dy-5 cassettes ) are thermolabile and activated for recognition of dsdna targets , but less so than regular invader probes with hotspots comprised of 2-n-(pyren-1-yl)methyl-2-n - methyl-2-aminouridine monomer x. in other words , close proximity of the two structural elements that normally activate pseudocomplementary dna and invader probes for dsdna - recognition i.e . , the weak base pairs between 2,6-diaminopurine and 2-thiouracil , and the destabilizing + 1 interstrand zipper arrangements of intercalating pyrene moieties does not provide a clear design benefit . in contrast , dsx probes , in which the two destabilizing structural motifs are separated , are strongly activated for recognition of mixed - sequence dsdna targets . thus , the use of chimeric pseudocomplementary invader probes presents itself as a promising strategy for mixed - sequence recognition of dsdna for applications in molecular biology and nucleic acid diagnostics , especially since we have recently demonstrated that conventional invader probes can recognize target regions in chromosomal dna . on concentrations were estimated using the following extinction coefficients for dna ( od/mol ) : g ( 12.01 ) , a ( 15.20 ) , t ( 8.40 ) , c ( 7.05 ) ; rna ( od/mol ) : g ( 13.70 ) , a ( 15.40 ) , u ( 10.00 ) , c ( 9.00 ) ; pyrene ( 22.4),d ( 8.5 ) , s ( 10.0 ) and y ( 32.4 ) .

cholangiocarcinoma ( cca ) is a highly malignant cancer , arising from ductular epithelium of biliary tree . according to anatomical location , this cancer can be divided into two major types including extrahepatic cca ( ecca ) and intrahepatic cca ( icca ) . cca is one of the highly aggressive malignant tumors and has been reported as a major cause of death from the primary liver cancer [ 1 , 3 ] . the incidence rate has been reported to be on a rise worldwide and the cumulative mortality rate has risen by 39% [ 46 ] . causes and risk factors for cca have not been fully clarified ; however some have been suggested to be involved in cca initiation . these include chronic inflammation of biliary epithelium that may involve hepatobiliary diseases such as primary sclerosing cholangitis ( psc ) , intrahepatic biliary stones , fibropolycystic liver disease , and viral hepatitis . genetic factors including several polymorphisms have also been recognized as critical risk factors for cca development . patients with cca mostly appear in late clinical presentation because of the lack of specific symptoms in early malignancies . therefore , it is difficult to diagnose cca at an early stage , resulting in high mortality with less than 5-year survival and poor prognosis . currently , there is no effective therapeutics ; however , it has been suggested that the only curative treatment is surgical resection , which may not be suitable for all cases . postoperative 5-year survival rate is very low , and treatment with radiotherapy and chemotherapy also carries a poor overall survival rate [ 9 , 10 ] . hence , novel biomarkers for early diagnosis , prognosis , and therapeutics are required to improve cca patient outcomes . at present , the food and drug administration ( fda ) has approved only 9 cancer biomarkers from serum for clinical routine detection . among those markers , carcinoembryonic antigen ( cea ) and carbohydrate antigen 19 - 9 ( ca 19 - 9 ) are well - known serum biomarkers that are routinely used for cca detection . however , these molecules are not cca specific and the specificity and sensitivity for screening have been reported to be low for cca as their levels are increased in cholestatic sera [ 1113 ] . genome derangement is frequently involved in carcinogenesis and may contribute to abnormalities in genes encoding proteins that have a critical role in key pathways related to cell growth and survival , leading to cancer development . therefore , identification of potential molecular biomarkers with high sensitivity and specificity would be beneficial for cca diagnosis and patient prognosis as well as targeting therapeutics [ 8 , 14 ] . omics approaches aim at the universal detection of genes ( genomics ) , mrna ( transcriptomics ) , proteins ( proteomics ) , and metabolites ( metabolomics ) . these techniques are useful for retrieving cancer biomarkers as they simultaneously investigate multiple molecules ( see figure 1 ) . genomics is a discipline in the systematic study of the structure , function , and expression of organism 's genome that involves dna sequencing and assembly as well as analysis of an annotation of structure and function of the gene . transcriptomics is a discipline to study global expression of rna including mrna , trna , and rrna as well as noncoding rna . conventionally , genes have been analyzed individually by single gene detection methods , but high throughput methods such as dna microarrays can analyze the expression of thousands of genes simultaneously . the single nucleotide polymorphism array ( snp array ) is a type of dna microarray that can be used to detect polymorphisms within the whole genome . next generation sequencing ( ngs ) has gained considerable attention for investigations at the nucleotide levels including both dna and rna sequences . proteomics is the large - scaled study of all expressed proteins that provides information about protein abundance and protein variation , modification , and interaction through pathway and network analysis [ 15 , 16 ] . two - dimensional polyacrylamide gel electrophoresis ( 2d - page ) that can separate a large amount of protein mixture based on the molecular weight and isoelectric point has initially been used to quantitate global changes of protein expression . mass spectrometry has been utilized to separate ions from proteins , peptides , or metabolites according to their mass - to - charge ratio ( m / z ) and to yield a result as mass spectrum that can be further analyzed to determine characteristics of molecular mass and structure . protein microarray has been developed to detect thousands of proteins based on specific antibody detection . metabolomics is a discipline that evaluates the profiles of metabolites , which can be useful in biomarker discovery because metabolites are usually stable end - products [ 1821 ] . metabolome analysis can be performed using a variety of techniques such as nuclear magnetic resonance ( nmr ) as well as mass spectrometry . these approaches offer high throughput screening of biomarkers for diagnosis , prognosis , and therapeutics that may also be useful for understanding of the changes in phenotypes associated with cancer compared to normal counterparts . this review summarizes molecular biomarkers based on their uses in early detection , prognosis , and therapeutics . in each section , biomarkers identified through genomics , transcriptomics , proteomics , and metabolomics as well as their potential molecular mechanisms and involvements underlying cca carcinogenesis will be discussed . hence , the cca patients are normally found at the late stage of cancer with low survival rate . however , a few clinical tools such as ultrasound , computed tomography ( ct ) , and routine cytology ( rc ) are commonly performed to screen and monitor any changes in bile ducts in the high - risk individuals with benign biliary strictures , psc , and hepatocarcinoma ( hcc ) [ 2 , 2224 ] . brush cytology during endoscopic retrograde cholangiopancreatography ( ercp ) is an initial procedure used to diagnose cca ; however , it is not of high specificity as similar appearance can be found in the biliary strictures . therefore , specific early diagnostic markers for cca are urgently needed to improve the disease prognosis . genetic alterations such as loss or gain in chromosomal fragments a cgh analysis revealed gain of chromosomal fragments 5q , 7p , 8q , 17q , and 20q and loss of chromosomal fragments 3p , 6q , 9p , and 17p , which were frequently found in cca in the absence of liver fluke infection [ 2528 ] . the alteration of these fragments has been correlated with activating mutations in certain oncogenes including egfr ( erbb1 ) on chromosomal fragment 7p12 , her2 ( erbb2 ) on 17q22 , and pdgfa on 7p22 . besides oncogenes , inactivating mutations were also frequently found in tumor suppressor genes including cdkn2a on 9p21q and tp53 on 17p13 [ 26 , 29 , 30 ] . in the liver fluke - associated cca , the pattern of chromosomal abnormalities is different from that of nonliver fluke - associated cca . the gain of chromosomal fragment 21q22 and loss of fragments 1p36 , 9p21 , 17q13 , and 22q12 were frequently found in cca tissues with liver fluke infection . for liver fluke - related cca , several genes within the abnormal chromosomal regions , including trefoil factor family 3 ( tff3 ) on 21q22.3 , run - related transcription factor 3 ( runx3 ) on 1p36 , cdkn2a on 9p21q and tp53 on 17p13 , and thymidine phosphorylate ( tp ) on 22q12 , which could be related to cca development or progression interestingly , the loss of 1p , 9p , and 17p chromosome regions which encode for cdkn2a and tp53 was found in cca both with and without liver fluke association . , the validation in larger cohorts of samples would be needed to prove this speculation . dna microarray technology was utilized to determine the genome - wide expression of gene related to cca carcinogenesis and sarcomatous transdifferentiation compared to normal epithelial cells . immunohistochemistry ( ihc ) and western immunoblotting analysis ( wb ) were performed in cca samples to validate the expression of secreted phosphoprotein 1 ( spp1 ) , ephrin - b2 ( efnb2 ) , iroquois - class homeodomain protein irx-3 ( irx3 ) , peroxisome proliferator - activated receptor gamma ( ppar ) , and insulin - like growth factor - binding protein 7 ( igfbp7 ) . spp1 is a cd44 ligand that binds to v - containing integrins , contributing to malignant cell attachment and tumor invasion . the oligonucleotide microarray revealed high expression of spp1 in icca [ 35 , 36 ] . efnb2 encodes for a member of the ephrin ( eph ) family , comprising receptor protein - tyrosine kinases , which is involved in a number of developmental processes . efnb2 has been shown to be preferentially expressed in cca , and overexpression of efnb2 has been related to the clinical stage in various types of cancer , suggesting the possible role of efnb2 as a novel diagnostic marker . irx3 encoding homeobox transcription factors which regulate early cellular development pathways including wnt and sonic hedgehog was found to be differentially expressed in cca [ 34 , 38 ] . ppar- is overexpressed in a number of cancers , including hcc , pancreatic cancer , and cca . the whole exome sequencing ( wes ) revealed several kras mutations , which are considered to be a potential diagnosis for cca [ 4143 ] . it has been shown that kras mutations are one of the most frequently altered genes in cca [ 4446 ] . sequencing analysis among chinese cca patients showing somatic mutations particularly kras and pik3ca mutations , but not braf , is associated with cca . the kras mutations would activate the raf - mek - erk - map kinase pathway to enhance gene transcription , cell cycle progression , and cell growth [ 47 , 48 ] . such kras mutations have also been identified in several cca cell lines . in animal models , kras gene activation , together with p53 activation , hotspots for pik3ca mutations in cca were found within exons 9 and 20 that encode helical and kinase domains of p110 involved in the pi3k / akt pathway . pik3ca mutations would affect cell proliferation by deregulating the pi3k / akt signaling pathway . from tissue microarray , the translation proteins eif4-e and phosphorylated 4e - bp1 were identified as targets for pi3k pathway activation in cca , hinting to the clues toward cca pathogenesis . gene expression profiles of cca tissues were compared to the normal counterparts to identify differentially coexpressed genes ( dcgs ) through microarrays and computational bioinformatics analysis . the results revealed that four transcription factors including forkhead box c1 ( foxc1 ) , zic family member 2 ( zic2 ) , nk2 transcription factor related , locus 2 ( nkx2 - 2 ) , and glucagon receptor ( gcgr ) are represented as hub nodes in the regulatory network . foxc1 is one of the forkhead transcription factor family contributing to ocular and cerebellar development . high expression of foxc1 has been associated with cell proliferation and migration in cca [ 53 , 55 ] . zic2 , a major zinc - finger transcription factor , plays a key role during developmental stage of embryo . nkx2 - 2 , a homeobox transcription factor , triggers central nervous system morphogenesis in normal condition . however , it induces oncogenic transformation in ewing 's sarcoma [ 5759 ] . it could be possible that these players may have a crucial role in cca development and therefore could be further investigated to find their potential use in diagnosis . cdna microarray approach was used to compare gene expression profiling of icca and normal liver tissues from patients in northeast thailand . real - time pcr ( rt - pcr ) was used to validate the overexpression of 7 genes including fxyd3 ( ion transporter ) , g protein - couple receptor family c group 5 member a ( gprc5a ) , carcinoembryonic antigen - related cell adhesion molecule 5 ( ceacam5 ) , mucin 13 ( muc13 ) , epithelial cell adhesion molecule ( epcam ) , transmembrane channel like 5 ( tmc5 ) , and ets homolog factor ( ehf ) and the downregulation of 3 genes including carbamoyl phosphate synthetase 1 ( cps1)/mitochondrial carbamoyl synthetase 1 , tyrosine amino transferase ( tat ) , and inter- globin inhibitor h1 ( itih1 ) . the results showed that most genes encoding proteins related to cell growth and metastasis increased , while those which control metabolic activities decreased . therefore , these exon - level expression profiles should be explored to identify genetic biomarkers for early detection in cca . another cdna microarray study reported differentially expressed genes in opisthorchiasis - associated cca . among 276 genes evaluated , 131 genes in cell proliferation , transformation , apoptosis , dna repair , and cytoskeleton structure were upregulated , whereas 145 genes correlated with metabolic enzymes , tumor suppressors , apoptosis , and oxidative response were downregulated . during early liver fluke infection ( within 1 month after infection ) , the expression of s100a6 , platelet derived growth factor - alpha ( pdgfa ) , neural proliferation differentiation and control protein 1 ( npdc1 ) , transcription factor jun - b ( junb ) , jund-1 , and nuclear factor kappa - light - chain - enhancer of activated b cells ( nf-b- ) was induced , while the expression of cytochrome p450 , succinate dehydrogenase , raf kinase inhibitor ( rkip ) , isocitrate dehydrogenase 2 ( idh2 ) , and glutathione s - transferase - alpha4 ( gsta4 ) was reduced . jund and jund1 are protooncogenes associated with cell proliferation , differentiation , transformation , and apoptosis . nfkb- is involved in nf-b signaling pathways directing transformation , proliferation , invasion , angiogenesis , and metastasis . altogether , the pattern of gene expression along with parasitic infection data provides the significance of these molecules with opisthorchiasis - associated cca carcinogenesis . ca19 - 9 is lewis blood - group antigen , which has been widely used as a serum marker for cca . however , it exhibits low sensitivity and specificity because it is normally produced by normal human pancreatic cells , biliary ductular cells , and gastric and colonic epithelial cells . ca19 - 9 is also elevated in pancreatic cancer , gastric cancer , and primary biliary cirrhosis . although there is no strong evidence supporting ca 19 - 9 as cca - specific biomarkers , a few studies attempted to identify the correlation of ca19 - 9 with cca [ 6770 ] . bile proteomics of cca patients has been performed to differentiate malignant phenotypes from benign biliary strictures . the alteration of protein expression in early stage of cca can be detected from extracellular fluid . the bile proteome revealed overexpression of carcinoembryonic antigen - related cell adhesion molecule 6 ( ceacam6 ) and mucin 1 ( muc1 ) in patients with malignant biliary stricture including cca , compared to the benign counterparts . recent proteomic investigation based on whole proteins from cca serum samples revealed the substantially higher expression of fam19a5 protein and rb - associated krab zinc - finger protein ( rbak ) compared to those samples with benign biliary tract diseases ( bbtds ) . secreted fam19a5 , a member of the tafa family , regularly functions as a brain - specific chemokine . commonly known as a transcription factor repressor , rbak expression is suggested to create optimal microenvironment for cca development by fibroblasts [ 72 , 73 ] . further clarification of these molecules is required for the development as novel diagnostic for cca . furthermore , capillary electrophoresis mass spectrometry analysis was used to identify and distinguish the disease - specific peptide patterns in choledocholithaisis and psc from cca through bile proteomic analysis . the differentiation from psc and cca was justified by 22 peptides , among which 12 were hemoglobin subunits , serum albumin , cytoplasmic actin , keratins , inter - alpha - trypsin inhibitors heavy chains , and 14 - 3 - 3/ protein . the expression of these peptide markers indicated the changes in molecular pathways involved in inflammation , apoptosis , proteolysis and protein catabolism , and epithelial cell transformation . an independent validation set of 18 patients showed specificity of 78% and sensitivity of 84% , suggesting the possible role of bile proteomic analysis as a diagnostic tool for early development of cca in patients with psc . based on bile proteomics , 14 - 3 - 3/ protein has been identified in cca , hinting to its involvement in carcinogenesis due to its function in cellular processes such as actin cytoskeletal organization , cell adhesion , and antiapoptosis . the expression of 14 - 3 - 3 proteins was first immunohistochemically evaluated on cca tissues . overexpression of 14 - 3 - 3 protein isoforms , , , , , and has been associated with cca . these five 14 - 3 - 3 isoforms could bind to cruciform dna and enhance dna replication , which favors cca carcinogenesis . using rna silencing ( sirna ) technique , 14 - 3 - 3 proteins were found to be associated with cancer as downregulation of 14 - 3 - 3 could lead to increased and unscheduled cell cycle progression . 14 - 3 - 3 proteins were also associated with epithelial mesenchymal transition ( emt ) and cell invasion in cca [ 7779 ] . these findings led to the study of 14 - 3 - 3 association to anoikis resistance of cca in vitro using sirna to silence 14 - 3 - 3 expression . anoikis resistance is a condition where the cells have ability to survive after detaching from extracellular matrix ( ecm ) prior to metastasis . the study revealed a significant role of 14 - 3 - 3 protein in anoikis resistance of cca cells , pointing to the potential use as an early diagnostic biomarker and a target for cca therapeutics . bile proteomes from cca patients were analyzed using 2d - page coupled with matrix assisted laser desorption / ionization - time of flight - mass spectrometry ( maldi - tof - ms ) . two potential biomarkers were identified including s100 calcium - binding protein a9 ( s100a9 ) and chaperonin - containing tcr1 , subunit 3 ( cct ) . more recent study also showed overexpression of s100p in ecca using the shotgun mass spectrometry analysis . upregulation of olfactomedin-4 ( olfm4 ) , an antiapoptotic factor , was also observed in 60% of ecca tissues . sds - page gel and liquid chromatography - mass spectrometry ( lc - ms / ms ) application also identified the differential protein abundances in benign and malignant biliary strictures through bile proteomics . the study clarified several proteins that were significantly elevated in cca patients compared to psc and benign cases such as -s - macroglobulin , apolipoprotein b-100 ( apo - b ) , ceruloplasmin , complement c3 , kininogen-1 ( kng1 ) isoform 2 , myeloperoxidase ( mpo ) , and inter--trypsin inhibitor heavy chain h4 ( itih4 ) . comparison of bile from patients with cca and benign biliary disease was studied using magnetic resonance spectroscopy ( mrs ) . mrs is a sensitive analytical method to evaluate chemical composition providing molecular structural information from nonhomogeneous biological samples . the results demonstrated that the levels of phosphatidylcholine ( ptc ) , bile acids , lipid , and cholesterol could be used to distinguish cca patients from benign groups with 88.9% sensitivity , 87.1% specificity , and 87.8% accuracy . differential patterns of bile components in cca patients may arise as a result of cancer cell proliferation and progression through deregulation of signaling pathways . further validations as well as assay development are warranted in order to find the efficient platform for cca diagnosis in terms of accuracy and cost effectiveness . recently , shotgun mass spectrometric analysis was carried out to identify differential protein expression in ecca tissues . of 1,992 proteins identified , newly prominent markers have been reported including metabolic enzymes , such as ornithine aminotransferase ( oat ) , fatty acid binding protein 1 , liver ( fabpl ) , and amine oxidase [ flavin - containing ] a ( aofa ) . elevated level of oat , a major enzyme in the proline biosynthesis , has been recorded in proliferative malignant tissues . fabp , a cytoplasmic transporter of fatty acids , plays an essential role in complex lipid synthesis and oxidation . the alteration of fabp expression has been associated with several cancer types [ 85 , 86 ] . technological advances have yielded a vast amount of information on molecular markers for predicting tumor progression . prognostic markers aim to beneficially assess the patient 's overall survival outcome , such as the probability of cancer recurrence after standard treatment . the presence or absence of prognostic markers can be useful for decision making process in therapeutic strategies . activating and inactivating mutations these mutations promote cell survival , cancer initiation , and progression , which greatly affect survival of patients . the cgh analysis revealed chromosomal abnormalities in icca at the regions encoding erbb2 gene ( chr17q12 ) and map2k2/mek2 gene ( chr19p13 ) [ 26 , 88 ] . the erbb receptor tyrosine kinase family consists of four cell surface receptors including erbb1 , erbb2 , erbb3 , and erbb4 . these receptors are activated by binding of the corresponding ligands , which induces dimerization of the receptors . the activated receptors relay the signals through key signaling pathways that regulate cell survival and motility . the activating mutations of erbb2 have been previously observed in many types of cancers such as lung , breast , and colon cancer . erbb2 mutations have been correlated with tumor progression as shown in the erbb-2/neu transformed rat cholangiocytes that exhibit similar phenotypes as found in human cca . the gain - of - function mutations and overexpression of the erbb genes have been associated with poor prognosis and cca progression [ 91 , 92 ] . wes has been performed on liver fluke - associated cca and matched normal tissues in order to identify somatic mutations that arise during carcinogenesis . the results revealed frequent somatic mutations in certain genes such as tp53 ( 44.4% ) , kras ( 16.7% ) , and smad4 ( 16.7% ) . the mutations in p53 have been described as the most common genetic alteration in cancer . the critical roles of p53 include the regulation of cell cycle and apoptosis as well as dna repair . several studies have shown that p53 mutations are generally associated with the development of cancer and survival rate in many types of cancer , indicating that p53 is a prognostic biomarker [ 9496 ] . a meta - analysis study revealed high expression of p53 related to adverse clinical features and poor prognosis in ecca patients . ras and raf gene families are oncogenes in the mitogen - activated protein kinases ( mapk ) family . mutations in ras gene have been associated with both icca and ecca [ 98 , 99 ] . activating mutation in the kras gene , which is downstream of epidermal growth factor receptor ( egfr or erbb1 ) , is one of the most frequent mutations found in icca [ 100 , 101 ] . kras gene mutations have been correlated with higher tumor stages ( stage i , 8% ; stage ii , 15% ; stage iii , 31% ; stage iv , 46% ) . moreover , activating mutation in one of the raf gene isoforms , braf , has been reported to involve icca development [ 103 , 104 ] . the low expression of smad4 protein has been observed in icca tissues and associated with poor differentiation and high lymph node metastasis , suggesting that smad4 may represent an adverse prognostic marker . idh , a metabolic enzyme in tricarboxylic acid ( tca ) cycle , functions in catalyzing the reversible conversion of isocitrate -kg and carbon dioxide . mutations in idh1 and idh2 produce a molecule that alters genetic programming in cells , resulting in enhanced cell proliferation . there are several hotspots for idh1 and idh2 mutations , which are gain - of - function mutations [ 107109 ] . exome sequencing of liver fluke - associated ccas also identified somatic mutations in both genes . it has been reported that a 3-year survival rate was significantly reduced in resected patients with idh gene mutation ( 33% ) compared to patients with normal idh gene ( 81% ) . recently , dna extracted from 75 cca tissues was used to address genetic aberration using ngs technology . in agreement with aforementioned studies , mutations in erbb2 , kras , tp53 , and smad4 furthermore , this study also identified mutations in c - met , bap1 , and fgfr pathways , which previously associated with cca prognosis . gain - of - function mutations in c - met , which is one of the growth factor receptors , are often found in biliary tract cancer and are also related to higher grade of invasiveness and poor prognosis [ 111113 ] . bap1 encodes brca1-associated protein 1 , known as a tumor suppressor and a metastasis suppressor . hence , loss of bap1 is associated with an aggressive metastatic behavior and related to adverse prognosis [ 114 , 115 ] . in this study , patients with fgfr mutations exhibited good prognosis and good response to chemotherapy over two years . however , the patients with fgfr - nol4 fusion coexisting with bap1 mutation had rapid cancer progression . inoculation of rats with low grade malignant rat bde1 cholangiocytes ( bdesp cells ) allowed early clinical stage to develop whereas injection with high grade malignant erbb-2/neu - transformed bde1 cholangiocytes ( bdeneu cells ) triggers advanced cca features . the results demonstrated that sox17 , krt20 , and erbb2 genes were overexpressed in bdeneu cells compared to bdesp cells , suggesting their potential use as prognostic molecular markers for cca . sox17 has not been directly linked to cholangiocarcinogenesis , but its function as an oncofetal transcription factor could possibly play a role in cell migration . expression of krt20 in bdeneu cells could accelerate the transition of cancer cells into stem cell - like phenotypes , leading to rapid cell proliferation that can promote cca development . moreover , mmp-7 gene was found overexpressed in bdeneu cells but not expressed in bdesp cells . elevated expression of matrix metalloproteinase ( mmp ) exhibits a critical role in enhancement of cancer metastasis . mmp-7 was shown with significant potential as a prognostic factor for poor survival in postoperative icca patients [ 118 , 119 ] . another study also used cdna microarray to compare gene expression pattern between the sarcomatoid cells ( sck ) and differentiated cells ( choi - ck ) . it has been associated with lymph node metastasis and adverse overall survival that strongly link to poor prognosis in cca patients . an increase in aberrant methylation and noncoding rna expression has been found to associate with downregulation of tumor suppressor genes , giving rise to cca progression . a genome - wide analysis of 28 cca using the illumina 27-k methylation array identified different expression of 1,610 cpg sites that involved 603 methylated genes . following gene enrichment analysis , a number of pathways , such as wnt , pi3k , mapk , and notch signaling , are commonly found to be altered in icca . these signaling pathways are well known in cell proliferation , cell metastasis , and apoptosis regulation . micrornas ( mirnas ) , small noncoding rnas , function as critical regulators of the genome , controlling key cellular properties [ 126 , 127 ] . mature mirnas regulate the expression of many genes that correlate with various cellular mechanisms . therefore , the differentially expressed mirnas probably serve as prognostic markers for cca . the first mirnas profiling using mirna array technique exhibited unique mirna signature comprising 27 members in cca cell lines including hucct1 and mec of cells . among several upregulated expressed mirnas , elevated expression of mir-21 and mir-200c was dominant in icca compared to normal bile duct . the high expression of mir-21 is associated with low expression of programmed cell death 4 ( pdcd4 ) and tissue inhibitor of metalloproteinase 3 ( timp3 ) . it was also found to regulate pten - dependent activation of pi3k , which in turn affects cca progression . analysis of mir-200c and gene expression profiling demonstrated the correlation between mir-200c and the expression of neural cell adhesion molecule 1 ( ncam1 ) . another study reported a genome - wide mirna expression pattern in 27 laser capture microdissected icca tissues compared to 10 normal tissues . the results revealed 38 mirnas that were differentially expressed between tumors and normal counterparts . from this study , mir-204 was shown to be associated with the level of ca 19 - 9 . mir-204 has been shown to play a critical role in modulating emt by regulating the expression of slug , e - cadherin , and vimentin . proteomics analysis of peripheral cca tissues and paired nontumoral liver tissues from the same patient has been performed to distinguish protein expression . increased levels of -smooth muscle actin ( -sma ) and periostin were shown in the stromal myofibroblasts surrounding tumor cells . -sma is a marker of stromal cell activation that correlates to poor prognosis in colon cancer [ 134 , 135 ] . periostin , known with a key role in cell adhesion , proliferation , and migration , may contribute to poor prognosis in cancer patients . the lc - ms / ms based proteomics identified 38 upregulation proteins in cancerous samples . among these proteins , 4 candidate markers actinin 1 , actinin 4 , protein dj-1 , and cathepsin b were validated by wb and ihc analysis . -actinin , an actin binding protein , is essential for remodeling of actin filament which promotes cell motility thus enhancing cancer cell metastasis . interestingly , overexpression of actinin 4 in cytoplasm is correlated to various clinicopathological parameters in certain human cancers . dj-1 has been described as an oncoprotein associated with hras and transforms cells by promoting cell proliferation and resistance to cell cycle arrest , resulting in poor prognosis [ 139 , 140 ] . cathepsin b is a member of cysteine protease family , which is normally found in lysosome . the normal function of cathepsin b involves cell proliferation , cell differentiation , and organogenesis as well as metabolism . in several cancers , cathepsin b involves degradation of ecm and promotes angiogenesis and metastatic capability inversely contributing to a decrease in survival rate of cca patients [ 141 , 142 ] . they also regulate several signaling pathways needed for cell cycle control and protection of cells against stress or apoptosis . aberrant hsps lead to protein dysfunction resulting in abnormality in cellular functions , thereby potentially promoting carcinogenesis and tumor progression . proteomic profiling of bile products revealed the upregulation of heat shock 60 kda protein 1 ( hsp60.1 ) in bile from cca patients . by using maldi tof / tof analysis , the carbonylated heat shock 70 kda protein 1 ( hsp70.1 ) was found to be significantly higher in tumor tissues than adjacent normal cells . carbonylation of hsp70.1 has been significantly correlated with poor prognosis in cca patients [ 145 , 146 ] . furthermore , the recent study revealed the high expression of hsp90 in both icca and ecca . overexpression of hsp90 was significantly associated with decreased overall and disease - free survival in both icca and ecca . high level of hsp90 expression was observed in poorly differentiated icca and was associated with metastatic cases , suggesting that hsp90 is a factor for cancer progression and metastasis in cca . proteomics analysis using maldi - tof - ms and electrospray ionization - tandem ms ( esi - ms / ms ) in hucc-1 cell line revealed particularly high expression of galectin 3 , a dominant protein in cell - to - cell and cell - ecm interaction . galectin 3 has been successfully used to predict metastasis and tumor progression [ 149152 ] . elevated expression level of -enolase , a glycolytic enzyme , was also observed in this study . moreover , expression of -enolase was also found in other cca cell lines including m156 , k100 , m139 , and m213 cells . the overexpression of -enolase was confirmed through ihc in 75% of cca patients with hyperplastic bile duct and the tumor compared to adjacent normal tissue region . moreover , the patients with high level of -enolase exhibited worse survival compared to those with low level of -enolase . abnormal synthesis of glycans and glycoproteins has been related to cancer progression in diverse cancerous cell types [ 154156 ] . based on proteomics , glycomics and glycol - proteomics technologies have been utilized to reveal significance of mucins as a glycol - biomarker in cca . mucins ( muc ) are a protein family characterized by heavy glycosylation produced from epithelial cells . these proteins can be divided into two subclasses , which are secreted form and transmembrane form . overexpression of transmembrane form in human malignancies has been reported to stimulate cellular signaling in epithelial cell polarity , cell growth , and survival . high muc1 level is linked to cell transformation and loss of cell polarity in various cancer types [ 159162 ] . it has been identified as a risk factor for poor prognosis in patients with mass - forming icca after surgery [ 163 , 164 ] . in addition , muc4 functions as intramembrane ligand binding and a modulator of erbb2 receptor tyrosine kinase pathway , resulting in antiapoptosis , thus encouraging tumor progression . it has been found that icca patients with the coexpression of muc4 and erbb2 correlated well with worse clinical outcome . secreted muc2 functions as a protective protein layer , lining epithelial surface of intestinal tract . unlike muc1 and muc4 , the mrs approach was utilized to investigate bile contents from patients with cca compared to patients with benign biliary tract diseases . a significant higher level of glycine - conjugated bile acid but lower phosphatidylcholine ( ptc ) was also observed in bile of cca patients compared to that of patients with benign biliary tract diseases . the absence of phospholipid transport into the bile leads to prolonged exposure of biliary epithelial cells to toxic bile , eventually influencing cca development [ 172 , 173 ] . it has also been revealed by proteomic profiling using mass spectrometry that several proteins involving metabolic pathways in hucca-1 cca cells were dysregulated including glutathione - s - transferase ( gst ) . gst possesses an antioxidant activity and its downregulation in cca would lead to accumulation of free radicals , causing genetic damage , which links to malignant transformation and cca progression . furthermore , overexpression of lactate dehydrogenase ( ldh - a ) and downregulation of glycine n - methyltransferase ( gnmt ) were identified in peripheral cca through nano - lc - ms / ms . lactate is produced from pyruvate through ldh - a , a hallmark reaction in the warburg effect for cancer cells [ 174 , 175 ] . the high level of lactate production refers to high rate of glycolysis , which is believed to subsequently fulfill the anabolic requirement for aberrant cancer cell growth . ldh - a was shown to be overexpressed in cca tissues and high levels of ldh - a transcripts were found in icca cells . using sirna for ldh - a knockdown , hucct-1 cells exhibited induced apoptosis and suppressed proliferation indicating the key role of lad - h in cancer progression . gnmt has been primarily in glycine , serine , and threonine metabolism , hinting that gnmt dysregulation in cca may result in metabolic shift , which would favor cca development . the absence of gnmt expression in cca tissues compared to normal cholangiocytes was associated with low survival rate . carbonylation of serotransferrin was detected and identified by mass spectrometric technique and the results showed high carbonylated serotransferrin in tumor tissues of cca patients . in addition , carbonylation of serotransferrin in tumor tissue had a significant correlation with a poor prognosis . serotransferrin is an iron ( fe ) transporter that generally carries ferric iron from digestive organs to all proliferating cells through body . dysfunction of this iron transporter may result in the iron accumulation , which may involve iron overload and participate in induction of oxidative stress in tumor tissues [ 145 , 180 ] . surgery is the only curative treatment in cca ; however the high recurrence and low survival rate are still evident . in most cases , tumors are unresectable and much effort in palliative procedures is needed to relieve the pain . currently , there is no effective therapeutics for cca ; therefore it is necessary to find more measures to suppress cancer progression to prolong survival rate in cca patients . research on potential inhibitors or drugs against target must be verified in cancer cells , animal models , and human clinical trials . there were chromosomal amplifications in 1q , 5q , 7q , and 17q in cca , while amplifications of 4p , 5p , 7p , 10p , 13q , 18q , and 20q were mostly found in icca patients . the deletions at 1p , 4q , 10q , 13q , 14q , and 18q were observed in cca , while deletion at 13q was mostly observed in icca . furthermore , the amplifications of erbb2 ( 17q12 ) , mek2 ( chr19p13 ) , mtor ( 1p36.2 ) , vegfr 3 ( 5q35.3 ) , and vegfa ( 6p12 ) genes were found to be correlated with cca , hence posing as potential targets for therapeutics [ 26 , 181184 ] . another study demonstrated the association of egfr and vascular endothelial growth factor ( vegf ) in cca . it also showed that egfr expression correlated to tumor progression and vegf expression was associated with haematogenic metastasis in cca [ 92 , 186 ] . these findings suggest that egfr and vegf can be candidates as therapeutic targets for cca [ 187190 ] . genetic alterations and epigenetic aberrant in aforementioned genes could interfere in cell proliferation , apoptosis , survival , and angiogenesis of cancer cells [ 187190 ] . vandetanib ( zd6474 , a tyrosine kinase inhibitor ) has been used to inhibit the egfr and vegfr signaling in cca cell lines and xenograft . however , cca cell lines with kras mutations were found resistant to vandetanib . in cca xenograft mouse model , vandetanib could decrease tumor growth and metastasis . another selective inhibitor of egfr , called zd1839 ( iressa ) , was found to stabilize p27 , a cyclin - dependent kinase inhibitor 1b thereby enhancing radiosensitivity in cca cell lines . currently , there are a few vegf inhibitors on clinical trials such as sunitinib , sorafenib , and regorafenib . sunitinib malate is an inhibitor of vegfr types 1 and 2 , fms - like tyrosine kinase 3 ( flt3 ) , and platelet - derived growth factor ( pdgf ) . it has direct antitumor and antiangiogenic properties in various cell lines [ 192 , 193 ] . with potential therapeutic activity for cca , it has been undertaken in clinical trial phase ii in patients with advanced cca ( clinicaltrials.gov identifier : nct01718327 ) . sorafenib has been shown to inhibit vegf receptors , pdgf receptors , flt3 , raf-1 , and braf in vitro . it has been currently under clinical trial phase ii in patients with gallbladder carcinoma and cca ( clinicaltrials.gov identifier : nct00238212 ) . with only distinction in fluoride atom at center phenyl ring , regorafenib shares a common feature with sorafenib , to function as a kinase inhibitor [ 194 , 195 ] . regorafenib showed antitumor growth as well as antiangiogenetic properties reducing tumor microvasculature by which its greater inhibitory effect compared to that of sorafenib was on vegfr2 and fgfr1 [ 196 , 197 ] . it could also inhibit vegfr1 , vegfr3 , and raf [ 196 , 198 ] . it was reported to inhibit tumor growth of liver metastases ; it is therefore currently under clinical trial phase ii in patients with advanced and metastatic biliary tract carcinoma / cca ( clinicaltrials.gov identifier : nct02053376 ) . as previously mentioned , wes identified gene mutation of idh1 and idh2 in cca [ 93 , 199 , 200 ] . it has been found that there were 14 idh1 mutations and 7 idh2 mutations and 90% of these mutations were observed in icca . idh1 mutation would result in gain - of - function activity , which causes accumulation of 2-hg . the excess of 2-hg is associated with idh1 and idh2 mutations by inhibiting the -kg from binding to dioxygenases . normally , canalization of the oxidative decarboxylation of isocitrate to -kg is carried out by idh enzymes . mutated idh would cause 2-hg accumulation , which in turn inhibits prolyl hydroxylase , which is used to stabilize hypoxia - inducible factor-1 ( hif-1 ) , leading to the absence of oxygen - dependent hydroxylation . hif-1 accumulation mediates activation of several pathways such as mmps and vegfr , involving cell growth , invasion , angiogenesis , and metastasis . idh1 and idh2 inhibitors are ag1 - 5198 and ag1 - 6780 , respectively [ 202 , 203 ] . ag1 - 5198 suppressed the 2-hg production in idh1-mutant gliomas cells while ag1 - 6780 blocked 2-hg production in idh2-mutant hematological cell lines [ 202 , 203 ] . currently , an idh1 inhibitor , ag-120 , is on clinical trial phase i in patients with cca and advanced solid tumors ( clinicaltrials.gov identifier : nct02073994 ) . based on microarray analysis , the connectivity map ( cmap ) tool was used to study the connection between gene signature of the disease and drug treatment . the microarray with cmap database was used to identify the potential drugs that had negative correlation to cca - related gene expression . hsp90 inhibitors including 17-aag ( tanespimycin ) , geldanamycin , and alvespimysin were identified as the potent drugs for cca . besides hsp90 , this study also revealed that hsp90 inhibitors , tanespimycin and nvp - auy922 ( a novel hsp90 inhibitor ) , could increase level of hsp70 which was found to have a low expression in cca patients [ 146 , 205 ] . these data indicated that hsp70 and hsp90 may act as therapeutic markers in cca that can be targeted by hsp90 inhibitors . bile proteomics using sds - page gel and lc - ms / ms identified differential protein abundances in benign and malignant biliary strictures [ 74 , 83 ] . upregulation of -s - macroglobulin , apo - b b100 , ceruloplasmin , complement c3 , kng1 isoform 2 , mpo , and itih4 was observed . the overexpression of tff2 was associated with cca invasiveness by regulating via egfr / mapk pathway . by using an egfr antagonist , pd153035 in ecca cell line could block the tff activation . however , tff2 expression is still controversial since other studies stated that tff-2 precursors proteins were less abundant in cca . these findings of tff in cca through bile proteomics suggest trefoil as possible target for cca . 2d - page and tandem mass spectrometry reported increased il-6 in biliary tract cancers including cca . il-6 is a proinflammatory cytokine , which could assist in cholangiocyte proliferation via a variety of pathways and signal transduction when it is aberrantly controlled . the ag490 , a mcl-1 inhibitor , can downregulate mcl-1 . therefore , targeting mcl-1 could be a potential candidate for therapeutics . furthermore , il-6 blockers such as sarilumab , alx-0061 , sirukumab , medi5117 , clazakizumab , and olokizumab are in early clinical trials for rheumatoid arthritis . however , their uses in cca patients need further investigations [ 209 , 210 ] . opb-31121 , a stat inhibitor , has been tested on various cell lines and in vivo . the study found that opb-31121 strongly suppressed stat3 and stat5 phosphorylation without inhibition of upstream kinases . it is currently on clinical trials in patients with progressive hepatocellular carcinoma ( clinicaltrials.gov identifier : nct01406574 ) . azd9150 is a 16-oligonucleotide antisense molecule ( aso ) , which targets the 3 untranslated part of stat3 , thereby preventing protein expression . a dose - dependent knockdown stat3 mrna and proteins were observed to affect tumor growth inhibition in xenograft in vivo . azd9150 is currently on clinical trial in patients with advanced hepatocellular carcinoma ( clinicaltrials.gov identifier : nct01839604 ) . as previously mentioned , mrs - based bile proteomes from patients with cca and benign biliary tract diseases were compared [ 84 , 213 ] . the data showed an increase in glycine - conjugated bile acids in cca patients compared to benign disease groups . while 7 primary bile acid was found to be increased , biliary ptc was reduced in bile from patients with cca compared to the gallstone groups . ptc can be transported to biliary duct via multidrug resistant protein 3 ( mdr3 ) . in mdr - knockout mice , abnormality in ptc secretion from liver could cause a decrease in phospholipid export into the bile , rendering biliary epithelium prone to toxic agents in bile . maintaining bile ptc and its transporters should be considered as cca therapeutic strategy in order to regulate healthy bile and reduce cell toxicity . as aforementioned protein , oat , was found to be expressed in ecca tissues by shotgun mass spectrometric analysis , oat is a crucial mitochondrial enzyme producing glutamate , which is needed for cell proliferation and energy . there are several oat inhibitors such as gabaculine and ( 1s,3s)-3-amino-4-(hexafluoropropan-2-ylidene ) cyclopentane-1-carboxylic acid , which have been shown to inhibit hcc progression . lack of effective therapeutics for cca urges the demand for specific and early diagnostic biomarkers to increase survival rate , for prognostic biomarkers to provide more precision of cca progression , and for therapeutic biomarkers to develop curative strategies . with advanced molecular techniques , along with genes and proteins that have been identified as molecular markers for cca , some of which have been practically used , more diverse and specific promising biomarkers in cca have been established including the mirna of mir-21 , mir-200 , mir-204 , and enzymes such as gstp , tff , idh , and mucins . the discovery of these molecules in the pathways involving cell proliferation , invasion , apoptosis , and tumor suppressor would hint us toward the molecular mechanism that gives rise to cca behaviors and characteristics . future directions include the exploration and validation of their potential use in diagnosis and prognosis , as well as therapeutics .

cholangiocarcinoma ( cca ) is an aggressive biliary tract malignancy arising from the epithelial bile duct . the lack of early diagnostic biomarkers as well as therapeutic measures results in severe outcomes and poor prognosis . thus , effective early diagnostic , prognostic , and therapeutic biomarkers are required to improve the prognosis and prolong survival rates in cca patients . recent advancement in omics technologies combined with the integrative experimental and clinical validations has provided an insight into the underlying mechanism of cca initiation and progression as well as clues towards novel biomarkers . this work highlights the discovery and validation of molecular markers in cca identified through omics approaches . the possible roles of these molecules in various cellular pathways , which render cca carcinogenesis and progression , will also be discussed . this paper can serve as a reference point for further investigations to yield deeper understanding in the complex feature of this disease , potentially leading to better approaches for diagnosis , prognosis , and therapeutics .

1. Introduction 2. Diagnostic Biomarkers 3. Prognostic Biomarkers 4. Therapeutic Targets 5. Concluding Remarks

cholangiocarcinoma ( cca ) is a highly malignant cancer , arising from ductular epithelium of biliary tree . cca is one of the highly aggressive malignant tumors and has been reported as a major cause of death from the primary liver cancer [ 1 , 3 ] . causes and risk factors for cca have not been fully clarified ; however some have been suggested to be involved in cca initiation . these include chronic inflammation of biliary epithelium that may involve hepatobiliary diseases such as primary sclerosing cholangitis ( psc ) , intrahepatic biliary stones , fibropolycystic liver disease , and viral hepatitis . patients with cca mostly appear in late clinical presentation because of the lack of specific symptoms in early malignancies . therefore , it is difficult to diagnose cca at an early stage , resulting in high mortality with less than 5-year survival and poor prognosis . hence , novel biomarkers for early diagnosis , prognosis , and therapeutics are required to improve cca patient outcomes . genome derangement is frequently involved in carcinogenesis and may contribute to abnormalities in genes encoding proteins that have a critical role in key pathways related to cell growth and survival , leading to cancer development . therefore , identification of potential molecular biomarkers with high sensitivity and specificity would be beneficial for cca diagnosis and patient prognosis as well as targeting therapeutics [ 8 , 14 ] . omics approaches aim at the universal detection of genes ( genomics ) , mrna ( transcriptomics ) , proteins ( proteomics ) , and metabolites ( metabolomics ) . these techniques are useful for retrieving cancer biomarkers as they simultaneously investigate multiple molecules ( see figure 1 ) . genomics is a discipline in the systematic study of the structure , function , and expression of organism 's genome that involves dna sequencing and assembly as well as analysis of an annotation of structure and function of the gene . transcriptomics is a discipline to study global expression of rna including mrna , trna , and rrna as well as noncoding rna . proteomics is the large - scaled study of all expressed proteins that provides information about protein abundance and protein variation , modification , and interaction through pathway and network analysis [ 15 , 16 ] . mass spectrometry has been utilized to separate ions from proteins , peptides , or metabolites according to their mass - to - charge ratio ( m / z ) and to yield a result as mass spectrum that can be further analyzed to determine characteristics of molecular mass and structure . metabolome analysis can be performed using a variety of techniques such as nuclear magnetic resonance ( nmr ) as well as mass spectrometry . these approaches offer high throughput screening of biomarkers for diagnosis , prognosis , and therapeutics that may also be useful for understanding of the changes in phenotypes associated with cancer compared to normal counterparts . this review summarizes molecular biomarkers based on their uses in early detection , prognosis , and therapeutics . in each section , biomarkers identified through genomics , transcriptomics , proteomics , and metabolomics as well as their potential molecular mechanisms and involvements underlying cca carcinogenesis will be discussed . however , a few clinical tools such as ultrasound , computed tomography ( ct ) , and routine cytology ( rc ) are commonly performed to screen and monitor any changes in bile ducts in the high - risk individuals with benign biliary strictures , psc , and hepatocarcinoma ( hcc ) [ 2 , 2224 ] . brush cytology during endoscopic retrograde cholangiopancreatography ( ercp ) is an initial procedure used to diagnose cca ; however , it is not of high specificity as similar appearance can be found in the biliary strictures . therefore , specific early diagnostic markers for cca are urgently needed to improve the disease prognosis . genetic alterations such as loss or gain in chromosomal fragments a cgh analysis revealed gain of chromosomal fragments 5q , 7p , 8q , 17q , and 20q and loss of chromosomal fragments 3p , 6q , 9p , and 17p , which were frequently found in cca in the absence of liver fluke infection [ 2528 ] . the alteration of these fragments has been correlated with activating mutations in certain oncogenes including egfr ( erbb1 ) on chromosomal fragment 7p12 , her2 ( erbb2 ) on 17q22 , and pdgfa on 7p22 . the gain of chromosomal fragment 21q22 and loss of fragments 1p36 , 9p21 , 17q13 , and 22q12 were frequently found in cca tissues with liver fluke infection . for liver fluke - related cca , several genes within the abnormal chromosomal regions , including trefoil factor family 3 ( tff3 ) on 21q22.3 , run - related transcription factor 3 ( runx3 ) on 1p36 , cdkn2a on 9p21q and tp53 on 17p13 , and thymidine phosphorylate ( tp ) on 22q12 , which could be related to cca development or progression interestingly , the loss of 1p , 9p , and 17p chromosome regions which encode for cdkn2a and tp53 was found in cca both with and without liver fluke association . dna microarray technology was utilized to determine the genome - wide expression of gene related to cca carcinogenesis and sarcomatous transdifferentiation compared to normal epithelial cells . immunohistochemistry ( ihc ) and western immunoblotting analysis ( wb ) were performed in cca samples to validate the expression of secreted phosphoprotein 1 ( spp1 ) , ephrin - b2 ( efnb2 ) , iroquois - class homeodomain protein irx-3 ( irx3 ) , peroxisome proliferator - activated receptor gamma ( ppar ) , and insulin - like growth factor - binding protein 7 ( igfbp7 ) . efnb2 has been shown to be preferentially expressed in cca , and overexpression of efnb2 has been related to the clinical stage in various types of cancer , suggesting the possible role of efnb2 as a novel diagnostic marker . it has been shown that kras mutations are one of the most frequently altered genes in cca [ 4446 ] . the kras mutations would activate the raf - mek - erk - map kinase pathway to enhance gene transcription , cell cycle progression , and cell growth [ 47 , 48 ] . in animal models , kras gene activation , together with p53 activation , hotspots for pik3ca mutations in cca were found within exons 9 and 20 that encode helical and kinase domains of p110 involved in the pi3k / akt pathway . from tissue microarray , the translation proteins eif4-e and phosphorylated 4e - bp1 were identified as targets for pi3k pathway activation in cca , hinting to the clues toward cca pathogenesis . the results revealed that four transcription factors including forkhead box c1 ( foxc1 ) , zic family member 2 ( zic2 ) , nk2 transcription factor related , locus 2 ( nkx2 - 2 ) , and glucagon receptor ( gcgr ) are represented as hub nodes in the regulatory network . real - time pcr ( rt - pcr ) was used to validate the overexpression of 7 genes including fxyd3 ( ion transporter ) , g protein - couple receptor family c group 5 member a ( gprc5a ) , carcinoembryonic antigen - related cell adhesion molecule 5 ( ceacam5 ) , mucin 13 ( muc13 ) , epithelial cell adhesion molecule ( epcam ) , transmembrane channel like 5 ( tmc5 ) , and ets homolog factor ( ehf ) and the downregulation of 3 genes including carbamoyl phosphate synthetase 1 ( cps1)/mitochondrial carbamoyl synthetase 1 , tyrosine amino transferase ( tat ) , and inter- globin inhibitor h1 ( itih1 ) . nfkb- is involved in nf-b signaling pathways directing transformation , proliferation , invasion , angiogenesis , and metastasis . altogether , the pattern of gene expression along with parasitic infection data provides the significance of these molecules with opisthorchiasis - associated cca carcinogenesis . ca19 - 9 is lewis blood - group antigen , which has been widely used as a serum marker for cca . ca19 - 9 is also elevated in pancreatic cancer , gastric cancer , and primary biliary cirrhosis . bile proteomics of cca patients has been performed to differentiate malignant phenotypes from benign biliary strictures . further clarification of these molecules is required for the development as novel diagnostic for cca . the expression of these peptide markers indicated the changes in molecular pathways involved in inflammation , apoptosis , proteolysis and protein catabolism , and epithelial cell transformation . an independent validation set of 18 patients showed specificity of 78% and sensitivity of 84% , suggesting the possible role of bile proteomic analysis as a diagnostic tool for early development of cca in patients with psc . based on bile proteomics , 14 - 3 - 3/ protein has been identified in cca , hinting to its involvement in carcinogenesis due to its function in cellular processes such as actin cytoskeletal organization , cell adhesion , and antiapoptosis . these five 14 - 3 - 3 isoforms could bind to cruciform dna and enhance dna replication , which favors cca carcinogenesis . the study revealed a significant role of 14 - 3 - 3 protein in anoikis resistance of cca cells , pointing to the potential use as an early diagnostic biomarker and a target for cca therapeutics . bile proteomes from cca patients were analyzed using 2d - page coupled with matrix assisted laser desorption / ionization - time of flight - mass spectrometry ( maldi - tof - ms ) . the study clarified several proteins that were significantly elevated in cca patients compared to psc and benign cases such as -s - macroglobulin , apolipoprotein b-100 ( apo - b ) , ceruloplasmin , complement c3 , kininogen-1 ( kng1 ) isoform 2 , myeloperoxidase ( mpo ) , and inter--trypsin inhibitor heavy chain h4 ( itih4 ) . the results demonstrated that the levels of phosphatidylcholine ( ptc ) , bile acids , lipid , and cholesterol could be used to distinguish cca patients from benign groups with 88.9% sensitivity , 87.1% specificity , and 87.8% accuracy . differential patterns of bile components in cca patients may arise as a result of cancer cell proliferation and progression through deregulation of signaling pathways . further validations as well as assay development are warranted in order to find the efficient platform for cca diagnosis in terms of accuracy and cost effectiveness . activating and inactivating mutations these mutations promote cell survival , cancer initiation , and progression , which greatly affect survival of patients . the activating mutations of erbb2 have been previously observed in many types of cancers such as lung , breast , and colon cancer . erbb2 mutations have been correlated with tumor progression as shown in the erbb-2/neu transformed rat cholangiocytes that exhibit similar phenotypes as found in human cca . the gain - of - function mutations and overexpression of the erbb genes have been associated with poor prognosis and cca progression [ 91 , 92 ] . the critical roles of p53 include the regulation of cell cycle and apoptosis as well as dna repair . a meta - analysis study revealed high expression of p53 related to adverse clinical features and poor prognosis in ecca patients . activating mutation in the kras gene , which is downstream of epidermal growth factor receptor ( egfr or erbb1 ) , is one of the most frequent mutations found in icca [ 100 , 101 ] . in agreement with aforementioned studies , mutations in erbb2 , kras , tp53 , and smad4 furthermore , this study also identified mutations in c - met , bap1 , and fgfr pathways , which previously associated with cca prognosis . gain - of - function mutations in c - met , which is one of the growth factor receptors , are often found in biliary tract cancer and are also related to higher grade of invasiveness and poor prognosis [ 111113 ] . the results demonstrated that sox17 , krt20 , and erbb2 genes were overexpressed in bdeneu cells compared to bdesp cells , suggesting their potential use as prognostic molecular markers for cca . it has been associated with lymph node metastasis and adverse overall survival that strongly link to poor prognosis in cca patients . following gene enrichment analysis , a number of pathways , such as wnt , pi3k , mapk , and notch signaling , are commonly found to be altered in icca . it was also found to regulate pten - dependent activation of pi3k , which in turn affects cca progression . mir-204 has been shown to play a critical role in modulating emt by regulating the expression of slug , e - cadherin , and vimentin . periostin , known with a key role in cell adhesion , proliferation , and migration , may contribute to poor prognosis in cancer patients . dj-1 has been described as an oncoprotein associated with hras and transforms cells by promoting cell proliferation and resistance to cell cycle arrest , resulting in poor prognosis [ 139 , 140 ] . the normal function of cathepsin b involves cell proliferation , cell differentiation , and organogenesis as well as metabolism . in several cancers , cathepsin b involves degradation of ecm and promotes angiogenesis and metastatic capability inversely contributing to a decrease in survival rate of cca patients [ 141 , 142 ] . carbonylation of hsp70.1 has been significantly correlated with poor prognosis in cca patients [ 145 , 146 ] . the overexpression of -enolase was confirmed through ihc in 75% of cca patients with hyperplastic bile duct and the tumor compared to adjacent normal tissue region . based on proteomics , glycomics and glycol - proteomics technologies have been utilized to reveal significance of mucins as a glycol - biomarker in cca . it has been identified as a risk factor for poor prognosis in patients with mass - forming icca after surgery [ 163 , 164 ] . unlike muc1 and muc4 , the mrs approach was utilized to investigate bile contents from patients with cca compared to patients with benign biliary tract diseases . a significant higher level of glycine - conjugated bile acid but lower phosphatidylcholine ( ptc ) was also observed in bile of cca patients compared to that of patients with benign biliary tract diseases . gst possesses an antioxidant activity and its downregulation in cca would lead to accumulation of free radicals , causing genetic damage , which links to malignant transformation and cca progression . gnmt has been primarily in glycine , serine , and threonine metabolism , hinting that gnmt dysregulation in cca may result in metabolic shift , which would favor cca development . carbonylation of serotransferrin was detected and identified by mass spectrometric technique and the results showed high carbonylated serotransferrin in tumor tissues of cca patients . dysfunction of this iron transporter may result in the iron accumulation , which may involve iron overload and participate in induction of oxidative stress in tumor tissues [ 145 , 180 ] . currently , there is no effective therapeutics for cca ; therefore it is necessary to find more measures to suppress cancer progression to prolong survival rate in cca patients . research on potential inhibitors or drugs against target must be verified in cancer cells , animal models , and human clinical trials . there were chromosomal amplifications in 1q , 5q , 7q , and 17q in cca , while amplifications of 4p , 5p , 7p , 10p , 13q , 18q , and 20q were mostly found in icca patients . the deletions at 1p , 4q , 10q , 13q , 14q , and 18q were observed in cca , while deletion at 13q was mostly observed in icca . sunitinib malate is an inhibitor of vegfr types 1 and 2 , fms - like tyrosine kinase 3 ( flt3 ) , and platelet - derived growth factor ( pdgf ) . regorafenib showed antitumor growth as well as antiangiogenetic properties reducing tumor microvasculature by which its greater inhibitory effect compared to that of sorafenib was on vegfr2 and fgfr1 [ 196 , 197 ] . as previously mentioned , wes identified gene mutation of idh1 and idh2 in cca [ 93 , 199 , 200 ] . idh1 mutation would result in gain - of - function activity , which causes accumulation of 2-hg . mutated idh would cause 2-hg accumulation , which in turn inhibits prolyl hydroxylase , which is used to stabilize hypoxia - inducible factor-1 ( hif-1 ) , leading to the absence of oxygen - dependent hydroxylation . besides hsp90 , this study also revealed that hsp90 inhibitors , tanespimycin and nvp - auy922 ( a novel hsp90 inhibitor ) , could increase level of hsp70 which was found to have a low expression in cca patients [ 146 , 205 ] . these data indicated that hsp70 and hsp90 may act as therapeutic markers in cca that can be targeted by hsp90 inhibitors . upregulation of -s - macroglobulin , apo - b b100 , ceruloplasmin , complement c3 , kng1 isoform 2 , mpo , and itih4 was observed . however , their uses in cca patients need further investigations [ 209 , 210 ] . azd9150 is a 16-oligonucleotide antisense molecule ( aso ) , which targets the 3 untranslated part of stat3 , thereby preventing protein expression . the data showed an increase in glycine - conjugated bile acids in cca patients compared to benign disease groups . lack of effective therapeutics for cca urges the demand for specific and early diagnostic biomarkers to increase survival rate , for prognostic biomarkers to provide more precision of cca progression , and for therapeutic biomarkers to develop curative strategies . with advanced molecular techniques , along with genes and proteins that have been identified as molecular markers for cca , some of which have been practically used , more diverse and specific promising biomarkers in cca have been established including the mirna of mir-21 , mir-200 , mir-204 , and enzymes such as gstp , tff , idh , and mucins . the discovery of these molecules in the pathways involving cell proliferation , invasion , apoptosis , and tumor suppressor would hint us toward the molecular mechanism that gives rise to cca behaviors and characteristics . future directions include the exploration and validation of their potential use in diagnosis and prognosis , as well as therapeutics .

following acute myocardial infarction ( ami ) , recovery of exercise capacity and control of coronary risk factors ( crfs ) are highly critical for prognosis post ami1 . long - term follow - up research has shown that the improvement of exercise capacity is negatively related to future cardiac events2 , and that the improvement of crfs is beneficial for preventing the development of atherosclerosis . supervised , hospital - based phase ii cardiac exercise therapy ( cet ) allows patients to exercise safely following personalized exercise prescriptions and monitoring of crfs . home - based phase iii cet following hospital - based phase ii continues to prepare patients with the ability and confidence to go back to the society as soon as possible . does supervised , hospital - based phase ii cet help patients to achieve levels of exercise capacity equal to those of age - matched normal subjects with patent coronary arteries ? this study used age - matched subjects with patent coronary arteries as a normal - control group ( ncg ) . it was designed to investigate the effects of phase ii and iii cet on exercise capacity , and on the long - term changes in crfs of patients with ami in comparison with age - matched normal subjects . the subjects were patients with ami who had been admitted to tzu - chi medical center , and had undergone coronary angiography ( cag ) for evaluation and treatment of coronary artery lesions . subjects in the experimental group ( eg ) were defined as those who were admitted to hospital after ami , who had participated in the phase ii hospital - based cet and continued to do the phase iii home - based exercise program . subjects in the control group ( cg ) were defined as those who were admitted to hospital after ami but did not participate in the hospital - based phase ii cet because of inconvenience of travel or for personal reasons . after discharge from hospital , eg patients participated in an 8-week cet program designed specifically for each patient by the same experienced physical therapist . there was no coronary artery bypass surgery ( cabg ) given to eg or cg , and no stroke during the period when the study was conducted . another group , the normal control group ( ncg ) , took part in this study . subjects in ncg were whose who had received cag and had proven patent coronary arteries . the reason for cag was either suspicion of coronary artery lesions or pre - examination for possible congenital heart disease . subjects in ncg were age - matched with the subjects with ami in eg and cg . the criterion for age matching was an age within 3 years of the target subject . in order to carefully understand how the home - based exercise was performed , we used a questionnaire to specifically ask subjects the mode , the frequency and duration of exercise . subjects who performed the exercise at least 3 times a week , 30 minutes a time and reached their target heart rate were defined as regular exercise performers . this study was approved by the institutional review board of tzu chi medical center , and written informed consent to participation in the study was obtained from each subject . data for duration of hospital stay , medication , cigarette smoking , hypertension ( htn ) , diabetes mellitus ( dm ) , body mass index ( bmi ) , and fasting plasma serum levels of total cholesterol ( t - chol ) , triglyceride ( tg ) , high density lipoprotein cholesterol ( hdl - c ) and low density lipoprotein cholesterol ( ldl - c ) were collected during hospitalization . severity of disease , such as numbers of stenotic vessels , with or without q wave , and infarcted wall are recorded shown in table 1table 1.test items examined at different times in the normal control group ( ncg ) , exercise group ( eg ) , and control group ( cg)periodacute stagephase ii cetphase iii cetduring admissionbeginningendhome exercisetest itemdiseaseseveritycrfsmedicationmetsmetscrfscrfsmetsmedicationquestionnairegroupncg-*-*------eg**********cg**********crfs , coronary risk factors ; mets , metabolic equivalents ; cet , cardiac exercise therapy . smoking habits , bmi and plasma serum lipids were re - evaluated at the times of the second ( the 2nd test ) and third examinations ( the 3rd test ) . the 2nd and 3rd tests were done at two and five months after discharge from hospital , respectively . crfs , coronary risk factors ; mets , metabolic equivalents ; cet , cardiac exercise therapy all three groups performed the symptom - limited maximal exercise test . subjects blood was collected to measure serum lipids , and anthropometric measurements were taken to calculate bmi . the maximal exercise test was performed by both eg and cg at the beginning of phase ii cet ( the 1st test ) , at the end of phase ii cet ( the 2nd test ) and at 3 months after the end of phase ii cet ( the 3rd test ) . ncg only performed the maximal exercise test at the time of the 1 test . eg and cg patients performed an exercise test on a treadmill ( marquette max1 , milwaukee wi , usa ) . the symptom - limited maximal exercise capacity , expressed in metabolic equivalent ( mets ) , was obtained at the end of the exercise test4 . the ecg was monitored continuously , with hr , bp and rate - pressure product ( rpp ) measured every three minutes during the exercise test , and throughout the five - minute recovery period . the borg scale of rating of perceived exertion ( rpe ) , which is reported to be highly related to maximal oxygen consumption and metabolic demand , were quantified at the same time . we requested the patients to abstain from food , coffee , and cigarettes for a minimum of two hours before each test . they watched a real test to learn how the maximal exercise test is conducted before they performed the test . before the maximal exercise test started , subjects were asked to lie supine for 3 minute so that we could collect measurements of their resting state . the bruce protocol starts at an intensity of 4.6 mets , and increases by 23 mets every 3 minutes , and it is considered unsuitable for patients with ami5 . therefore , the modified naughton protocol was adopted as the 1st test for its lower beginning intensity ( 3 mets ) and smaller increment ( 1 met every 3 minutes ) . criteria for terminating the 1st exercise test included ischemic signs and/or symptoms , inability to continue the exercise due to fatigue , hr reaching 140 bpm for subjects under 40 years old , or hr reaching 130 bpm for subjects over 40 years old . for the 2nd and 3rd exercise tests , criteria for terminating the exercise test included ventricular arrhythmia ( such as ventricular tachycardia and fibrillation ) , st segment depression ( 2 mm ) or elevation ( 1 mm ) , target hr exceeded ( 90% of the predicted age - adjusted maximal hr ) , or bp over 220 /hg or lower than that determined at the beginning of the test . venous blood samples were collected after a 12-hour fast and used for the determination of lipids ( serum t chol , tg , hdl - c , and ldl - c ) . serum t chol was measured using the enzymatic method and automatic multi - channel chemical analyzer ( 747 automatic analyzer , hitachi , tokyo , japan ) , with the au500/550 t - chol reagent kit ( katayama , osaka , japan ) . serum hdl - c levels were determined using the precipitation method with phosphotungstic acid and magnesium chloride6 . serum ldl - c was calculated using the values of t - chol , tg and hdl - c in the following formula : ldl - c= t - chol ( ( 1/5tg)+ hdl - c ) phase ii cet began 19(9 ) ( range 639 ) days after ami , and continued for eight weeks at a frequency of two times per week . a warm - up was performed of 1015 minutes of calisthenics ( gentle stretching ) . the main exercise was performed on a treadmill , at an exercise intensity individually prescribed on the basis of baseline data , and the graded treadmill exercise test results . the exercise intensity was gradually increased to a goal of 60 to 79% of the predicted age - adjusted maximum heart rate ( hr ) , and the exercise continued for 20 minutes at an intensity below that which would cause symptomatic or silent ischemia . a cool - down , consisting of ten minutes of calisthenics was also performed . during the period of cet , subjects were often given real - time feedback of hr as exercise proceeded , and learned to monitor their own hr . after phase ii cet finished , phase iii cet programs were designed specifically for each individual , and data were collected for 3 months . during phase iii cet , patients were asked to continue leisure - time physical activities chosen by themselves , with an exercise formula prescribed according to the results of the second of the exercise tests performed at the end of phase ii cet . based on the over - riding emphasis on safety , an exercise type familiar to each individual was chosen for phase iii cet . all eg patients were telephoned by the same investigator each month to provide relevant information and personal support . subjects in cg performed the 1st and 2nd maximal exercise test and were given an exercise prescription based on the results of the tests . subjects were telephoned by the same investigator each month to provide relevant information and personal support , and if needed , to change the exercise intensity to ensure its effectiveness and safety . subjects in cg were advised to exercise regularly after discharge , and were followed for the status of home - based exercise during phase ii and phase iii by questionnaire . two - way repeated measures anova was used to compare the differences in crfs for group effect ( two levels : eg vs. cg ) and training effect ( three levels : 1st test , 2nd test and 3rd test ) with repeated measures of the second factor . significant interaction effects were analyzed using univariate repeated measures and mean comparisons with the lsd method . sixty subjects completed the study , 20 ami patients in eg ( age : 55.310.6 years ) , 10 ami patients in cg ( age : 56.69.5 years ) , and the 30 age - matched normal subjects with patent coronary arteries in ncg ( age : 57.19.6 years ) . table 2table 2.hemodynamic parameters , exercise capacity and crfs of each groupvariablesncg ( n=30)eg ( n=20)cg ( n=10)1st test2nd test3rd test1st test2nd test3rd testage ( yrs)57.19.655.310.6 - -56.69.5 - -exercise capacitymets9.42.16.51.510.01.49.71.57.11.28.51.79.81.0hemodynamic parametersrpp*100024.55.119.36.522.34.124.44.921.83.026.03.725.11.6maximal hr140.120.7126.820.4139.913.1149.516.5137.68.1146.516.5148.910.4maximal bp174.422.4149.928.0159.123.6162.822.7159.114.3181.615.2171.018.1borg scale-13.80.814.11.014.30.813.81.013.91.114.51.1crfssmoking ( % ) 3378617562222 dm ( % ) 1722 - -22 - -htn ( % ) 5033 - -36 - -total cholesterol ( mg / dl)179.945.2184.043.4178.430.9196.649.3208.855.4207.557.0207.048.6triglyceride ( mg / dl)178.4110.4164.282.0182.9134.5155.1103.9154.6120.3178.0157.1153.6121.2hdl - c ( mg / dl)43.212.438.110.838.49.142.510.344.910.046.710.346.27.3ldl - c ( mg / dl)103.343.8113.036.4102.835.5124.551.8139.551.1126.260.8133.146.3bmi27.54.325.24.025.24.125.33.925.44.824.23.324.53.6disease severityadmission days-10.45.5 - -8.73.3 - -number of stenotic vessels01.50.8 - -2.00.9 - -ekgq wave ( n)-16 - -8 - -non - q wave ( n)-4 - -2 - -infarcted wallanterior ( n)-8 - -3 - -lower / posterior ( n)-7 - -5 - -lateral ( n)-1 - -0 - -angioplastyphase i ( n)-9 - -5 - -phase ii ( n)-0 - -0 - -phase iii ( n)-2 - -1 - -crfs : coronary risk factors , phase i : acute stage during the admission , phase ii : supervised , hospital - based phase ii cardiac exercise therapy , phase iii : home - based phase iii cardiac exercise therapy . * : significantly lower than ncg ( p<0.05 ) : : significantly higher than ncg ( p<0.05 ) . : significantly higher than the 1st test ( p<0.05 ) ; & : significantly lower than the 1st test ( p<0.05 ) ; : significantly higher than the 2nd test ( p<0.05 ) ; # : significant difference between groups ( p<0.05 ) . shows the subjects , anthropometric characteristics . mets , rpp and systolic bp of ncg were significantly higher than those of eg and cg . the percentage of smokers was lower in ncg than in the other two groups . crfs : coronary risk factors , phase i : acute stage during the admission , phase ii : supervised , hospital - based phase ii cardiac exercise therapy , phase iii : home - based phase iii cardiac exercise therapy . * : significantly lower than ncg ( p<0.05 ) : : significantly higher than ncg ( p<0.05 ) . : significantly higher than the 1st test ( p<0.05 ) ; & : significantly lower than the 1st test ( p<0.05 ) ; : significantly higher than the 2nd test ( p<0.05 ) ; # : significant difference between groups ( p<0.05 ) . as shown in table 2 , exercise capacity increased 54% ( 6.5 vs. 10.0 mets , p<0.01 ) in eg from the 1st to the 2nd test without any increase in rpe . both eg and cg showed improvements in their exercise capacities ( eg : 6.5 to 9.7 mets ; cg : 7.1 to 9.8 mets ) from the 1st to the 3rd test , with improvements of approximately 49% and 38% , respectively . the only significant difference found between eg and cg appeared in systolic bp at the stop point of the maximal exercise test . the 2nd test of hdl - c is significantly higher than the first in eg . there were no differences in blocker , ace - i , diuretics , ca channel blocker and hypolipemic drug use among the groups . in eg , six subjects chose walking as their home exercise , six chose walking / jogging , five chose cycling and one each chose table - tennis , golf and rope skipping . a well designed phase ii cet not only increases the confidence of patients to proceed to phase iii , but also increases compliance and safety during phase iii . mets are calculated from oxygen consumption during exercise and they are easy to convert to the energy consumption for activities . the values of mets are related to genetics , fitness , disease level , age , and gender , and it is one of the best indexes for measuring exercise capacity and cardiovascular function8 . mets and hr / sbp at the point of maximal exercise are highly correlated with left ventrical perfusion and abnormal left ventrical function , which have been proven to be highly important in predicting the prognosis after ami9 . the hemodynamic parameter , rpp , the product of hr and sbp , can be used to evaluate both dynamic and static exercise , and is not influenced by the use of blockers . it is agreed that hr and sbp are increased by the increase of exercise intensity ; however , the degree of increase can be reduced by proper exercise therapy10 . as shown in table 2 , phase ii cet also stabilizes the autonomic nervous system and this is reflected in hemodynamic parameters11 . in the present study , , there was no significant difference in maximal mets , medication or the successful rate of percutaneous coronary intervention . however , between the 1st and the 2nd test , mets increased only in eg and without any change in rpe or rpp . cg did not show an improvement in mets until the 3rd test , while rpp increased in the 2nd test . without the intervention of phase ii cet , the hemodynamic parameters at the maximal mets in cg were poorer than those of eg , especially sbp at the point of maximal exercise . this shows that sustainable regular exercise is helpful for the improvement of mets , and for the same reason , that phase ii cet successfully shortens the natural recovery time of exercise capacity . besides , hr is representative of the physical load exacted on the heart11 . in the present study , 6079% of maximal target hr was determined based on the results of ecg , hemodynamic parameters , exercise capacity and rpe obtained in the symptom - limited maximal exercise test . this helped to ensure the phase iii home exercise program was feasible and safe . cet , based on exercise training , has changed the emphasis from long - term prognosis to short - term effects . however , long - term prognosis is highly correlated to lifelong regular exercise and control of crfs12 . eg chose an exercise activity they were familiar with , and trained at 6079% of their maximal hr in the phase iii home cet . one of the authors called all the subjects every month to ask if they had any difficulty in continuing their exercise program . found that the benefits of a 12-week phase ii cet were not maintained after 6 months13 . in the present study , subjects in eg continued to show improvements in their maximal mets until 3 months after the end of phase ii cet . it would have been better to have performed a longer follow - up , but it is difficult to control subjects in long - term clinical studies . the authors wish to further investigate phase iii cet in the future , and hopefully as time proceeds , there will be more subjects and longer periods of follow - up providing data which will allow a better understanding of the mechanism behind the outcomes . we found that maximal mets in cg increased during the phase iii cet and eventually reached the same level as the shown by ncg in the 1st test . using an exercise that subjects are already familiar with can provide fitness and wellbeing . it has been reported to be of physiological benefit14 . according to the questionnaire regarding exercise status , 67% of subjects in eg chose to do walking - related activities , such as walking ( 6 subjects ) , and brisk walking/ jogging ( 6 subjects ) . all the subjects in cg chose to walk as their home exercise program ( walking : 7 , brisk walk : 1 , jogging : 1 , and treadmill walking : 1 ) . we found that moderate intensity of home exercise , 3 times a week with a duration of at least 30 minutes , was safe , flexible and did not discourage the subjects so it would be well accepted by most people . previous research has shown that walking can prevent recurrence of heart attack and has been used as a secondary prevention method for patients with ami15 . there were no reinfarction or cardiac events in either eg or cg during phases ii and iii cet , and maximal mets were improved and maintained , the effects of exercise therapy on total cholesterol ( t - cho ) and low density lipoprotein ( ldl - c ) are controversial16 , but it is agreed that exercise is mostly beneficial for controlling triglyceride no matter how long the training period is . possible mechanisms for this include lowering the synthesis of very low density lipoprotein ( vldl ) , speeding up catabolism , and greater metabolism of free fatty acids to provide energy source for skeletal muscles16 . we note that neither eg nor cg had high triglyceride to begin with , and this is likely why there was not much exercise effect on triglyceride . possible reasons why sustainable walking training increased high density lipoprotein cholesterol ( hdl - c ) by 2030% are overactive lipoprotein lipase and lecithin - cholesterol - acyltransferase induce greater catabolism of vldl , and lower the activity of hepatic lipase . in the present study , there was no significant change in hdl - c although it did increase in eg . in taiwan , the impact of crfs on cad is similar to that reported for the united states and japan17 . especially , hdl - c has been shown to be highly correlated with cad18 . hdl - c is influenced by gender , age , medication , smoking , obesity , and the parameters of the exercise prescription19 . nicotine increases epinephrine and norepinephrine in the circulation speeding up the development of atherosclerosis . compared to non - smokers , smokers have 5.7% lower hdl - c and quitting smoking can make hdl - c more normal20 . our results show that the cessation of smoking rate increased at the 2nd test , and continued to increase at the 3rd test . however , a significant increase in hdl - c was only seen in the 3rd test . this shows that quitting smoking alone is not enough to explain the increase in hdl - c . our results show that the maximal mets increased in eg during the phase ii cet , but bmi did not change . this could explain why hdl - c did not increase during the phase ii cet . in addition to smoking and bmi , the intensity , duration , and frequency of exercise during cet also affect hdl - c22 , 23 . mendoza et al . showed that 312 months of exercise are needed to elicit an increase in hdl - c16 . berg et al . showed that at least 3 months is required to improve hdl - c . therefore , the reason that hdl - c did not improve during the phase ii cet might be because exercise , 2 times a week for a total of 8 weeks is insufficient . in contrast , exercise , 2 times a week elicited a difference in exercise capacity in the present study . after phase ii and phase iii cet possible reasons for this could be that exercise capacity continued to improve and the cessation of smoking rate was maintained , with no change in bmi . it is also possible that the combination of phase ii and iii cet was beneficial for maintaining cessation of smoking and bmi , creating an environment in which an increase of hdl - c could occur . hdl - c in cg did not change with improvement of exercise capacity , despite the fact that bmi was maintained and there was a stop in the smoking cessation rate . the small sample size could be another reason why no change in hdl - c was observed . the authors hope to enroll more subjects in the future to better investigate the temporal change of hdl - c during phase iii cet . one limitation of our research was the relatively large number of days before exercise was initiated after myocardial infarction . we recognize that this makes the interpretation of the training effect more difficult . however , considering the substantial area and rugged travel conditions of hualien county and the difficulty of making transportation arrangements for some patients , this was perhaps the best result achievable . it is our hope that this limitation is offset by the fact that this research presents a contribution to clinical medicine from an under - represented corner of taiwan and of the world . this study pioneered the enrolment of subjects with patent coronary arteries as a normal control group , and we believe it will provide valuable insights for clinical medicine . in conclusion , a supervised , hospital - based phase ii cet performed twice a week for 8 weeks shortened the recovery time of exercise capacity in patients with ami . successful and regular exercise during phase iii home - based cet maintained the exercise capacity gains of phase ii , improved hdl - c , and also continued to increase exercise capacity in the control group .

[ purpose ] to investigate the effects of phase ii cardiac exercise therapy ( cet ) on exercise capacity and changes in coronary risk factors ( crfs ) of patients with acute myocardial infarction ( ami ) . [ subjects ] thirty male subjects with ami were divided into an experimental group ( eg ) and a control group ( cg ) . another 30 age - matched subjects with patent coronary arteries served as a normal - control group ( ncg ) . [ methods ] subjects in eg ( n=20 ) trained using a stationary bicycle for 30 min at their target heart rate twice a week for 8 weeks . exercise capacity was defined as the maximal metabolic equivalents ( mets ) that subjects reached during the symptom - limited maximal exercise test . hr , bp and rpp were recorded . subjects in eg and cg received exercise tests and screening for crfs at the beginning of , end of , and 3 months after phase ii cet , while subjects in ncg participated only in the 1st test . [ results ] mets of cg did not improve until the 3rd test , while rpp at the 2nd test showed a significant increase . however , eg showed increased mets at the 2nd test without increase of rpp , and increased their high density lipoprotein cholesterol ( hdl - c ) during the follow - up period between the 2nd and 3rd tests . [ conclusion ] phase ii cet shortens the recovery time of exercise capacity , helps to maintain the gained exercise capacity and increases hdl - c in phase iii .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

following acute myocardial infarction ( ami ) , recovery of exercise capacity and control of coronary risk factors ( crfs ) are highly critical for prognosis post ami1 . long - term follow - up research has shown that the improvement of exercise capacity is negatively related to future cardiac events2 , and that the improvement of crfs is beneficial for preventing the development of atherosclerosis . supervised , hospital - based phase ii cardiac exercise therapy ( cet ) allows patients to exercise safely following personalized exercise prescriptions and monitoring of crfs . home - based phase iii cet following hospital - based phase ii continues to prepare patients with the ability and confidence to go back to the society as soon as possible . does supervised , hospital - based phase ii cet help patients to achieve levels of exercise capacity equal to those of age - matched normal subjects with patent coronary arteries ? this study used age - matched subjects with patent coronary arteries as a normal - control group ( ncg ) . it was designed to investigate the effects of phase ii and iii cet on exercise capacity , and on the long - term changes in crfs of patients with ami in comparison with age - matched normal subjects . the subjects were patients with ami who had been admitted to tzu - chi medical center , and had undergone coronary angiography ( cag ) for evaluation and treatment of coronary artery lesions . subjects in the experimental group ( eg ) were defined as those who were admitted to hospital after ami , who had participated in the phase ii hospital - based cet and continued to do the phase iii home - based exercise program . subjects in the control group ( cg ) were defined as those who were admitted to hospital after ami but did not participate in the hospital - based phase ii cet because of inconvenience of travel or for personal reasons . another group , the normal control group ( ncg ) , took part in this study . subjects in ncg were whose who had received cag and had proven patent coronary arteries . subjects in ncg were age - matched with the subjects with ami in eg and cg . subjects who performed the exercise at least 3 times a week , 30 minutes a time and reached their target heart rate were defined as regular exercise performers . data for duration of hospital stay , medication , cigarette smoking , hypertension ( htn ) , diabetes mellitus ( dm ) , body mass index ( bmi ) , and fasting plasma serum levels of total cholesterol ( t - chol ) , triglyceride ( tg ) , high density lipoprotein cholesterol ( hdl - c ) and low density lipoprotein cholesterol ( ldl - c ) were collected during hospitalization . severity of disease , such as numbers of stenotic vessels , with or without q wave , and infarcted wall are recorded shown in table 1table 1.test items examined at different times in the normal control group ( ncg ) , exercise group ( eg ) , and control group ( cg)periodacute stagephase ii cetphase iii cetduring admissionbeginningendhome exercisetest itemdiseaseseveritycrfsmedicationmetsmetscrfscrfsmetsmedicationquestionnairegroupncg-*-*------eg**********cg**********crfs , coronary risk factors ; mets , metabolic equivalents ; cet , cardiac exercise therapy . smoking habits , bmi and plasma serum lipids were re - evaluated at the times of the second ( the 2nd test ) and third examinations ( the 3rd test ) . the 2nd and 3rd tests were done at two and five months after discharge from hospital , respectively . crfs , coronary risk factors ; mets , metabolic equivalents ; cet , cardiac exercise therapy all three groups performed the symptom - limited maximal exercise test . the maximal exercise test was performed by both eg and cg at the beginning of phase ii cet ( the 1st test ) , at the end of phase ii cet ( the 2nd test ) and at 3 months after the end of phase ii cet ( the 3rd test ) . ncg only performed the maximal exercise test at the time of the 1 test . eg and cg patients performed an exercise test on a treadmill ( marquette max1 , milwaukee wi , usa ) . the symptom - limited maximal exercise capacity , expressed in metabolic equivalent ( mets ) , was obtained at the end of the exercise test4 . the ecg was monitored continuously , with hr , bp and rate - pressure product ( rpp ) measured every three minutes during the exercise test , and throughout the five - minute recovery period . they watched a real test to learn how the maximal exercise test is conducted before they performed the test . before the maximal exercise test started , subjects were asked to lie supine for 3 minute so that we could collect measurements of their resting state . the bruce protocol starts at an intensity of 4.6 mets , and increases by 23 mets every 3 minutes , and it is considered unsuitable for patients with ami5 . therefore , the modified naughton protocol was adopted as the 1st test for its lower beginning intensity ( 3 mets ) and smaller increment ( 1 met every 3 minutes ) . for the 2nd and 3rd exercise tests , criteria for terminating the exercise test included ventricular arrhythmia ( such as ventricular tachycardia and fibrillation ) , st segment depression ( 2 mm ) or elevation ( 1 mm ) , target hr exceeded ( 90% of the predicted age - adjusted maximal hr ) , or bp over 220 /hg or lower than that determined at the beginning of the test . venous blood samples were collected after a 12-hour fast and used for the determination of lipids ( serum t chol , tg , hdl - c , and ldl - c ) . serum ldl - c was calculated using the values of t - chol , tg and hdl - c in the following formula : ldl - c= t - chol ( ( 1/5tg)+ hdl - c ) phase ii cet began 19(9 ) ( range 639 ) days after ami , and continued for eight weeks at a frequency of two times per week . the exercise intensity was gradually increased to a goal of 60 to 79% of the predicted age - adjusted maximum heart rate ( hr ) , and the exercise continued for 20 minutes at an intensity below that which would cause symptomatic or silent ischemia . during the period of cet , subjects were often given real - time feedback of hr as exercise proceeded , and learned to monitor their own hr . after phase ii cet finished , phase iii cet programs were designed specifically for each individual , and data were collected for 3 months . during phase iii cet , patients were asked to continue leisure - time physical activities chosen by themselves , with an exercise formula prescribed according to the results of the second of the exercise tests performed at the end of phase ii cet . subjects in cg performed the 1st and 2nd maximal exercise test and were given an exercise prescription based on the results of the tests . subjects in cg were advised to exercise regularly after discharge , and were followed for the status of home - based exercise during phase ii and phase iii by questionnaire . two - way repeated measures anova was used to compare the differences in crfs for group effect ( two levels : eg vs. cg ) and training effect ( three levels : 1st test , 2nd test and 3rd test ) with repeated measures of the second factor . sixty subjects completed the study , 20 ami patients in eg ( age : 55.310.6 years ) , 10 ami patients in cg ( age : 56.69.5 years ) , and the 30 age - matched normal subjects with patent coronary arteries in ncg ( age : 57.19.6 years ) . table 2table 2.hemodynamic parameters , exercise capacity and crfs of each groupvariablesncg ( n=30)eg ( n=20)cg ( n=10)1st test2nd test3rd test1st test2nd test3rd testage ( yrs)57.19.655.310.6 - -56.69.5 - -exercise capacitymets9.42.16.51.510.01.49.71.57.11.28.51.79.81.0hemodynamic parametersrpp*100024.55.119.36.522.34.124.44.921.83.026.03.725.11.6maximal hr140.120.7126.820.4139.913.1149.516.5137.68.1146.516.5148.910.4maximal bp174.422.4149.928.0159.123.6162.822.7159.114.3181.615.2171.018.1borg scale-13.80.814.11.014.30.813.81.013.91.114.51.1crfssmoking ( % ) 3378617562222 dm ( % ) 1722 - -22 - -htn ( % ) 5033 - -36 - -total cholesterol ( mg / dl)179.945.2184.043.4178.430.9196.649.3208.855.4207.557.0207.048.6triglyceride ( mg / dl)178.4110.4164.282.0182.9134.5155.1103.9154.6120.3178.0157.1153.6121.2hdl - c ( mg / dl)43.212.438.110.838.49.142.510.344.910.046.710.346.27.3ldl - c ( mg / dl)103.343.8113.036.4102.835.5124.551.8139.551.1126.260.8133.146.3bmi27.54.325.24.025.24.125.33.925.44.824.23.324.53.6disease severityadmission days-10.45.5 - -8.73.3 - -number of stenotic vessels01.50.8 - -2.00.9 - -ekgq wave ( n)-16 - -8 - -non - q wave ( n)-4 - -2 - -infarcted wallanterior ( n)-8 - -3 - -lower / posterior ( n)-7 - -5 - -lateral ( n)-1 - -0 - -angioplastyphase i ( n)-9 - -5 - -phase ii ( n)-0 - -0 - -phase iii ( n)-2 - -1 - -crfs : coronary risk factors , phase i : acute stage during the admission , phase ii : supervised , hospital - based phase ii cardiac exercise therapy , phase iii : home - based phase iii cardiac exercise therapy . : significantly higher than the 1st test ( p<0.05 ) ; & : significantly lower than the 1st test ( p<0.05 ) ; : significantly higher than the 2nd test ( p<0.05 ) ; # : significant difference between groups ( p<0.05 ) . the percentage of smokers was lower in ncg than in the other two groups . crfs : coronary risk factors , phase i : acute stage during the admission , phase ii : supervised , hospital - based phase ii cardiac exercise therapy , phase iii : home - based phase iii cardiac exercise therapy . : significantly higher than the 1st test ( p<0.05 ) ; & : significantly lower than the 1st test ( p<0.05 ) ; : significantly higher than the 2nd test ( p<0.05 ) ; # : significant difference between groups ( p<0.05 ) . as shown in table 2 , exercise capacity increased 54% ( 6.5 vs. 10.0 mets , p<0.01 ) in eg from the 1st to the 2nd test without any increase in rpe . both eg and cg showed improvements in their exercise capacities ( eg : 6.5 to 9.7 mets ; cg : 7.1 to 9.8 mets ) from the 1st to the 3rd test , with improvements of approximately 49% and 38% , respectively . the only significant difference found between eg and cg appeared in systolic bp at the stop point of the maximal exercise test . the 2nd test of hdl - c is significantly higher than the first in eg . a well designed phase ii cet not only increases the confidence of patients to proceed to phase iii , but also increases compliance and safety during phase iii . the values of mets are related to genetics , fitness , disease level , age , and gender , and it is one of the best indexes for measuring exercise capacity and cardiovascular function8 . it is agreed that hr and sbp are increased by the increase of exercise intensity ; however , the degree of increase can be reduced by proper exercise therapy10 . however , between the 1st and the 2nd test , mets increased only in eg and without any change in rpe or rpp . cg did not show an improvement in mets until the 3rd test , while rpp increased in the 2nd test . without the intervention of phase ii cet , the hemodynamic parameters at the maximal mets in cg were poorer than those of eg , especially sbp at the point of maximal exercise . this shows that sustainable regular exercise is helpful for the improvement of mets , and for the same reason , that phase ii cet successfully shortens the natural recovery time of exercise capacity . in the present study , 6079% of maximal target hr was determined based on the results of ecg , hemodynamic parameters , exercise capacity and rpe obtained in the symptom - limited maximal exercise test . cet , based on exercise training , has changed the emphasis from long - term prognosis to short - term effects . eg chose an exercise activity they were familiar with , and trained at 6079% of their maximal hr in the phase iii home cet . in the present study , subjects in eg continued to show improvements in their maximal mets until 3 months after the end of phase ii cet . it would have been better to have performed a longer follow - up , but it is difficult to control subjects in long - term clinical studies . the authors wish to further investigate phase iii cet in the future , and hopefully as time proceeds , there will be more subjects and longer periods of follow - up providing data which will allow a better understanding of the mechanism behind the outcomes . we found that maximal mets in cg increased during the phase iii cet and eventually reached the same level as the shown by ncg in the 1st test . according to the questionnaire regarding exercise status , 67% of subjects in eg chose to do walking - related activities , such as walking ( 6 subjects ) , and brisk walking/ jogging ( 6 subjects ) . there were no reinfarction or cardiac events in either eg or cg during phases ii and iii cet , and maximal mets were improved and maintained , the effects of exercise therapy on total cholesterol ( t - cho ) and low density lipoprotein ( ldl - c ) are controversial16 , but it is agreed that exercise is mostly beneficial for controlling triglyceride no matter how long the training period is . possible reasons why sustainable walking training increased high density lipoprotein cholesterol ( hdl - c ) by 2030% are overactive lipoprotein lipase and lecithin - cholesterol - acyltransferase induce greater catabolism of vldl , and lower the activity of hepatic lipase . in the present study , there was no significant change in hdl - c although it did increase in eg . hdl - c is influenced by gender , age , medication , smoking , obesity , and the parameters of the exercise prescription19 . our results show that the cessation of smoking rate increased at the 2nd test , and continued to increase at the 3rd test . however , a significant increase in hdl - c was only seen in the 3rd test . our results show that the maximal mets increased in eg during the phase ii cet , but bmi did not change . this could explain why hdl - c did not increase during the phase ii cet . in addition to smoking and bmi , the intensity , duration , and frequency of exercise during cet also affect hdl - c22 , 23 . showed that 312 months of exercise are needed to elicit an increase in hdl - c16 . showed that at least 3 months is required to improve hdl - c . therefore , the reason that hdl - c did not improve during the phase ii cet might be because exercise , 2 times a week for a total of 8 weeks is insufficient . in contrast , exercise , 2 times a week elicited a difference in exercise capacity in the present study . after phase ii and phase iii cet possible reasons for this could be that exercise capacity continued to improve and the cessation of smoking rate was maintained , with no change in bmi . it is also possible that the combination of phase ii and iii cet was beneficial for maintaining cessation of smoking and bmi , creating an environment in which an increase of hdl - c could occur . hdl - c in cg did not change with improvement of exercise capacity , despite the fact that bmi was maintained and there was a stop in the smoking cessation rate . the authors hope to enroll more subjects in the future to better investigate the temporal change of hdl - c during phase iii cet . this study pioneered the enrolment of subjects with patent coronary arteries as a normal control group , and we believe it will provide valuable insights for clinical medicine . in conclusion , a supervised , hospital - based phase ii cet performed twice a week for 8 weeks shortened the recovery time of exercise capacity in patients with ami . successful and regular exercise during phase iii home - based cet maintained the exercise capacity gains of phase ii , improved hdl - c , and also continued to increase exercise capacity in the control group .

stabilized rice bran is a unique whole food that naturally contains protein , vitamins , minerals , complex carbohydrates , phytonutrients , phospholipids , essential fatty acids , and more than 120 antioxidants.(1 ) dietary rice bran intake and rice bran components have demonstrated chronic disease fighting activity , particularly for protection against cardiovascular disease and certain cancers . we and others have shown that rice varieties are not equal in content and composition of bioactive rice bran components . how these phytochemicals are altered by microbial fermentation and metabolism is an emerging area of research that merits scientific investigation when assessing bioactivity and health benefits . a few studies have evaluated rice bran as a dietary supplement or functional food ingredient ; however , little is known about how chemical content changes with and without fermentation . boulardii ( s. boulardii ) , has probiotic activity and is widely used as a dietary supplement for intestinal disease prevention and treatment . the spectrum of biomedical activities and food processing applications reported with s. boulardii has significantly grown over the past decade and includes , but is not limited to , protection against enteric pathogens , modification of lymphocyte proliferation , and differential release of plant secondary metabolites from foods such as wine , sourdough and cheese.sacchromyces boulardii has been shown to be beneficial for modification of food components such as breakdown of dietary phytate and biofortification of folate to improve the nutritional value and health properties of food . the health benefit of s. boulardii both as a probiotic and for fermented foods was recently reviewed , and a meta - analysis of placebo - controlled treatment trials supports its safety and efficacy for protection against several types of diarrhea . protection against specific enteric bacterial pathogens by s. boulardii may , in part , be due to anti - inflammatory actions and effects on immunity.in vitro studies using mammalian cell cultures have shown that s. boulardii modifies host cell signaling pathways associated with proinflammatory responses , and that the mechanism may be based on blocking activation of nuclear factor - kappa b ( nf-b ) and mitogen activated protein kinase ( mapk ) . inhibition of these cell - signaling pathways is also an important mechanism for reducing cancer cell growth . rice bran components have been reported to inhibit activation and promote apoptosis of malignant lymphocytes and to inhibit growth of intestinal cancers . in this report , we examined the effects of s. boulardii fermented rice bran across rice varieties on viability of normal human blood lymphocytes and b lymphoma in vitro . rice bran chemical contents and the compounds altered by fermentation have not been previously assessed for effects on human b lymphomas , and were assessed using global and targeted metabolite profiling techniques . a significant lack of knowledge exists regarding the ability of probiotics to alter the phytochemistry of rice bran for health benefits , and global metabolite profiling represents a novel approach to detect changes in rice bran phytochemical content due to fermentation without a bias toward certain chemical classes . a metabolite profiling approach based on gas chromatographymass spectrometry ( gcms ) was recently used to investigate time - dependent metabolic changes during the germination of rice,(28 ) and more targeted studies have sought to identify bioactive and volatile compounds from rice bran oil or in bran polished from red and black rice varieties . bran from three rice varieties was hypothesized herein to vary in bioactive chemical contents after fermentation with s. boulardii and to differentially inhibit human b lymphoma viability . caffeic acid , p - coumaric acid , ferulic acid , salicylic acid , -sitosterol , and -tocopherol standards were purchased from sigma - aldrich ( st . louis , mo ) . cultures were maintained on yeast nitrogen base ( ynb ) amended with 0.5% ( w / v ) ammonium sulfate and 2% ( w / v ) dextrose . whole blood from healthy volunteers was collected into 8 ml cell preparation tubes ( cpt ) with sodium citrate as an anticoagulent ( becton dickinson vacutainer systems , franklin lakes , nj ) . cpt tubes were centrifuged at 1500 g for 30 min for separation and enrichment of normal human peripheral blood lymphocytes ( pbl ) . pbl were washed two times with 1 phosphate buffered saline solution prior to resuspension in cell culture medium . raji b lymphomas and freshly isolated normal pbl were cultured in rpmi medium supplemented with 10% fetal bovine serum , 2 mm l - glutamine , 10 mg / ml penicillin , 10,000 iu / ml streptomycin , 25 mg / ml amphotericin , 1 mm sodium pyruvate , and 1 mem nonessential amino acids . three rice varieties selected for investigation were neptune , wells and the near - isogenic line , red wells(30 ) ( table 1 ) . anna mcclung at the united states department of agriculture , rice research center ( stuttgart , arkansas ) . bran was isolated by standard milling process , heat stabilized at 110 c for 3 min , and stored at 20 c . rice bran , water and probiotic yeast fermentations were carried out using a modification of the methods described in refs ( 31 ) and ( 32 ) . briefly , 1.6 g of rice bran was added to 11.4 ml of sterilized water in the presence and absence of s. boulardii concentration of 6 10 cells ml , and samples were incubated at 37 c for 24 h with gentle shaking ( n = 3 ) . metabolites were extracted using two separate solvents : either ( a ) isopropanol : acetonitrile : water ( 3:2:2 ) for metabolite profiling or ( b ) methanol : water ( 80:20 ) for measuring bioactivity on lymphoma or peripheral blood lymphocytes in vitro . solvent a was used for metabolite profiling and was previously shown to extract both lipids and organic acids . solvent b was used to standardize cell culture treatments and conditions , as a similar single - phase aqueous - alcohol solvent was previously used to assess effects of rice bran compounds . after 24 h of fermentation in water , either isopropanol : acetonitrile or methanol was added to the culture for final 3:2:2 or 80:20 ratios , respectively . samples were vortexed and incubated at room temperature for five minutes , and bran material and yeast cells were pelleted using centrifugation ( 1500 g ) for ten minutes followed by filtration . the supernatant was collected and stored at 80 c until further chemical and biological analyses . rice bran metabolites were detected by transferring 500 l of extract to a new tube and dried using a vacuum centrifuge . the extract was derivatized by first adding 50 l of a solution containing 20 mg / ml of methoxyamine hydrochloride in pyridine and incubating at 37 c for two hours . next , 50 l of n - methyl - n - trimethylsilyltrifluoroacetamide with 1% trimethylchlorosilane ( mstfa + 1% tmcs ) ( thermo scientific ) was added and the reaction was incubated at 37 c for 60 min . samples were centrifuged at 3000 g for 5 min , and 80 l of the supernatant was used for gcms analysis . caffeic acid , coumaric acid , ferulic acid , salicylic acid , -sitosterol , and -tocopherol standards ( sigma - aldrich , st . louis , mo ) were dissolved in an isopropanol / acetonitrile / water solution ( 3:2:2 ) , evaporated , and derivatized under identical conditions . the derivatized samples were equilibrated to room temperature , transferred to a 200 l glass insert , and analyzed using a trace gc ultra coupled to a thermo dsq ii scanning from m / z 50650 at a rate of 5 scans / s in electron impact mode . samples were injected at a 10:1 split ratio , and the inlet and transfer line were held at 280 c . separation was achieved on a 30 m tg-5ms column ( thermo scientific , 0.25 mm i.d . , 0.25 m film thickness ) using a temperature program of 80 c for 0.5 min , then ramped at 15 c per minute to 330 c and held for 8 min , at a constant flow of 1.2 ml per minute . a single feature or known metabolite was defined by a given metabolite s retention time and mass , and the peak area was used to determine the relative quantity of each feature or known metabolite . raji b lymphomas and normal peripheral blood lymphocytes were plated to a density of 2.5 10 cells per ml . rice bran extracts were dried in a vacuum centrifuge and resuspended in cell culture medium , and cells were incubated in the presence of rice bran extract for 24 h. cells were centrifuged at 1500 g for 5 min , resuspended in a solution consisting of cell culture medium and 1% resazurin sodium salt , and incubated at 37 c for one hour . fluorescence was measured at 765 nm , and viability was expressed as percent fluorescence relative to the vehicle control . saccharomyces boulardii cultures were maintained in ynb ( mp biomedicals , solon , oh ) with 0.5% ammonium sulfate and 2% dextrose at 37 c . a liquid growth medium containing 5% rice bran and water was made with each rice variety . s. boulardii was added to the rice bran / water mixture at a final od600 of 0.02 ( approximately 6 10 cells ml ) . cultures were incubated at 37 c and sampled at 24 , 48 , and 72 h. yeast cells were enumerated by drop plating serial dilutions on ynb plates to determine total colony forming units ( cfus ) . chromatographic peaks between 2 and 25 min were detected by gcms and aligned using markerlynx software ( waters , millford , ma , usa ) with a retention time error window of 0.05 min . masses used for analyses ranged between 50 and 650 m / z with a mass error tolerance of 0.4 m / z . multivariate statistical analysis was performed using simca p+ ( v 12.0 , umetrics , ume , sweden ) . mean centering and pareto scaling were applied for all principal component , partial least - squares , and orthoganol projection to latent structures ( opls ) analyses . each feature was analyzed independently in a linear mixed - effects model to determine the significance and percent variance attributed to fermentation ( fixed effect ) , or variety and varietyfermentation interactions ( random effects ) . significance was determined with a p - value threshold of 0.05 , and percent variation was determined using the sum of squares partitions of each random effect relative to the total sum of squares of the model . fold changes due to fermentation were calculated for each feature using the peak areas of fermented divided by the nonfermented . bioactive compounds were compared among varieties by one - way anova ( p < 0.05 ) , and z - scores were calculated for metabolites from fermented varieties based on the mean and standard deviation of the nonfermented control . effects on lymphoma viability and increased growth of s. boulardii on rice bran varieties were determined using a one - way anova and tukey s hsd . significant differences between treatments ( rice bran varieties ) and controls ( ynb ) were confirmed by a dunnet s 2-tailed comparison . these tests were performed using r software ( v2.11.1 ) , graphpad prism ( v 5.0 , graphpad software , inc . , la jolla , ca ) and xlstat - pro ( addinsoft usa , new york , ny ) . bran from three rice varieties ( table 1 ) was extracted for metabolite profiling and analyzed by gas chromatography coupled to mass spectrometry ( gcms ) . semidwarf varieties , and red wells is isogenic to wells apart from a single deleted base pair in the proanthocyanidin gene rc . this mutation results in the production of red pigment in the bran layer of the seed.(30 ) principal component analysis was used to elucidate varietal differences in the metabolome of nonfermented rice bran ( figure 1a ) . varietal differences were largely explained by the first component ( 56% ) , and the second component differentiated between biological replicates ( 14% ) . rice bran was then incubated in the presence and absence of s. boulardii and metabolites were extracted and detected by gcms . the gcms chromatograms of nonfermented rice bran , s. boulardii fermented rice bran , and s. boulardii extracts showed unique differences among the treatments ( figure 1b ) . partial least squares discriminant analysis ( pls - da ) was used to detect differences in the metabolome among all three varieties and with or without fermentation with s. boulardii ( figure 1c ) . the first two components of the pls - da model explained 66% and 19% of the variation , respectively . metabolite profiling of rice bran from three varieties with and without fermentation by s. boulardii . ( a ) principal component analysis ( pca ) of bran extracts from three rice varieties ( neptune , wells and red wells ) show diversity in metabolite profiles . the first principal component separated the three varieties , and the second component was mostly composed of variation among replicates within a single variety . ( b ) a representative portion of a gcms chromatograph showed change in metabolites in fermented rice bran . neptune rice variety alone ( top ) , s. boulardii extract alone ( middle ) and bran from neptune variety fermented with s. boulardii ( bottom ) . some peaks are present in only one sample , and others are present in both but vary in quantity , as indicated by arrows above differential peaks . ( c ) pls - da model of three varieties nonfermented ( black shading ) or fermented with s. boulardii ( white ) . the first component separated each variety from its fermented counterpart , and the second component separated neptune from wells and red wells . this model demonstrates the ability to apply metabolite - profiling techniques to differentiate chemical contents of fermented rice bran from nonfermented rice bran , irrespective of the rice variety tested . s. boulardii fermentation induced changes in metabolite content for all three varieties were determined by quantitative analysis of peak areas from 10,260 gcms derived features . for neptune , wells , and red wells , 448 , 127 , and 311 features varied due to fermentation , respectively ( student s t test , p < 0.05 ) . the mean percent variance explained by the linear mixed model for all features was 14.8% . genotype and genotypefermentation effects explained a mean of 10% and 4.8% of the total variation , respectively . a series of pls - da and opls models were applied to each rice variety to determine metabolites with the most significant changes due to fermentation ( figure 2 ) . the effect of fermentation on each variety s metabolome was explained by the first component in each pls - da model . metabolites of interest contained p(1 ) and p(corr ) values greater than 0.02 and 0.8 , respectively , and were analyzed for quantitative differences between samples by fold - change due to fermentation and by student s t test ( p < 0.05 ) ( table 2 ) . mass spectra of significant peaks were screened in the national institute of technology standards metabolite database for probable matches . rice varieties differed in candidate metabolites altered by s. boulardii fermentation and by chemical classes . for the three varieties , there was wide variation in both the relative quantities of metabolites increased and the types of predicted metabolites . s. boulardii fermentation of rice bran differs with regard to variety and was next evaluated for impact on anticancer properties of rice bran . pls - da and opls models to determine metabolite variation induced by fermentation with s. boulardii . ( a ) neptune pls - da showed the metabolome differs between unfermented ( black ) and fermented ( white ) samples . ( b ) the neptune opls analysis showed metabolites that highly differ based on fermentation , indicated by the dashed box for quadrant 2 ( nonfermented ) and quadrant 3 ( fermented ) . p(corr ) values correspond to deviation across replicates , and p(1 ) values are proportional to the quantity of metabolite . wells ( c and d ) and red wells ( e and f ) also showed altered metabolite content induced by s. boulardii . polar rice bran extracts were previously shown to inhibit tumor promotion of lymphoblastoid b cells , and rice bran agglutinin inhibited growth of monoblastic leukemia u937 cells.(26 ) methanol - soluble metabolites from neptune rice bran were screened for dose dependent effects on viability of normal human peripheral blood lymphocytes ( pbl ) and malignant human b - cell lymphoma ( figure 3a ) . viability was measured by resazurin stain after 24 h of incubation with fermented and nonfermented rice bran extracts . the rice bran extracts did not affect the viability of normal pbl ( figure 3a ) . a significant reduction in lymphoma viability was demonstrated at the 500 g ml dose of neptune rice bran extract , while the 125 and 250 g ml were not significantly reduced from vehicle control ( figure 3a ) . the 500 g ml dose of rice bran extract was next used to examine effects of both nonfermented and fermented rice bran extracts across varieties on normal pbl and lymphoma . none of the rice bran extracts altered the viability of normal pbl ( figure 3b ) . the s. boulardii - fermented rice bran significantly inhibited lymphoma viability compared to vehicle controls for all varieties tested ( figure 3c ) . the nonfermented neptune rice bran extracts showed a 23% reduction in viability , and s. boulardii - fermented neptune rice bran extracts reduced viability by 85% compared to control . at 500 g ml , unfermented extracts of wells and red wells had no effect on lymphoma viability relative to the control , however fermented extracts inhibited viability by 75% and 51% , respectively ( figure 3c ) . the percent reduction in viability differed among varieties of the three fermented extracts ( anova , tukey post hoc , p < 0.05 ) . the differential reduction in viability by fermented rice bran among rice varieties supports that variation in metabolite contents as detected in figure 1 may be important for bioactivity . the isopropanol : acetonitrile : water ( 3:2:2 ) solvent used for metabolite profiling of fermented bran extracts ( figure 2 ) was also examined for effects on lymphoma viability , however this solvent demonstrated suboptimal background activity as a vehicle control and was therefore not utilized to compare effects across rice varieties ( data not shown ) . ( a ) different doses of nonfermented methanolic rice bran extracts ( neptune ) were added to normal human peripheral blood lymphocytes ( pbl ) and raji b lymphoma cultures for 24 h. values are expressed as the mean percent viable cells relative to the vehicle control sem . extracts of nonfermented neptune reduced lymphoma viability at 500 g ml ( student s t test , p < 0.05 ) . ( b ) fermented and nonfermented extracts of all three varieties at 500 g ml had no effect on viability of normal pbl . ( c ) fermented and nonfermented extracts of all three varieties at 500 g ml differentially affected lymphoma viability , as measured by cell fluorescence after the addition of resaruzin ( anova , tukey post hoc , p < 0.05 ) . significance from vehicle control is represented by an asterisk , and statistical groupings are denoted by the letters a , b , and c. given the varietal differences in anticancer activity of s. boulardii fermented rice bran extracts , a number of bioactive rice bran compounds were selected for relative quantification . rice bran contains a number of metabolites with reported anticancer effects , notably phenolics and phytosterols . salicylic , p - coumaric , ferulic , and caffeic acid , and also -tocopherol and -sitosterol were detected in nonfermented and fermented rice bran from each of the three varieties by comparing the initial gcms chromatograms to purchased standards ( figure 4 ) . nonfermented extracts from wells contained a greater quantity of salicylic acid than both red wells and neptune ( figure 4a ) . red wells contained higher amounts of ferulic acid than neptune , and neptune contained significantly less -sitosterol than both wells and red wells ( anova , tukey post hoc , p < 0.05 ) . a z - score analysis was conducted to determine significant changes in metabolite quantity due to fermentation with s. boulardii , using nonfermented rice bran as a control . the data shown in figure 4c supports that s. boulardii fermentation reduced the quantity of p - coumaric acid in red wells , and increased ferulic acid in neptune ( figure 4c ) . relative quantification of metabolites without fermentation ( a ) and with fermentation ( b ) was based on the area of the gcms chromatograph . metabolites showing a significant difference by relative quantity and between two varieties were indicated by an asterisk ( anova , tukey post hoc , p < 0.05 ) . ( c ) z - score for metabolites from fermented extracts using the nonfermented as a control . significant changes in metabolite quantity are indicated by z - score values outside of the shaded region . increased ferulic acid was detected in s. boulardii fermented neptune rice bran , and decreased p - coumaric acid was detected from fermented red wells compared to nonfermented . the ability of s. boulardii to utilize and quantitatively alter chemical components of rice bran was confirmed by measuring its growth on rice bran as a sole carbon source . overnight cultures of s. boulardii inoculated into medium containing 5% rice bran from each of the varieties grew significantly better than cultures inoculated into ynb broth with dextrose as the primary carbon source ( figure 5 ) . in addition , s. boulardii cultures maintained viability and cell numbers for 3 days on rice bran medium , while the number of cells in the ynb cultures steadily declined . no significant differences in the growth of s. boulardii were detected among the three rice varieties ( anova , tukey post hoc , p < 0.05 ) . rice bran from all three rice varieties significantly increased the growth of s. boulardii compared to ynb medium alone after 24 , 48 , and 72 h. an asterisk indicates difference in the quantity of yeast colonies compared to the ynb control ( one way anova , tukey post hoc , p < 0.05 ) . this study demonstrates the utility of integrating global and targeted metabolite profiling for analysis of rice bran phytochemicals in the presence and absence of s. boulardii fermentation , and has advanced our knowledge about how probiotic fermentation of rice bran can enhance the bioactivity of extracts . metabolomics is one strategy used to measure the wide array of phytochemicals that are typically evaluated in the free forms from food extracts , as these small molecules dissolve quickly and are immediately absorbed into the bloodstream . this high throughput , yet sensitive , approach is also useful to assess the bound forms of rice molecules , which are attached to the plant cell walls and must be released by microbes during digestion before they can be absorbed . these findings set the stage for developing metabolomics as a tool for investigating rice bran phytochemical diversity and digestion by probiotics . rice varieties neptune , wells , and red wells ( figure 1a , table 1 ) . this cluster was expected given the near - isogenic state of the wells varieties and provided strong rationale for investigating differential bioactive properties . rice bran fermentation with the s. boulardii probiotic enhanced metabolite diversity ( figure 1c ) , and showed rice varietal differences in bran extract - mediated reduction of lymphoma growth ( figure 3b ) . no apparent toxicity was demonstrated for s. boulardii fermented rice bran on normal peripheral blood lymphocytes . candidate metabolites that were significantly increased postfermentation also differed among the three varieties ( table 2 ) . the ability of s. boulardii to utilize rice bran as a sole carbon source substrate for cellular metabolism and growth ( figure 5 ) suggests that rice bran contains unique prebiotic characteristics . although no differences were detected among the neptune , wells and red wells rice varieties to increase probiotic growth , these findings warrant further investigation of rice bran prebiotic components and the synergistic effects of prebiotic / probiotic combinations on human health . to our knowledge , data from these studies demonstrate that rice varieties with pigmented seed coat also exhibit differential activity when compared to nonpigmented . the findings presented in this report suggest that some of the inconsistent results of past rice bran investigations on cancer cell growth may be due to the rice variety tested and not just those chemicals responsible for pigment . another plausible explanation for inconclusive data on rice bran is not only differences in metabolite content among varieties but also the influence of probiotics altering the bioavailability of cancer - protective compounds in select tissues . the ability of phenols , particularly ferulic , salicylic , caffeic , and p - coumaric acids and -tocopherol ( a lipid - soluble antioxidant ) found in rice bran , to scavenge free radicals , alter enzymes , affect biochemical pathways , and interfere with gene expression has attracted the attention of researchers in search of cancer - fighting agents . the efficacy of ferulic acid , which remains in the bloodstream longer than other known antioxidants and therefore may provide more protection , is dependent on its bioavailability and dosage.(31 ) however , plant phenols are often found in a biologically unavailable form due to an ester - bond to cell wall polysaccharides . therefore , the optimal dose of rice bran required to achieve cancer - fighting levels of ferulic acid is unknown . humans and rats have been shown to release diferulic acid from bran fiber using gastrointestinal esterases found in the large and small intestines , thus enhancing the bioavailability of this compound.(32 ) the data shown in figure 4c supports that the neptune rice variety may exhibit higher probiotic - induced ferulic acid release and bioavailability than the other two varieties , and that consuming the whole food postfermentation with s. boulardii may be a viable alternative for achieving enhanced levels of this compound without losing the benefits of the others . yeast cells typically only maintain viability for several hours after they have reached stationary phase and depleted their carbon source . our results show that rice bran medium allows s. boulardii cells to maintain viability over several days , suggesting that secondary fermentation by the yeast may be occurring and may further alter the phytochemical content of the rice bran ( figure 5 ) . thus , it will be necessary to optimize fermentation times to advance our understanding of the kinetics of rice bran phytochemical metabolism and release by s. boulardii . emerging evidence supports additive and/or synergistic effects of rice bran components for protection against certain cancers , however few studies have examined differences in phytochemical contents in commercially available rice varieties . our data support that many rice bran components were fermented by the yeast probiotic ( figure 2 ) and these components work together to enhance probiotic growth ( figure 5 ) . one study examined a yeast fermentation of rice bran for changes in the stability , palatability , and nutritional status ( carbohydrate , methionine , calcium , and ash content ) of the bran , but did not address the alteration of potentially bioactive phytochemicals.(42 ) given the evidence for cancer fighting activities of rice bran phytochemicals , the data presented herein support that s. boulardii - fermented rice bran should be next tested for bioavailability of bioactive components and for reducing lymphoma viability in vivo . chemopreventive single agent compounds found in rice bran include , but are not limited to , tocopherols , polyphenols , inositol hexaphosphate ( ip6 ) , nonstarchy polysaccharides , -oryzanol and phytosterols . our metabolite profile analysis of fermented rice bran revealed extensive rice bran chemical diversity , and can be used to further the identities of novel combinations of bioactive compounds that display phytochemical teamwork . whole rice bran consumption is undoubtedly recognized as important for providing more comprehensive protection against cancer cells when compared to supplementation with isolated ingredients , and the metabolite profiling techniques and chemical analyses presented herein support further interrogation of rice bran effects on intestinal microbe interactions as well as probiotic growth and metabolism . the metabolomics strategy applied herein has advanced our understanding of the health importance of rice bran phytochemical diversity in the presence and absence of fermentation and for disease fighting activity . single agent nutritional magic bullets too often fail to achieve the health benefits indicated by cell - based assays . available methodologies have also limited the scientific investigations of rice bran to these reductive approaches . by utilizing global metabolomic profiling , we can now more holistically approach complex mixtures of small molecules in rice bran and improve studies linking bioactive food components and human health .

emerging evidence supporting chronic disease fighting properties of rice bran has advanced the development of stabilized rice bran for human use as a functional food and dietary supplement . a global and targeted metabolomic investigation of stabilized rice bran fermented with saccharomyces boulardii was performed in three rice varieties . metabolites from s. boulardii - fermented rice bran were detected by gas chromatographymass spectrometry ( gcms ) and assessed for bioactivity compared to nonfermented rice bran in normal and malignant lymphocytes . global metabolite profiling revealed significant differences in the metabolome that led to discovery of candidate compounds modulated by s. boulardii fermentation . fermented rice bran extracts from three rice varieties reduced growth of human b lymphomas compared to each variety s nonfermented control and revealed that fermentation differentially altered bioactive compounds . these data support that integration of global and targeted metabolite analysis can be utilized for assessing health properties of rice bran phytochemicals that are enhanced by yeast fermentation and that differ across rice varieties .

Introduction Materials and Methods Results Discussion

stabilized rice bran is a unique whole food that naturally contains protein , vitamins , minerals , complex carbohydrates , phytonutrients , phospholipids , essential fatty acids , and more than 120 antioxidants. (1 ) dietary rice bran intake and rice bran components have demonstrated chronic disease fighting activity , particularly for protection against cardiovascular disease and certain cancers . we and others have shown that rice varieties are not equal in content and composition of bioactive rice bran components . how these phytochemicals are altered by microbial fermentation and metabolism is an emerging area of research that merits scientific investigation when assessing bioactivity and health benefits . a few studies have evaluated rice bran as a dietary supplement or functional food ingredient ; however , little is known about how chemical content changes with and without fermentation . boulardii ( s. boulardii ) , has probiotic activity and is widely used as a dietary supplement for intestinal disease prevention and treatment . the spectrum of biomedical activities and food processing applications reported with s. boulardii has significantly grown over the past decade and includes , but is not limited to , protection against enteric pathogens , modification of lymphocyte proliferation , and differential release of plant secondary metabolites from foods such as wine , sourdough and cheese.sacchromyces boulardii has been shown to be beneficial for modification of food components such as breakdown of dietary phytate and biofortification of folate to improve the nutritional value and health properties of food . the health benefit of s. boulardii both as a probiotic and for fermented foods was recently reviewed , and a meta - analysis of placebo - controlled treatment trials supports its safety and efficacy for protection against several types of diarrhea . protection against specific enteric bacterial pathogens by s. boulardii may , in part , be due to anti - inflammatory actions and effects on immunity.in vitro studies using mammalian cell cultures have shown that s. boulardii modifies host cell signaling pathways associated with proinflammatory responses , and that the mechanism may be based on blocking activation of nuclear factor - kappa b ( nf-b ) and mitogen activated protein kinase ( mapk ) . rice bran components have been reported to inhibit activation and promote apoptosis of malignant lymphocytes and to inhibit growth of intestinal cancers . in this report , we examined the effects of s. boulardii fermented rice bran across rice varieties on viability of normal human blood lymphocytes and b lymphoma in vitro . rice bran chemical contents and the compounds altered by fermentation have not been previously assessed for effects on human b lymphomas , and were assessed using global and targeted metabolite profiling techniques . a significant lack of knowledge exists regarding the ability of probiotics to alter the phytochemistry of rice bran for health benefits , and global metabolite profiling represents a novel approach to detect changes in rice bran phytochemical content due to fermentation without a bias toward certain chemical classes . a metabolite profiling approach based on gas chromatographymass spectrometry ( gcms ) was recently used to investigate time - dependent metabolic changes during the germination of rice,(28 ) and more targeted studies have sought to identify bioactive and volatile compounds from rice bran oil or in bran polished from red and black rice varieties . bran from three rice varieties was hypothesized herein to vary in bioactive chemical contents after fermentation with s. boulardii and to differentially inhibit human b lymphoma viability . raji b lymphomas and freshly isolated normal pbl were cultured in rpmi medium supplemented with 10% fetal bovine serum , 2 mm l - glutamine , 10 mg / ml penicillin , 10,000 iu / ml streptomycin , 25 mg / ml amphotericin , 1 mm sodium pyruvate , and 1 mem nonessential amino acids . three rice varieties selected for investigation were neptune , wells and the near - isogenic line , red wells(30 ) ( table 1 ) . rice bran , water and probiotic yeast fermentations were carried out using a modification of the methods described in refs ( 31 ) and ( 32 ) . briefly , 1.6 g of rice bran was added to 11.4 ml of sterilized water in the presence and absence of s. boulardii concentration of 6 10 cells ml , and samples were incubated at 37 c for 24 h with gentle shaking ( n = 3 ) . solvent a was used for metabolite profiling and was previously shown to extract both lipids and organic acids . solvent b was used to standardize cell culture treatments and conditions , as a similar single - phase aqueous - alcohol solvent was previously used to assess effects of rice bran compounds . rice bran metabolites were detected by transferring 500 l of extract to a new tube and dried using a vacuum centrifuge . rice bran extracts were dried in a vacuum centrifuge and resuspended in cell culture medium , and cells were incubated in the presence of rice bran extract for 24 h. cells were centrifuged at 1500 g for 5 min , resuspended in a solution consisting of cell culture medium and 1% resazurin sodium salt , and incubated at 37 c for one hour . saccharomyces boulardii cultures were maintained in ynb ( mp biomedicals , solon , oh ) with 0.5% ammonium sulfate and 2% dextrose at 37 c . s. boulardii was added to the rice bran / water mixture at a final od600 of 0.02 ( approximately 6 10 cells ml ) . chromatographic peaks between 2 and 25 min were detected by gcms and aligned using markerlynx software ( waters , millford , ma , usa ) with a retention time error window of 0.05 min . bioactive compounds were compared among varieties by one - way anova ( p < 0.05 ) , and z - scores were calculated for metabolites from fermented varieties based on the mean and standard deviation of the nonfermented control . effects on lymphoma viability and increased growth of s. boulardii on rice bran varieties were determined using a one - way anova and tukey s hsd . significant differences between treatments ( rice bran varieties ) and controls ( ynb ) were confirmed by a dunnet s 2-tailed comparison . , la jolla , ca ) and xlstat - pro ( addinsoft usa , new york , ny ) . bran from three rice varieties ( table 1 ) was extracted for metabolite profiling and analyzed by gas chromatography coupled to mass spectrometry ( gcms ) . semidwarf varieties , and red wells is isogenic to wells apart from a single deleted base pair in the proanthocyanidin gene rc . this mutation results in the production of red pigment in the bran layer of the seed. (30 ) principal component analysis was used to elucidate varietal differences in the metabolome of nonfermented rice bran ( figure 1a ) . rice bran was then incubated in the presence and absence of s. boulardii and metabolites were extracted and detected by gcms . the gcms chromatograms of nonfermented rice bran , s. boulardii fermented rice bran , and s. boulardii extracts showed unique differences among the treatments ( figure 1b ) . partial least squares discriminant analysis ( pls - da ) was used to detect differences in the metabolome among all three varieties and with or without fermentation with s. boulardii ( figure 1c ) . metabolite profiling of rice bran from three varieties with and without fermentation by s. boulardii . ( a ) principal component analysis ( pca ) of bran extracts from three rice varieties ( neptune , wells and red wells ) show diversity in metabolite profiles . ( b ) a representative portion of a gcms chromatograph showed change in metabolites in fermented rice bran . neptune rice variety alone ( top ) , s. boulardii extract alone ( middle ) and bran from neptune variety fermented with s. boulardii ( bottom ) . ( c ) pls - da model of three varieties nonfermented ( black shading ) or fermented with s. boulardii ( white ) . the first component separated each variety from its fermented counterpart , and the second component separated neptune from wells and red wells . this model demonstrates the ability to apply metabolite - profiling techniques to differentiate chemical contents of fermented rice bran from nonfermented rice bran , irrespective of the rice variety tested . s. boulardii fermentation induced changes in metabolite content for all three varieties were determined by quantitative analysis of peak areas from 10,260 gcms derived features . a series of pls - da and opls models were applied to each rice variety to determine metabolites with the most significant changes due to fermentation ( figure 2 ) . the effect of fermentation on each variety s metabolome was explained by the first component in each pls - da model . metabolites of interest contained p(1 ) and p(corr ) values greater than 0.02 and 0.8 , respectively , and were analyzed for quantitative differences between samples by fold - change due to fermentation and by student s t test ( p < 0.05 ) ( table 2 ) . rice varieties differed in candidate metabolites altered by s. boulardii fermentation and by chemical classes . s. boulardii fermentation of rice bran differs with regard to variety and was next evaluated for impact on anticancer properties of rice bran . pls - da and opls models to determine metabolite variation induced by fermentation with s. boulardii . ( a ) neptune pls - da showed the metabolome differs between unfermented ( black ) and fermented ( white ) samples . wells ( c and d ) and red wells ( e and f ) also showed altered metabolite content induced by s. boulardii . polar rice bran extracts were previously shown to inhibit tumor promotion of lymphoblastoid b cells , and rice bran agglutinin inhibited growth of monoblastic leukemia u937 cells. (26 ) methanol - soluble metabolites from neptune rice bran were screened for dose dependent effects on viability of normal human peripheral blood lymphocytes ( pbl ) and malignant human b - cell lymphoma ( figure 3a ) . viability was measured by resazurin stain after 24 h of incubation with fermented and nonfermented rice bran extracts . the rice bran extracts did not affect the viability of normal pbl ( figure 3a ) . a significant reduction in lymphoma viability was demonstrated at the 500 g ml dose of neptune rice bran extract , while the 125 and 250 g ml were not significantly reduced from vehicle control ( figure 3a ) . the 500 g ml dose of rice bran extract was next used to examine effects of both nonfermented and fermented rice bran extracts across varieties on normal pbl and lymphoma . none of the rice bran extracts altered the viability of normal pbl ( figure 3b ) . the s. boulardii - fermented rice bran significantly inhibited lymphoma viability compared to vehicle controls for all varieties tested ( figure 3c ) . the nonfermented neptune rice bran extracts showed a 23% reduction in viability , and s. boulardii - fermented neptune rice bran extracts reduced viability by 85% compared to control . the differential reduction in viability by fermented rice bran among rice varieties supports that variation in metabolite contents as detected in figure 1 may be important for bioactivity . the isopropanol : acetonitrile : water ( 3:2:2 ) solvent used for metabolite profiling of fermented bran extracts ( figure 2 ) was also examined for effects on lymphoma viability , however this solvent demonstrated suboptimal background activity as a vehicle control and was therefore not utilized to compare effects across rice varieties ( data not shown ) . ( a ) different doses of nonfermented methanolic rice bran extracts ( neptune ) were added to normal human peripheral blood lymphocytes ( pbl ) and raji b lymphoma cultures for 24 h. values are expressed as the mean percent viable cells relative to the vehicle control sem . significance from vehicle control is represented by an asterisk , and statistical groupings are denoted by the letters a , b , and c. given the varietal differences in anticancer activity of s. boulardii fermented rice bran extracts , a number of bioactive rice bran compounds were selected for relative quantification . rice bran contains a number of metabolites with reported anticancer effects , notably phenolics and phytosterols . salicylic , p - coumaric , ferulic , and caffeic acid , and also -tocopherol and -sitosterol were detected in nonfermented and fermented rice bran from each of the three varieties by comparing the initial gcms chromatograms to purchased standards ( figure 4 ) . a z - score analysis was conducted to determine significant changes in metabolite quantity due to fermentation with s. boulardii , using nonfermented rice bran as a control . the data shown in figure 4c supports that s. boulardii fermentation reduced the quantity of p - coumaric acid in red wells , and increased ferulic acid in neptune ( figure 4c ) . relative quantification of metabolites without fermentation ( a ) and with fermentation ( b ) was based on the area of the gcms chromatograph . ( c ) z - score for metabolites from fermented extracts using the nonfermented as a control . increased ferulic acid was detected in s. boulardii fermented neptune rice bran , and decreased p - coumaric acid was detected from fermented red wells compared to nonfermented . the ability of s. boulardii to utilize and quantitatively alter chemical components of rice bran was confirmed by measuring its growth on rice bran as a sole carbon source . overnight cultures of s. boulardii inoculated into medium containing 5% rice bran from each of the varieties grew significantly better than cultures inoculated into ynb broth with dextrose as the primary carbon source ( figure 5 ) . in addition , s. boulardii cultures maintained viability and cell numbers for 3 days on rice bran medium , while the number of cells in the ynb cultures steadily declined . no significant differences in the growth of s. boulardii were detected among the three rice varieties ( anova , tukey post hoc , p < 0.05 ) . rice bran from all three rice varieties significantly increased the growth of s. boulardii compared to ynb medium alone after 24 , 48 , and 72 h. an asterisk indicates difference in the quantity of yeast colonies compared to the ynb control ( one way anova , tukey post hoc , p < 0.05 ) . this study demonstrates the utility of integrating global and targeted metabolite profiling for analysis of rice bran phytochemicals in the presence and absence of s. boulardii fermentation , and has advanced our knowledge about how probiotic fermentation of rice bran can enhance the bioactivity of extracts . metabolomics is one strategy used to measure the wide array of phytochemicals that are typically evaluated in the free forms from food extracts , as these small molecules dissolve quickly and are immediately absorbed into the bloodstream . this high throughput , yet sensitive , approach is also useful to assess the bound forms of rice molecules , which are attached to the plant cell walls and must be released by microbes during digestion before they can be absorbed . these findings set the stage for developing metabolomics as a tool for investigating rice bran phytochemical diversity and digestion by probiotics . rice bran fermentation with the s. boulardii probiotic enhanced metabolite diversity ( figure 1c ) , and showed rice varietal differences in bran extract - mediated reduction of lymphoma growth ( figure 3b ) . no apparent toxicity was demonstrated for s. boulardii fermented rice bran on normal peripheral blood lymphocytes . the ability of s. boulardii to utilize rice bran as a sole carbon source substrate for cellular metabolism and growth ( figure 5 ) suggests that rice bran contains unique prebiotic characteristics . although no differences were detected among the neptune , wells and red wells rice varieties to increase probiotic growth , these findings warrant further investigation of rice bran prebiotic components and the synergistic effects of prebiotic / probiotic combinations on human health . to our knowledge , data from these studies demonstrate that rice varieties with pigmented seed coat also exhibit differential activity when compared to nonpigmented . another plausible explanation for inconclusive data on rice bran is not only differences in metabolite content among varieties but also the influence of probiotics altering the bioavailability of cancer - protective compounds in select tissues . the efficacy of ferulic acid , which remains in the bloodstream longer than other known antioxidants and therefore may provide more protection , is dependent on its bioavailability and dosage. therefore , the optimal dose of rice bran required to achieve cancer - fighting levels of ferulic acid is unknown . humans and rats have been shown to release diferulic acid from bran fiber using gastrointestinal esterases found in the large and small intestines , thus enhancing the bioavailability of this compound. (32 ) the data shown in figure 4c supports that the neptune rice variety may exhibit higher probiotic - induced ferulic acid release and bioavailability than the other two varieties , and that consuming the whole food postfermentation with s. boulardii may be a viable alternative for achieving enhanced levels of this compound without losing the benefits of the others . our results show that rice bran medium allows s. boulardii cells to maintain viability over several days , suggesting that secondary fermentation by the yeast may be occurring and may further alter the phytochemical content of the rice bran ( figure 5 ) . thus , it will be necessary to optimize fermentation times to advance our understanding of the kinetics of rice bran phytochemical metabolism and release by s. boulardii . emerging evidence supports additive and/or synergistic effects of rice bran components for protection against certain cancers , however few studies have examined differences in phytochemical contents in commercially available rice varieties . our data support that many rice bran components were fermented by the yeast probiotic ( figure 2 ) and these components work together to enhance probiotic growth ( figure 5 ) . one study examined a yeast fermentation of rice bran for changes in the stability , palatability , and nutritional status ( carbohydrate , methionine , calcium , and ash content ) of the bran , but did not address the alteration of potentially bioactive phytochemicals. (42 ) given the evidence for cancer fighting activities of rice bran phytochemicals , the data presented herein support that s. boulardii - fermented rice bran should be next tested for bioavailability of bioactive components and for reducing lymphoma viability in vivo . our metabolite profile analysis of fermented rice bran revealed extensive rice bran chemical diversity , and can be used to further the identities of novel combinations of bioactive compounds that display phytochemical teamwork . whole rice bran consumption is undoubtedly recognized as important for providing more comprehensive protection against cancer cells when compared to supplementation with isolated ingredients , and the metabolite profiling techniques and chemical analyses presented herein support further interrogation of rice bran effects on intestinal microbe interactions as well as probiotic growth and metabolism . the metabolomics strategy applied herein has advanced our understanding of the health importance of rice bran phytochemical diversity in the presence and absence of fermentation and for disease fighting activity . available methodologies have also limited the scientific investigations of rice bran to these reductive approaches .

the murine model of lqt3 used in our study has been previously described in detailand was kindly donated by peter carmeliet . heterozygous kpq - scn5a knock - in tg mice were characterized and compared with wild - type ( wt ) littermate mice . all of the animals were adult males ( average age 6 months , weight 40 g ) . the mice were anesthetized with avertin 0.015 ml / g intraperitoneal ( ip ) injection , and the experiments were terminal . ecg recording started 2 minutes after onset of anesthesia while the mice were placed on a heating pad with continuous monitoring of body temperature . five needle electrodes ( one electrode implanted subcutaneously in each limb and one placed in the precordial position ) connected to ad instruments ltd , ( oxford , uk ) amplifiers set with bandpass filtering between 0.03 and 1 khz were used for six - lead ecg recording . ecg parameters ( heart rate [ hr ] , rr and qt intervals ) were analyzed blindly to genotype . measurements were performed using the signal - averaged ecg ( mouse saecg v1.2 program , ad instruments ) through a template - matching algorithm . qt interval ( from onset of qrs complex to return to baseline of t wave ) was measured in lead i. the remaining leads were mainly used to validate what was observed in lead i ( i.e. , ventricular arrhythmias , prolonged qt , etc . ) . cardiac arrhythmias were classified by the most severe episode as absent , minor ( isolated ventricular premature beats [ vpbs ] , couples of vpbs , ventricular bigeminy ) , or major ( ventricular tachycardia [ vt ] , ventricular fibrillation [ vf ] ) . our investigation follows the guidelines for the care and use of laboratory animals published by the national institutes of health ( nih publication no . 85 - 23 , revised 1996 ) and was approved by the ethics review board of the italian ministry of health . all procedures were performed in accordance with the animal care guidelines of the federation of laboratory animals science associations . after baseline ecg variables were recorded , all mice , still under anesthesia , were assigned to one of the two arms of the study . the mice either were injected with the cholinergic agonist carbachol or were pretreated with propranolol followed 2 to 3 minutes later by carbachol injection . all animals were monitored continuously by ecg for up to 30 minutes after the administration of carbachol until the end of the experiment . all drugs used were provided by sigma - aldrich srl , milan , italy and astrazeneca spa , basiglio , italy . the cholinergic agonist carbachol ( carbamylcholine chloride 0.5 mg / kg ) was administered ip in all mice . within 1 to 2 minutes of injection we selected a 2-minute good - quality ecg tracing to obtain hr and qt measurements following carbachol . ecg monitoring continued up to 30 minutes to assess the potential occurrence of arrhythmic events . after the baseline ecg variables had been recorded , propranolol 0.1 mg / kg was injected ip two to three minutes after the injection , a 6% to 9% decrement in hr was achieved , and hr and qt measurements on propranolol were made . carbachol was then injected , and ecg monitoring continued for up to 30 minutes to observe arrhythmic events . genotyping was performed using a specific custom taqman assay ( life technologies italia , monza , italy ) to discriminate between wt and mutant scn5a sequence on a 7900 ht fast real - time polymerase chain reaction instrument ( life technologies ) . continuous variables are given as mean sd and were compared among groups defined by genetic status and treatment protocol using the unpaired student t test or analysis of variance , as appropriate . whenever assumptions of normality and homogeneity of variance were questionable , the equivalent nonparametric tests ( mann - whitney or kruskal - wallis test for independent samples ) were used . similarly , changes of basal ecg parameters after drug exposure within genetic groups were analyzed with t test or wilcoxon signed rank test for paired samples . categorical variables are expressed as absolute and relative frequencies and were analyzed by or fisher exact test . post - treatment survival to the primary endpoint of vt / vf was described by kaplan - meier cumulative estimates , with comparison performed by the log rank test . all analyses were made using spss statistics ( version 19 , ibm italia spa , italy ) . ecg recording started 2 minutes after onset of anesthesia while the mice were placed on a heating pad with continuous monitoring of body temperature . five needle electrodes ( one electrode implanted subcutaneously in each limb and one placed in the precordial position ) connected to ad instruments ltd , ( oxford , uk ) amplifiers set with bandpass filtering between 0.03 and 1 khz were used for six - lead ecg recording . ecg parameters ( heart rate [ hr ] , rr and qt intervals ) were analyzed blindly to genotype . measurements were performed using the signal - averaged ecg ( mouse saecg v1.2 program , ad instruments ) through a template - matching algorithm . qt interval ( from onset of qrs complex to return to baseline of t wave ) was measured in lead i. the remaining leads were mainly used to validate what was observed in lead i ( i.e. , ventricular arrhythmias , prolonged qt , etc . ) . cardiac arrhythmias were classified by the most severe episode as absent , minor ( isolated ventricular premature beats [ vpbs ] , couples of vpbs , ventricular bigeminy ) , or major ( ventricular tachycardia [ vt ] , ventricular fibrillation [ vf ] ) . our investigation follows the guidelines for the care and use of laboratory animals published by the national institutes of health ( nih publication no . 85 - 23 , revised 1996 ) and was approved by the ethics review board of the italian ministry of health . all procedures were performed in accordance with the animal care guidelines of the federation of laboratory animals science associations . after baseline ecg variables were recorded , all mice , still under anesthesia , were assigned to one of the two arms of the study . the mice either were injected with the cholinergic agonist carbachol or were pretreated with propranolol followed 2 to 3 minutes later by carbachol injection . all animals were monitored continuously by ecg for up to 30 minutes after the administration of carbachol until the end of the experiment . all drugs used were provided by sigma - aldrich srl , milan , italy and astrazeneca spa , basiglio , italy . the cholinergic agonist carbachol ( carbamylcholine chloride 0.5 mg / kg ) was administered ip in all mice . within 1 to 2 minutes of injection , hr decreased from baseline . we selected a 2-minute good - quality ecg tracing to obtain hr and qt measurements following carbachol . ecg monitoring continued up to 30 minutes to assess the potential occurrence of arrhythmic events . after the baseline ecg variables had been recorded , propranolol 0.1 mg / kg was injected ip two to three minutes after the injection , a 6% to 9% decrement in hr was achieved , and hr and qt measurements on propranolol were made . carbachol was then injected , and ecg monitoring continued for up to 30 minutes to observe arrhythmic events . genotyping was performed using a specific custom taqman assay ( life technologies italia , monza , italy ) to discriminate between wt and mutant scn5a sequence on a 7900 ht fast real - time polymerase chain reaction instrument ( life technologies ) . continuous variables are given as mean sd and were compared among groups defined by genetic status and treatment protocol using the unpaired student t test or analysis of variance , as appropriate . whenever assumptions of normality and homogeneity of variance were questionable , the equivalent nonparametric tests ( mann - whitney or kruskal - wallis test for independent samples ) were used . similarly , changes of basal ecg parameters after drug exposure within genetic groups were analyzed with t test or wilcoxon signed rank test for paired samples . categorical variables are expressed as absolute and relative frequencies and were analyzed by or fisher exact test . post - treatment survival to the primary endpoint of vt / vf was described by kaplan - meier cumulative estimates , with comparison performed by the log rank test . all analyses were made using spss statistics ( version 19 , ibm italia spa , italy ) . the study population , divided into subgroups by pharmacologic intervention and genotype , is shown in figure 1 . phenotypic characterization confirmed that , compared to their wt littermates , tg mice had a significantly more prolonged qt interval ( 77 12 ms vs 61 6 ms , p < .001 ) and a significantly lower mean basal hr ( 340 52 bpm vs 371 47 bpm , p = .005 ) . given the observed difference in basal hr between tg and wt animals and the established relationship between rr and qt intervals , we also compared the qt at fixed ranges of rr intervals ( 140159 ms , 160179 ms , 180199 ms , and 200220 ms ) to control for qt adaptation , as it is done in infants . as expected , at all predefined rr ranges , tg mice showed a significantly ( p < .001 for all comparisons ) longer qt interval compared to wt mice ( 69 8 ms , 76 9 ms , 79 10 ms , and 83 10 ms vs 58 5 ms , 60 6 ms , 65 5 ms , and 68 8 ms , respectively ; table 1 and figure 2 ) . fifty - eight mice ( 39 tg and 19 wt ) underwent pharmacologic experiments to evaluate the proarrhythmic effect of carbachol and the potential protective role of propranolol pretreatment . specifically , 20 tg and 9 wt mice were treated with carbachol alone ( groups 1 and 3 ) , whereas 19 tg and 10 wt mice were pretreated with propranolol and then injected with carbachol ( groups 2 and 4 ; figure 1 ) . whereas mean basal hr was confirmed to be significantly lower in the 39 tg mice compared to the 19 wt animals ( 352 52 bpm vs 383 49 bpm , p < .05 ) , no significant difference in hr was observed across the four groups ( p = .10 ; table 2 ) . mean values of basal qt interval were almost identical within both tg subgroups ( 76 15 ms in group 1 and 76 12 ms in group 2 ) and within both wt groups ( 60 6 ms in group 3 vs 62 5 ms in group 4 ) . as expected , each tg subgroup had a significantly ( p < .05 ) longer qt interval compared to its corresponding wt counterpart . compared to baseline values , carbachol treatment significantly decreased hr and prolonged qt interval in all mice ( table 2 ) . the relative ( % ) change from baseline of both hr and qt following carbachol was rather similar across the four subgroups , independent of genotype or propranolol pre - treatment ( hr : 47% 11% , 53% 16% , 54% 12% , 59% 10% , p = .07 ; qt : 39% 29% , 54% 34% , 69% 37% , 64% 31% , p = .1 ) . the occurrence of cardiac arrhythmias according to genotype and drug exposure is summarized in figure 3 . in the tg kpq - scn5a mice , the overall incidence of drug - induced arrhythmias was high ( 27/39 [ 69% ] ) but with striking differences between groups 1 and 2 . specifically , in group 1 , of the 20 tg mice injected only with carbachol , 4 ( 20% ) had no arrhythmias , 5 ( 25% ) showed minor events ( vpbs ) , and 11 ( 55% ) developed major arrhythmias ( vt and/or vf ) , which occurred at a median time of 11 minutes ( interquartile range 921 ) after carbachol injection . by contrast , in group 2 , among the 19 tg mice pretreated with propranolol and then injected with carbachol , none developed major arrhythmias ; isolated vpbs were observed in 11 mice ( 58% ) , and 8 ( 42% ) had no cardiac arrhythmias ( figure 3a ) . therefore , pre - treatment with propranolol had a marked and significant ( p < .001 ) effect on the occurrence of major arrhythmias among tg mice as it was associated with the total absence of vt / vf compared to tg mice not pretreated with propranolol ( i.e. , with 100% cumulative survival to vt / vf compared to 39% ; figure 4 ) . among the wt mice overall , only minor arrhythmias were observed after the pharmacologic interventions , and this occurred in 9 of 19 ( 47% ) . specifically , in group 3 , of the nine mice treated with carbachol , 5 ( 56% ) had isolated vpbs whereas none developed major arrhythmias ( vt / vf ; figure 3b ) . similarly , in group 4 none of the 10 wt mice pretreated with propranolol and then injected with carbachol had major arrhythmias , whereas four developed isolated vpbs and six had no arrhythmias at all ( figure 3b ) . thus , among the wt mice , all carbachol - induced arrhythmias were minor and unaffected by propranolol ( p = .66 ; figure 3 ) . phenotypic characterization confirmed that , compared to their wt littermates , tg mice had a significantly more prolonged qt interval ( 77 12 ms vs 61 6 ms , p < .001 ) and a significantly lower mean basal hr ( 340 52 bpm vs 371 47 bpm , p = .005 ) . given the observed difference in basal hr between tg and wt animals and the established relationship between rr and qt intervals , we also compared the qt at fixed ranges of rr intervals ( 140159 ms , 160179 ms , 180199 ms , and 200220 ms ) to control for qt adaptation , as it is done in infants . as expected , at all predefined rr ranges , tg mice showed a significantly ( p < .001 for all comparisons ) longer qt interval compared to wt mice ( 69 8 ms , 76 9 ms , 79 10 ms , and 83 10 ms vs 58 5 ms , 60 6 ms , 65 5 ms , and 68 8 ms , respectively ; table 1 and figure 2 ) . fifty - eight mice ( 39 tg and 19 wt ) underwent pharmacologic experiments to evaluate the proarrhythmic effect of carbachol and the potential protective role of propranolol pretreatment . specifically , 20 tg and 9 wt mice were treated with carbachol alone ( groups 1 and 3 ) , whereas 19 tg and 10 wt mice were pretreated with propranolol and then injected with carbachol ( groups 2 and 4 ; figure 1 ) . whereas mean basal hr was confirmed to be significantly lower in the 39 tg mice compared to the 19 wt animals ( 352 52 bpm vs 383 49 bpm , p < .05 ) , no significant difference in hr mean values of basal qt interval were almost identical within both tg subgroups ( 76 15 ms in group 1 and 76 12 ms in group 2 ) and within both wt groups ( 60 6 ms in group 3 vs 62 5 ms in group 4 ) . as expected , each tg subgroup had a significantly ( p < .05 ) longer qt interval compared to its corresponding wt counterpart . compared to baseline values , carbachol treatment significantly decreased hr and prolonged qt interval in all mice ( table 2 ) . the relative ( % ) change from baseline of both hr and qt following carbachol was rather similar across the four subgroups , independent of genotype or propranolol pre - treatment ( hr : 47% 11% , 53% 16% , 54% 12% , 59% 10% , p = .07 ; qt : 39% 29% , 54% 34% , 69% 37% , 64% 31% , p = .1 ) . the occurrence of cardiac arrhythmias according to genotype and drug exposure is summarized in figure 3 . in the tg kpq - scn5a mice , the overall incidence of drug - induced arrhythmias was high ( 27/39 [ 69% ] ) but with striking differences between groups 1 and 2 . specifically , in group 1 , of the 20 tg mice injected only with carbachol , 4 ( 20% ) had no arrhythmias , 5 ( 25% ) showed minor events ( vpbs ) , and 11 ( 55% ) developed major arrhythmias ( vt and/or vf ) , which occurred at a median time of 11 minutes ( interquartile range 921 ) after carbachol injection . by contrast , in group 2 , among the 19 tg mice pretreated with propranolol and then injected with carbachol , none developed major arrhythmias ; isolated vpbs were observed in 11 mice ( 58% ) , and 8 ( 42% ) had no cardiac arrhythmias ( figure 3a ) . therefore , pre - treatment with propranolol had a marked and significant ( p < .001 ) effect on the occurrence of major arrhythmias among tg mice as it was associated with the total absence of vt / vf compared to tg mice not pretreated with propranolol ( i.e. , with 100% cumulative survival to vt / vf compared to 39% ; figure 4 ) . among the wt mice overall , only minor arrhythmias were observed after the pharmacologic interventions , and this occurred in 9 of 19 ( 47% ) . specifically , in group 3 , of the nine mice treated with carbachol , 5 ( 56% ) had isolated vpbs whereas none developed major arrhythmias ( vt / vf ; figure 3b ) . similarly , in group 4 none of the 10 wt mice pretreated with propranolol and then injected with carbachol had major arrhythmias , whereas four developed isolated vpbs and six had no arrhythmias at all ( figure 3b ) . thus , among the wt mice , all carbachol - induced arrhythmias were minor and unaffected by propranolol ( p = .66 ; figure 3 ) . the present study demonstrates that propranolol effectively prevents carbachol - induced life - threatening arrhythmias in an established animal model for lqt3 . the cholinergic stimulation produced by carbachol causes bradycardia and cardiac arrhythmias in vivo in this reliable experimental preparation , which presents the classic features of lqt3 in the clinic . the striking protection afforded by propranolol mirrors the latest clinical observations in lqt3 patients and forces a reassessment of the previous assumptions that have caused so much harm to some of these patients . identification of the major lqts genes in the mid-1990s has had a major impact on clinical management . a negative consequence has been the pressure on all investigators ( present ones included ) to publish , as soon as possible , their own data on genotype phenotype correlation . genotyped patients providing data on the effect of therapy initially were scanty , and this resulted in publications with very limited numbers but nonetheless conveying important messages . in 2000 , moss et al reported the effect of beta - blockers in 869 lqts patients , of whom 139 had been genotyped . of the 28 lqt3 patients , only four had cardiac events before therapy , and the conclusion was although the number of patients with lqt3 is quite small and the event rates in this genotype are quite low , no beneficial beta - blocker effect was evident in lqt3 . this was followed by two larger studies that conveyed the same message . in 2001 , schwartz et al reported on 670 genotyped patients , all with cardiac events ; 65 ( 9.7% ) were lqt3 patients . among those on beta - blocker therapy , mortality was 4% for both lqt1 and lqt2 patients but reached 17% for lqt3 patients . this raises concerns , not yet supported by evidence , regarding the use of beta - blockers because of the attendant reduction in heart rate . in 2004 , priori et al reported on 335 genotyped lqts patients , of whom 28 ( 8.3% ) were lqt3 . cardiac arrests on beta - blocker therapy were more frequent ( 14% ) in lqt3 than lqt1 ( 1% ) and lqt2 patients ( 7% ) . prophylactic defibrillator therapy may be a reasonable addition to beta - blockers in patients with lqt2 or lqt3 genotypes . these convergent messages , despite all being based on limited numbers and expressed cautiously , led many cardiologists to the unwarranted conclusion that beta - blockers were not protecting lqt3 patients . the unfortunate consequence has been the high number of lqt3 patients who received a prophylactic icd despite being asymptomatic and without having ever been treated with beta - blockers . eventually , we realized that this concept was not fitting with our personal clinical experience , and the analysis of individual outcomes unmasked an important and striking difference : whereas patients with cardiac events in the first year of life had a very poor prognosis and died soon , those who were asymptomatic in their first year were very well protected by beta - blockers . our breakthrough has just been confirmed by the largest study ever performed in lqt3 patients ( n = 403 ) , which demonstrates that among those without events in the first year of life mortality on beta - blockers is just close to 3% . thus , the available clinical data indicate clearly that beta - blockers are very effective for lqt3 patients as well , with the exception of the small subgroup of symptomatic infants who need an icd and/or left cardiac sympathetic denervation . while rumors about the lack of efficacy of beta - blockers for lqt3 patients were spreading in the cardiology community , two experimental studies appeared and claimed to closely mirror the clinical experience and to confirm the clinical observation that lqt3 patients do not benefit from beta - blocker therapy . shimizu and antzelevitch used their arterially perfused wedge of canine ventricle and , having modeled lqt3 , reported beneficial effects of sympathetic stimulation ( by isoproterenol ) that were abolished by propranolol . -blockade might be contraindicated in lqt3 had a significant impact on the clinical decisions of the subsequent years . the problem with their study was the assumption that isoproterenol , a nonbiologic substance , would mimic what happens in real life when there is a sudden increase in sympathetic activity . as occurs during exercise , blood - borne epinephrine has a dominant effect and , because of its simultaneous effect on all cardiac cells , reduces electrical heterogeneity and increases electrical stability . what happens experimentally with isoproterenol is similar , but stimulation is limited to the beta - receptors . by contrast , sudden centrally mediated sympathetic activation elicits a mostly localized release of norepinephrine from neural terminals , which increases both regional heterogeneity and cardiac electrical instability . this explains the profound difference in the correlation between sudden death and hr responses during exercise and mental stress . thus , it had to be expected that uniform beta - adrenergic stimulation by isoproterenol perfusion would reduce the dispersion of repolarization caused by anthopleurin ( which augments late ina ) , and that this beneficial effect produced by stimulation of beta - adrenergic receptors would be prevented by propranolol , a drug whose function is blocking the beta - receptors . we do not believe that the interaction between isoproterenol and propranolol in isolated tissue preparations justifies assumptions on the antiarrhythmic efficacy of propranolol in patients exposed to sudden neurally mediated release of norepinephrine . fabritz et al used the same model of the present study and reported that propranolol did not prevent the arrhythmias induced by carbachol . we do not have a ready explanation for these different results , as sometimes occurs among laboratories . we can only point to some experimental difference , such as propranolol assumed by drinking water vs injected ip and an ambulatory vs anesthetized state . however , a major and potentially important difference is the number of tg animals tested with carbachol plus propranolol , which was 19 mice in the present experiments and only four in those by fabritz et al . when experimental results differ , it is an old and good rule to verify what happens in the patients . it is now evident , based on adequate numbers , that beta - blockers are very effective in protecting lqt3 patients from life - threatening arrhythmias . the exception represented by infants with cardiac arrest in the first year of life does not impact on the concept . we used a well - validated animal model for lqt3 , confirmed its characteristic qt prolongation , reproduced vt / vf by the muscarinic agonist carbachol , and observed that propranolol - pretreated mice did not develop vt / vf after carbachol . we repeated these interventions in wt mice and observed modest effects of carbachol essentially unmodified by propranolol . the efficacy of propranolol likely reflects the combination of beta - adrenergic blockade with its well - known membrane stabilizing effect , which includes significant reductions in both peak and late na current . indeed , we believe that for lqt3 patients , propranolol should be regarded the beta - blocker of choice . the decrease in hr produced by propranolol and its modest effect on qt interval were as expected given that we looked at the qt as measured and not at the qtc to avoid the overcorrection associated with very short rr intervals . when carbachol produced vt / vf in the tg mice , most of the arrhythmias occurred within 15 min from the injection . the kaplan meier curve provides a graphic evidence of the protective effect of propranolol in these lqt3 animals . even though the ip dose of propranolol per kilogram is the same as that given intravenously in man , it is fair to remember that beta - blocker therapy in lqts patients is administered orally . therefore , some difference in the magnitude of the effects can not be ruled out . also , we studied the effect of propranolol in one specific , albeit fairly representative , scn5a mutation ; accordingly , we can not exclude that with different mutations one could observe different degrees of protection , as we already reported . they should help dispel , once and for all , the concern that beta - blockade provides no protection against vt / vf occurring in experimental models for lqt3 and thereby also in patients . furthermore , when taken together with the most recent clinical data , these observations send a clear and unequivocal message to practicing cardiologists that lqt3 patients should be placed on beta - blocker therapy as first choice without hesitation , with preference given to propranolol . identification of the major lqts genes in the mid-1990s has had a major impact on clinical management . a negative consequence has been the pressure on all investigators ( present ones included ) to publish , as soon as possible , their own data on genotype phenotype correlation . genotyped patients providing data on the effect of therapy initially were scanty , and this resulted in publications with very limited numbers but nonetheless conveying important messages . in 2000 , moss et al reported the effect of beta - blockers in 869 lqts patients , of whom 139 had been genotyped . of the 28 lqt3 patients , only four had cardiac events before therapy , and the conclusion was although the number of patients with lqt3 is quite small and the event rates in this genotype are quite low , no beneficial beta - blocker effect was evident in lqt3 . this was followed by two larger studies that conveyed the same message . in 2001 , schwartz et al reported on 670 genotyped patients , all with cardiac events ; 65 ( 9.7% ) were lqt3 patients . among those on beta - blocker therapy , mortality was 4% for both lqt1 and lqt2 patients but reached 17% for lqt3 patients . this raises concerns , not yet supported by evidence , regarding the use of beta - blockers because of the attendant reduction in heart rate . in 2004 , priori et al reported on 335 genotyped lqts patients , of whom 28 ( 8.3% ) were lqt3 . cardiac arrests on beta - blocker therapy were more frequent ( 14% ) in lqt3 than lqt1 ( 1% ) and lqt2 patients ( 7% ) . prophylactic defibrillator therapy may be a reasonable addition to beta - blockers in patients with lqt2 or lqt3 genotypes . these convergent messages , despite all being based on limited numbers and expressed cautiously , led many cardiologists to the unwarranted conclusion that beta - blockers were not protecting lqt3 patients . the unfortunate consequence has been the high number of lqt3 patients who received a prophylactic icd despite being asymptomatic and without having ever been treated with beta - blockers . eventually , we realized that this concept was not fitting with our personal clinical experience , and the analysis of individual outcomes unmasked an important and striking difference : whereas patients with cardiac events in the first year of life had a very poor prognosis and died soon , those who were asymptomatic in their first year were very well protected by beta - blockers . our breakthrough has just been confirmed by the largest study ever performed in lqt3 patients ( n = 403 ) , which demonstrates that among those without events in the first year of life mortality on beta - blockers is just close to 3% . thus , the available clinical data indicate clearly that beta - blockers are very effective for lqt3 patients as well , with the exception of the small subgroup of symptomatic infants who need an icd and/or left cardiac sympathetic denervation . while rumors about the lack of efficacy of beta - blockers for lqt3 patients were spreading in the cardiology community , two experimental studies appeared and claimed to closely mirror the clinical experience and to confirm the clinical observation that lqt3 patients do not benefit from beta - blocker therapy . shimizu and antzelevitch used their arterially perfused wedge of canine ventricle and , having modeled lqt3 , reported beneficial effects of sympathetic stimulation ( by isoproterenol ) that were abolished by propranolol . their conclusion that -blockade might be contraindicated in lqt3 had a significant impact on the clinical decisions of the subsequent years . the problem with their study was the assumption that isoproterenol , a nonbiologic substance , would mimic what happens in real life when there is a sudden increase in sympathetic activity . as occurs during exercise , blood - borne epinephrine has a dominant effect and , because of its simultaneous effect on all cardiac cells , reduces electrical heterogeneity and increases electrical stability . what happens experimentally with isoproterenol is similar , but stimulation is limited to the beta - receptors . by contrast , sudden centrally mediated sympathetic activation elicits a mostly localized release of norepinephrine from neural terminals , which increases both regional heterogeneity and cardiac electrical instability . this explains the profound difference in the correlation between sudden death and hr responses during exercise and mental stress . thus , it had to be expected that uniform beta - adrenergic stimulation by isoproterenol perfusion would reduce the dispersion of repolarization caused by anthopleurin ( which augments late ina ) , and that this beneficial effect produced by stimulation of beta - adrenergic receptors would be prevented by propranolol , a drug whose function is blocking the beta - receptors . we do not believe that the interaction between isoproterenol and propranolol in isolated tissue preparations justifies assumptions on the antiarrhythmic efficacy of propranolol in patients exposed to sudden neurally mediated release of norepinephrine . fabritz et al used the same model of the present study and reported that propranolol did not prevent the arrhythmias induced by carbachol . we do not have a ready explanation for these different results , as sometimes occurs among laboratories . we can only point to some experimental difference , such as propranolol assumed by drinking water vs injected ip and an ambulatory vs anesthetized state . however , a major and potentially important difference is the number of tg animals tested with carbachol plus propranolol , which was 19 mice in the present experiments and only four in those by fabritz et al . when experimental results differ , it is an old and good rule to verify what happens in the patients . it is now evident , based on adequate numbers , that beta - blockers are very effective in protecting lqt3 patients from life - threatening arrhythmias . the exception represented by infants with cardiac arrest in the first year of life does not impact on the concept . we used a well - validated animal model for lqt3 , confirmed its characteristic qt prolongation , reproduced vt / vf by the muscarinic agonist carbachol , and observed that propranolol - pretreated mice did not develop vt / vf after carbachol . we repeated these interventions in wt mice and observed modest effects of carbachol essentially unmodified by propranolol . the efficacy of propranolol likely reflects the combination of beta - adrenergic blockade with its well - known membrane stabilizing effect , which includes significant reductions in both peak and late na current . indeed , we believe that for lqt3 patients , propranolol should be regarded the beta - blocker of choice . the decrease in hr produced by propranolol and its modest effect on qt interval were as expected given that we looked at the qt as measured and not at the qtc to avoid the overcorrection associated with very short rr intervals . when carbachol produced vt / vf in the tg mice , most of the arrhythmias occurred within 15 min from the injection . the kaplan meier curve provides a graphic evidence of the protective effect of propranolol in these lqt3 animals . even though the ip dose of propranolol per kilogram is the same as that given intravenously in man , it is fair to remember that beta - blocker therapy in lqts patients is administered orally . therefore , some difference in the magnitude of the effects can not be ruled out . also , we studied the effect of propranolol in one specific , albeit fairly representative , scn5a mutation ; accordingly , we can not exclude that with different mutations one could observe different degrees of protection , as we already reported . these experimental results carry clinical implications . they should help dispel , once and for all , the concern that beta - blockade provides no protection against vt / vf occurring in experimental models for lqt3 and thereby also in patients . furthermore , when taken together with the most recent clinical data , these observations send a clear and unequivocal message to practicing cardiologists that lqt3 patients should be placed on beta - blocker therapy as first choice without hesitation , with preference given to propranolol .

backgroundthe efficacy of beta - blockers for treatment of patients with long qt syndrome type 3 ( lqt3 ) has been repeatedly questioned , and it has been suggested that they might be detrimental for this genetic subgroup of patients with long qt syndrome ( lqts ) . the disquieting consequence has been that cardiologists confronted with lqt3 patients often do not even attempt pharmacologic therapy and implant cardioverter - defibrillators as first - choice treatment . however , the most recent clinical data indicate high efficacy of beta - blocker therapy in lqt3 patients.objectivethe purpose of this study was to test the antiarrhythmic efficacy of beta - blockers in an established experimental model for lqt3.methodsafter phenotypic validation of 65 kpq - scn5a knock - in transgenic ( tg ) mice compared to 32 wild - type ( wt ) mice , we tested the effect of the arrhythmogenic cholinergic muscarinic agonist carbachol in 19 wt and 39 tg anesthetized mice , with and without pretreatment with propranolol given intraperitoneally.resultsat the same heart rates , tg mice had a markedly longer qt interval than wt mice . whereas carbachol had minor arrhythmic effects in the wt mice , it produced ventricular tachycardia ( vt ) and ventricular fibrillation ( vf ) in 55% of 20 tg mice . by contrast , in none of 19 tg mice pretreated with propranolol did vt / vf occur after carbachol injection.conclusionthese experimental data indicate that , contrary to previous reports , beta - blockade effectively prevents vt / vf in a validated lqt3 model . together with the most recent clinical data , these findings indicate that there is no reason for not initiating protective therapy with beta - blockers in lqt3 patients .

Methods ECG recordings and measurements Study protocol Pharmacologic interventions Statistical analysis Results Basal ECG Effect of drugs on ECG Arrhythmic events Discussion Previous clinical data Previous experimental data The present experiments Study limitations Clinical implications

heterozygous kpq - scn5a knock - in tg mice were characterized and compared with wild - type ( wt ) littermate mice . cardiac arrhythmias were classified by the most severe episode as absent , minor ( isolated ventricular premature beats [ vpbs ] , couples of vpbs , ventricular bigeminy ) , or major ( ventricular tachycardia [ vt ] , ventricular fibrillation [ vf ] ) . the mice either were injected with the cholinergic agonist carbachol or were pretreated with propranolol followed 2 to 3 minutes later by carbachol injection . cardiac arrhythmias were classified by the most severe episode as absent , minor ( isolated ventricular premature beats [ vpbs ] , couples of vpbs , ventricular bigeminy ) , or major ( ventricular tachycardia [ vt ] , ventricular fibrillation [ vf ] ) . the mice either were injected with the cholinergic agonist carbachol or were pretreated with propranolol followed 2 to 3 minutes later by carbachol injection . post - treatment survival to the primary endpoint of vt / vf was described by kaplan - meier cumulative estimates , with comparison performed by the log rank test . phenotypic characterization confirmed that , compared to their wt littermates , tg mice had a significantly more prolonged qt interval ( 77 12 ms vs 61 6 ms , p < .001 ) and a significantly lower mean basal hr ( 340 52 bpm vs 371 47 bpm , p = .005 ) . as expected , at all predefined rr ranges , tg mice showed a significantly ( p < .001 for all comparisons ) longer qt interval compared to wt mice ( 69 8 ms , 76 9 ms , 79 10 ms , and 83 10 ms vs 58 5 ms , 60 6 ms , 65 5 ms , and 68 8 ms , respectively ; table 1 and figure 2 ) . fifty - eight mice ( 39 tg and 19 wt ) underwent pharmacologic experiments to evaluate the proarrhythmic effect of carbachol and the potential protective role of propranolol pretreatment . specifically , 20 tg and 9 wt mice were treated with carbachol alone ( groups 1 and 3 ) , whereas 19 tg and 10 wt mice were pretreated with propranolol and then injected with carbachol ( groups 2 and 4 ; figure 1 ) . whereas mean basal hr was confirmed to be significantly lower in the 39 tg mice compared to the 19 wt animals ( 352 52 bpm vs 383 49 bpm , p < .05 ) , no significant difference in hr was observed across the four groups ( p = .10 ; table 2 ) . as expected , each tg subgroup had a significantly ( p < .05 ) longer qt interval compared to its corresponding wt counterpart . in the tg kpq - scn5a mice , the overall incidence of drug - induced arrhythmias was high ( 27/39 [ 69% ] ) but with striking differences between groups 1 and 2 . specifically , in group 1 , of the 20 tg mice injected only with carbachol , 4 ( 20% ) had no arrhythmias , 5 ( 25% ) showed minor events ( vpbs ) , and 11 ( 55% ) developed major arrhythmias ( vt and/or vf ) , which occurred at a median time of 11 minutes ( interquartile range 921 ) after carbachol injection . by contrast , in group 2 , among the 19 tg mice pretreated with propranolol and then injected with carbachol , none developed major arrhythmias ; isolated vpbs were observed in 11 mice ( 58% ) , and 8 ( 42% ) had no cardiac arrhythmias ( figure 3a ) . therefore , pre - treatment with propranolol had a marked and significant ( p < .001 ) effect on the occurrence of major arrhythmias among tg mice as it was associated with the total absence of vt / vf compared to tg mice not pretreated with propranolol ( i.e. , with 100% cumulative survival to vt / vf compared to 39% ; figure 4 ) . among the wt mice overall , only minor arrhythmias were observed after the pharmacologic interventions , and this occurred in 9 of 19 ( 47% ) . specifically , in group 3 , of the nine mice treated with carbachol , 5 ( 56% ) had isolated vpbs whereas none developed major arrhythmias ( vt / vf ; figure 3b ) . similarly , in group 4 none of the 10 wt mice pretreated with propranolol and then injected with carbachol had major arrhythmias , whereas four developed isolated vpbs and six had no arrhythmias at all ( figure 3b ) . phenotypic characterization confirmed that , compared to their wt littermates , tg mice had a significantly more prolonged qt interval ( 77 12 ms vs 61 6 ms , p < .001 ) and a significantly lower mean basal hr ( 340 52 bpm vs 371 47 bpm , p = .005 ) . as expected , at all predefined rr ranges , tg mice showed a significantly ( p < .001 for all comparisons ) longer qt interval compared to wt mice ( 69 8 ms , 76 9 ms , 79 10 ms , and 83 10 ms vs 58 5 ms , 60 6 ms , 65 5 ms , and 68 8 ms , respectively ; table 1 and figure 2 ) . fifty - eight mice ( 39 tg and 19 wt ) underwent pharmacologic experiments to evaluate the proarrhythmic effect of carbachol and the potential protective role of propranolol pretreatment . specifically , 20 tg and 9 wt mice were treated with carbachol alone ( groups 1 and 3 ) , whereas 19 tg and 10 wt mice were pretreated with propranolol and then injected with carbachol ( groups 2 and 4 ; figure 1 ) . whereas mean basal hr was confirmed to be significantly lower in the 39 tg mice compared to the 19 wt animals ( 352 52 bpm vs 383 49 bpm , p < .05 ) , no significant difference in hr mean values of basal qt interval were almost identical within both tg subgroups ( 76 15 ms in group 1 and 76 12 ms in group 2 ) and within both wt groups ( 60 6 ms in group 3 vs 62 5 ms in group 4 ) . as expected , each tg subgroup had a significantly ( p < .05 ) longer qt interval compared to its corresponding wt counterpart . in the tg kpq - scn5a mice , the overall incidence of drug - induced arrhythmias was high ( 27/39 [ 69% ] ) but with striking differences between groups 1 and 2 . specifically , in group 1 , of the 20 tg mice injected only with carbachol , 4 ( 20% ) had no arrhythmias , 5 ( 25% ) showed minor events ( vpbs ) , and 11 ( 55% ) developed major arrhythmias ( vt and/or vf ) , which occurred at a median time of 11 minutes ( interquartile range 921 ) after carbachol injection . by contrast , in group 2 , among the 19 tg mice pretreated with propranolol and then injected with carbachol , none developed major arrhythmias ; isolated vpbs were observed in 11 mice ( 58% ) , and 8 ( 42% ) had no cardiac arrhythmias ( figure 3a ) . therefore , pre - treatment with propranolol had a marked and significant ( p < .001 ) effect on the occurrence of major arrhythmias among tg mice as it was associated with the total absence of vt / vf compared to tg mice not pretreated with propranolol ( i.e. , with 100% cumulative survival to vt / vf compared to 39% ; figure 4 ) . among the wt mice overall , only minor arrhythmias were observed after the pharmacologic interventions , and this occurred in 9 of 19 ( 47% ) . specifically , in group 3 , of the nine mice treated with carbachol , 5 ( 56% ) had isolated vpbs whereas none developed major arrhythmias ( vt / vf ; figure 3b ) . similarly , in group 4 none of the 10 wt mice pretreated with propranolol and then injected with carbachol had major arrhythmias , whereas four developed isolated vpbs and six had no arrhythmias at all ( figure 3b ) . thus , among the wt mice , all carbachol - induced arrhythmias were minor and unaffected by propranolol ( p = .66 ; figure 3 ) . the present study demonstrates that propranolol effectively prevents carbachol - induced life - threatening arrhythmias in an established animal model for lqt3 . the striking protection afforded by propranolol mirrors the latest clinical observations in lqt3 patients and forces a reassessment of the previous assumptions that have caused so much harm to some of these patients . in 2000 , moss et al reported the effect of beta - blockers in 869 lqts patients , of whom 139 had been genotyped . of the 28 lqt3 patients , only four had cardiac events before therapy , and the conclusion was although the number of patients with lqt3 is quite small and the event rates in this genotype are quite low , no beneficial beta - blocker effect was evident in lqt3 . among those on beta - blocker therapy , mortality was 4% for both lqt1 and lqt2 patients but reached 17% for lqt3 patients . this raises concerns , not yet supported by evidence , regarding the use of beta - blockers because of the attendant reduction in heart rate . cardiac arrests on beta - blocker therapy were more frequent ( 14% ) in lqt3 than lqt1 ( 1% ) and lqt2 patients ( 7% ) . prophylactic defibrillator therapy may be a reasonable addition to beta - blockers in patients with lqt2 or lqt3 genotypes . these convergent messages , despite all being based on limited numbers and expressed cautiously , led many cardiologists to the unwarranted conclusion that beta - blockers were not protecting lqt3 patients . the unfortunate consequence has been the high number of lqt3 patients who received a prophylactic icd despite being asymptomatic and without having ever been treated with beta - blockers . eventually , we realized that this concept was not fitting with our personal clinical experience , and the analysis of individual outcomes unmasked an important and striking difference : whereas patients with cardiac events in the first year of life had a very poor prognosis and died soon , those who were asymptomatic in their first year were very well protected by beta - blockers . our breakthrough has just been confirmed by the largest study ever performed in lqt3 patients ( n = 403 ) , which demonstrates that among those without events in the first year of life mortality on beta - blockers is just close to 3% . thus , the available clinical data indicate clearly that beta - blockers are very effective for lqt3 patients as well , with the exception of the small subgroup of symptomatic infants who need an icd and/or left cardiac sympathetic denervation . while rumors about the lack of efficacy of beta - blockers for lqt3 patients were spreading in the cardiology community , two experimental studies appeared and claimed to closely mirror the clinical experience and to confirm the clinical observation that lqt3 patients do not benefit from beta - blocker therapy . -blockade might be contraindicated in lqt3 had a significant impact on the clinical decisions of the subsequent years . thus , it had to be expected that uniform beta - adrenergic stimulation by isoproterenol perfusion would reduce the dispersion of repolarization caused by anthopleurin ( which augments late ina ) , and that this beneficial effect produced by stimulation of beta - adrenergic receptors would be prevented by propranolol , a drug whose function is blocking the beta - receptors . we do not believe that the interaction between isoproterenol and propranolol in isolated tissue preparations justifies assumptions on the antiarrhythmic efficacy of propranolol in patients exposed to sudden neurally mediated release of norepinephrine . fabritz et al used the same model of the present study and reported that propranolol did not prevent the arrhythmias induced by carbachol . we used a well - validated animal model for lqt3 , confirmed its characteristic qt prolongation , reproduced vt / vf by the muscarinic agonist carbachol , and observed that propranolol - pretreated mice did not develop vt / vf after carbachol . the efficacy of propranolol likely reflects the combination of beta - adrenergic blockade with its well - known membrane stabilizing effect , which includes significant reductions in both peak and late na current . indeed , we believe that for lqt3 patients , propranolol should be regarded the beta - blocker of choice . when carbachol produced vt / vf in the tg mice , most of the arrhythmias occurred within 15 min from the injection . even though the ip dose of propranolol per kilogram is the same as that given intravenously in man , it is fair to remember that beta - blocker therapy in lqts patients is administered orally . also , we studied the effect of propranolol in one specific , albeit fairly representative , scn5a mutation ; accordingly , we can not exclude that with different mutations one could observe different degrees of protection , as we already reported . they should help dispel , once and for all , the concern that beta - blockade provides no protection against vt / vf occurring in experimental models for lqt3 and thereby also in patients . furthermore , when taken together with the most recent clinical data , these observations send a clear and unequivocal message to practicing cardiologists that lqt3 patients should be placed on beta - blocker therapy as first choice without hesitation , with preference given to propranolol . genotyped patients providing data on the effect of therapy initially were scanty , and this resulted in publications with very limited numbers but nonetheless conveying important messages . in 2000 , moss et al reported the effect of beta - blockers in 869 lqts patients , of whom 139 had been genotyped . of the 28 lqt3 patients , only four had cardiac events before therapy , and the conclusion was although the number of patients with lqt3 is quite small and the event rates in this genotype are quite low , no beneficial beta - blocker effect was evident in lqt3 . among those on beta - blocker therapy , mortality was 4% for both lqt1 and lqt2 patients but reached 17% for lqt3 patients . this raises concerns , not yet supported by evidence , regarding the use of beta - blockers because of the attendant reduction in heart rate . cardiac arrests on beta - blocker therapy were more frequent ( 14% ) in lqt3 than lqt1 ( 1% ) and lqt2 patients ( 7% ) . prophylactic defibrillator therapy may be a reasonable addition to beta - blockers in patients with lqt2 or lqt3 genotypes . these convergent messages , despite all being based on limited numbers and expressed cautiously , led many cardiologists to the unwarranted conclusion that beta - blockers were not protecting lqt3 patients . the unfortunate consequence has been the high number of lqt3 patients who received a prophylactic icd despite being asymptomatic and without having ever been treated with beta - blockers . eventually , we realized that this concept was not fitting with our personal clinical experience , and the analysis of individual outcomes unmasked an important and striking difference : whereas patients with cardiac events in the first year of life had a very poor prognosis and died soon , those who were asymptomatic in their first year were very well protected by beta - blockers . our breakthrough has just been confirmed by the largest study ever performed in lqt3 patients ( n = 403 ) , which demonstrates that among those without events in the first year of life mortality on beta - blockers is just close to 3% . thus , the available clinical data indicate clearly that beta - blockers are very effective for lqt3 patients as well , with the exception of the small subgroup of symptomatic infants who need an icd and/or left cardiac sympathetic denervation . while rumors about the lack of efficacy of beta - blockers for lqt3 patients were spreading in the cardiology community , two experimental studies appeared and claimed to closely mirror the clinical experience and to confirm the clinical observation that lqt3 patients do not benefit from beta - blocker therapy . their conclusion that -blockade might be contraindicated in lqt3 had a significant impact on the clinical decisions of the subsequent years . thus , it had to be expected that uniform beta - adrenergic stimulation by isoproterenol perfusion would reduce the dispersion of repolarization caused by anthopleurin ( which augments late ina ) , and that this beneficial effect produced by stimulation of beta - adrenergic receptors would be prevented by propranolol , a drug whose function is blocking the beta - receptors . we do not believe that the interaction between isoproterenol and propranolol in isolated tissue preparations justifies assumptions on the antiarrhythmic efficacy of propranolol in patients exposed to sudden neurally mediated release of norepinephrine . fabritz et al used the same model of the present study and reported that propranolol did not prevent the arrhythmias induced by carbachol . it is now evident , based on adequate numbers , that beta - blockers are very effective in protecting lqt3 patients from life - threatening arrhythmias . we used a well - validated animal model for lqt3 , confirmed its characteristic qt prolongation , reproduced vt / vf by the muscarinic agonist carbachol , and observed that propranolol - pretreated mice did not develop vt / vf after carbachol . indeed , we believe that for lqt3 patients , propranolol should be regarded the beta - blocker of choice . when carbachol produced vt / vf in the tg mice , most of the arrhythmias occurred within 15 min from the injection . even though the ip dose of propranolol per kilogram is the same as that given intravenously in man , it is fair to remember that beta - blocker therapy in lqts patients is administered orally . also , we studied the effect of propranolol in one specific , albeit fairly representative , scn5a mutation ; accordingly , we can not exclude that with different mutations one could observe different degrees of protection , as we already reported . they should help dispel , once and for all , the concern that beta - blockade provides no protection against vt / vf occurring in experimental models for lqt3 and thereby also in patients . furthermore , when taken together with the most recent clinical data , these observations send a clear and unequivocal message to practicing cardiologists that lqt3 patients should be placed on beta - blocker therapy as first choice without hesitation , with preference given to propranolol .

interferon - alfa ( ifn- ) monotherapy has been found with normalization of alanine aminotransferase ( alt ) levels in a few patients diagnosed as non - a , non - b hepatitis even before hepatitis c virus ( hcv ) was identified as the chief etiologic agent in this diagnosis . in 1989 , the first cases of successful treatment of documented chronic hepatitis c ( chc ) with ifn- monotherapy were reported , but relapse after the cessation of treatment was common [ 2 , 3 ] . the introduction of combination therapy with ifn- and ribavirin has markedly improved treatment response . nevertheless , less than one - half of patients with chc were able to experience a favorable response to the combination therapy [ 46 ] . since 2000 , the attachment of inert polyethylene glycol to conventional ifn- , pegylated ifn- ( pegifn- ) , reduced degradation and clearance , prolonging the half - life of ifn and permitting less frequent , weekly dosing while maintaining higher sustained ifn levels ( compared with 3 times weekly for conventional ifn ) . now , pegifn--ribavirin combination treatment has been recommended for all patients infected with hcv . for patients infected with hcv genotype 1 ( hcv-1 ) , the recommended treatment duration is 48 weeks , whereas for patients infected with hcv-2 or hcv-3 , the recommended treatment duration is 24 weeks . there are two distinct but complementary mechanisms for the antiviral effects of ifn- : ( a ) induction of a non - virus - specific antiviral state in infected cells , resulting in direct inhibition of viral replication , and ( b ) immunomodulatory effects that enhance the host 's specific antiviral immune responses and may accelerate the death of infected cells . food and drug administration ( fda ) has approved 3 ifn preparations for treatment of hcv : ( a ) 3 million units ( mus ) ifn--2a 3 times weekly ; ( b ) 3 mus of ifn--2b 3 times weekly ; and ( c ) 9 gs of ifn alfacon-1 twice weekly , or 15 g 3 times weekly in nonresponders . pegifn is a product of pegylation to conventional ifn ( the attachment of inert polyethylene glycol ( peg ) polymers to a therapeutic protein such as ifn ) . the larger molecular size of the compound results in a longer half - life due to reduced clearance , while retaining biological activity , and allows more convenient once - weekly dosing . two pegifns [ 11 , 12 ] were studied : ( a ) pegifn--2a , a 40 kda branched molecule with a terminal half - life of 80 hours ( range : 50140 hours ) and a mean clearance of 22 ml / hrkg administered at a fixed 180 g per week and ( b ) pegifn--2b , a 12 kda linear molecule with a mean terminal half - life of 40 hours ( range : 2260 hours ) and a mean clearance of 94 ml / hr kg , administered on the basis of weight ( 1.5 g / kg / week ) . maximal serum concentrations ( c max ) occur between 15 and 44 hours post dose and are sustained for up to 4872 hours . these two pegifns much improved the rates of svr in comparison with their nonpegylated counterparts [ 11 , 12 ] . ribavirin ( 1--d - ribofuranosyl-1,2,4-triazole-3-carboxamide ) is an oral purine nucleoside analogue with broad activity against viral pathogens . the mechanism of action of ribavirin in chc remains controversial . among the suggested , but not proven , roles of ribavirin in the treatment of chc are an immunologic modulation through switching the t - cell phenotype from type 2 to type 1 ; inhibition of host inosine monophosphate dehydrogenase activity ; depletion of intracellular guanosine triphosphate pools ; induction of mutational catastrophe ; or a moderate , transient , early direct antiviral effect . ribavirin may lead to rapid and lethal mutation of virions or depletion of intracellular guanosine triphosphate , which is necessary for viral rna synthesis . additionally , ribavirin may act synergistically with ifn by upregulating the activity of double - stranded rna - activated protein kinase and enhances the action of interferon - alfa against hepatitis c virus . the most interesting clinical observation is that ribavirin monotherapy had a minimal effect on hcv viremia , despite the fact that serum alt levels were reduced significantly in a considerable proportion of patients with chronic hcv infection . however , the combination of ribavirin and ifn provides a clinically synergistic anti - hcv effect . hence it was proposed that ribavirin may exert its effect on the host immune response . several studies on virus - specific t - cell reactivity in association with ifn treatment have found increased numbers of patients with chc with demonstrable hcv - specific th responses either during treatment or after a sustained therapeutic response . these findings raise the possibility that enhancement of hcv - specific t - cell reactivity may be one mechanism for successful antiviral treatment . hcv - specific t - cell reactivity was uncommon at baseline but increased markedly during antiviral therapy , peaking around treatment weeks 48 . the main difference in t - cell reactivity of patients treated with ifn - ribavirin was a significant decrease of the expression of ifn- , whereas inf- expression was similar to that in patients receiving ifn monotherapy . the greater efficacy of ribavirin may exert an anti - inflammatory effect and may help reducing ifn--driven t - cell activation and liver damage . in earlier studies , the primary end point for hcv therapies was a biochemical response , defined as the normalization of alt levels [ 2 , 3 ] . the introduction of virologic assays to detect hcv rna further allows the assessment of a virologic response , defined as polymerase chain reaction- ( pcr- ) seronegative ( 50 iu / ml , or 100 copies / ml ) for hcv rna . histological response has been assessed in some clinical studies , but there is little indication for posttreatment biopsy in clinical practice . four on - treatment and three patterns of off - treatment virological responses to antiviral therapy for hepatitis c have emerged over the past decade [ 2123 ] . they include the following : rapid virologic response ( rvr ) : pcr - seronegative of hcv rna at week 4 ; early virologic response ( evr ) : there are two kinds stratifications of evr : complete evr ( cevr ) : pcr - seronegative of hcv rna at week 12;partial evr ( pevr ) : decrease of hcv rna by > 2 log from baseline values at week 12 ; complete evr ( cevr ) : pcr - seronegative of hcv rna at week 12 ; partial evr ( pevr ) : decrease of hcv rna by > 2 log from baseline values at week 12 ; end - of - treatment virologic response ( etvr ) : pcr - seronegative of hcv rna at the end of therapy ; virologic breakthrough : hcv rna reappearance in serum while still on treatment ; sustained virologic response ( svr ) : pcr - seronegative of hcv rna 6 months after completing therapy ; virologic relapse : pcr - seronegative of hcv rna at the end of therapy , with return of circulating virus after completion of therapy ; nonresponders : persistently seropositive for hcv rna throughout treatment . more than 97% of patients with svr remain nonviremic by pcr for the subsequent 514 years [ 24 , 25 ] . these patients are regarded as having a high probability of a durable biochemical , virologic , and histological response . until the 1990s , the only therapy of proven benefit for patients with chc was ifn-. initially , a 6-month course of 3 weekly injections of 3 mus of ifn- was approved for treatment of chc , and a biochemical response , defined as the normalization of alt levels , was assigned as the primary end point [ 2 , 3 ] . ifn- monotherapy suppresses serum hcv rna to undetectable levels and normalizes the alt level in 25% to 40% of chc patients , usually within the first 2 - 3 months of treatment . however , these initial responses to ifn- monotherapy are usually transient , and sustained response is documented in only about 8% to 9% of patients . when virologic assays for detection of hcv rna became available , the virological response rates were observed to be lower than those reported with biochemical end points . in the meta - analysis of ifn- monotherapy , normalization of alt levels at the end of treatment and 6 months after stopping treatment was seen in 47% and 23% of treated patients , respectively . etvr and svr , however , were observed in only 29% and 8% of treated patients , respectively . improvement of efficacy on chc could be achieved with higher doses and/or a longer duration of ifn- monotherapy . a doubling of the duration of therapy to 12 months increased the frequency of svrs to approximately 20% . the best efficacy / risk ratio was in favor of 3 mus of ifn- 3 times weekly for at least 12 months in treatment - nave patients with chc . the introduction of ribavirin in combination with ifn- was a major breakthrough in the treatment of chc . even though ribavirin monotherapy was shown to be ineffective , the rate of svrs was 43% and 6% for the ifn--2a with and without ribavirin combination , respectively , and 36% and 18% for the ifn--2b with and without ribavirin combination . a meta - analysis in 1995 showed that the svr rate was significantly higher for ifn - ribavirin combination therapy than for ifn or ribavirin monotherapy ( odds ratio [ or ] : ifn - ribavirin versus ifn = 9.8 ; 95% confidence interval [ ci ] = l.950 ) . several landmark studies then followed and consistently demonstrated the dramatically improved responses to combination therapy , especially for hcv-2 or hcv-3 patients . in 1998 , two multicenter randomized controlled trials ( rcts ) ( one u.s . study and one international study ) totaling 1,744 previously untreated patients with compensated chc compared 24- and 48-week drug regimens of ifn--2b monotherapy ( 3 mus 3 times weekly ) with those of ifn--2b and ribavirin ( 1.000 mg / day or 1.200 mg / day for patients weighing < 75 kg or > 75 kg , resp . ) combination therapy followed by 24 weeks of off - therapy followup [ 5 , 6 ] . the overall svr rates for 24 and 48 weeks of therapy were 33% and 41% , respectively , for patients receiving ifn--2b - ribavirin , compared with svr rates of 6% at 24 weeks and 16% at 48 weeks ifn--2b monotherapy . in addition to definitively showing the benefit of combination therapy over ifn alone , these studies made several other important clinical points . second , the likelihood of response to treatment was related to pretreatment virus level and genotype . svrs to 48 or 24 weeks of combination therapy occurred in 29% and 17% of hcv-1 patients , respectively , and in 65% and 66% of hcv-2 or hcv-3 patients . the two studies reinforced the importance of longer duration therapy for 48 weeks in patients with hcv-1 infection . similarly , svrs to 48 or 24 weeks of combination therapy occurred in 38% and 27% of patients with pretreatment hcv rna levels of greater than 2 10 copies / ml , respectively , but the svr rates were no different for those with lower levels ( 45% and 43% , resp . ) . a systematic review in 2001 included data from 15 trials in which patients received ifn- monotherapy or ifn--ribavirin combination therapy . in comparison with ifn- monotherapy , combination therapy reduced the nonresponse rate ( absence of svr ) by 26% in treatment - nave patients ( relative risk = 0.74 ; 95% ci = 0.700.78 ) . morbidity and mortality showed a nonsignificant trend during treatment in favor of combination therapy . in 1998 , the fda approved the combination of ifn- and ribavirin for patients with chronic hcv infection . in 1999 , the easl international consensus conference on hepatitis c recommended that , for patients with chc who have not been previously treated , ( a ) standard therapy should consist of ifn- and ribavirin in combination for 24 weeks and that ( b ) treatment should be extended to 48 weeks in patients with both hcv-1 and hcv rna levels greater than 2 10 copies / ml . four rcts compared the efficacy and safety of once - weekly pegifn- monotherapy compared with ifn- monotherapy three times per week for the treatment of chronic hcv infection in treatment - nave patients [ 11 , 12 , 32 , 33 ] . the recommended dose of pegifn--2a monotherapy , administered fixed at 180 g / week for 48 weeks , achieved higher svr rates compared with ifn--2a monotherapy ( 30% to 39% versus 8% to 19% ) [ 12 , 32 , 33 ] ; the pegifn--2b monotherapy , administered according to body weight at 1.5 g / kg / week for 48 weeks , achieved an svr rate of 23% , compared to 12% with ifn--2b monotherapy . of note , conducted the first substantive prospective study confined to patients with compensated cirrhosis or advanced fibrosis . cirrhosis has been a poor predictor of responsiveness and is associated with a high risk of leucopenia and thrombocytopenia [ 5 , 6 ] . this study , however , showed that pegifn monotherapy was both well tolerated and effective in cirrhotic chc patients , with an svr rate of 30% . pegifn monotherapy has been recommended for patients with contraindications to ribavirin , such as those with renal insufficiency , hemoglobinopathies , and ischemic cardiovascular disease . some clinical trials have been reported to date in these populations [ 34 , 35 ] . for patients with contraindications to ribavirin but who have indications for antiviral therapy , pegifn represents the best option of treatment . the results of pegifn- monotherapy encouraged more clinical trials to go on and anticipation that combination therapy with pegifn- and ribavirin would be even more effective . the earlier two large rcts were applied with fixed durations of 48 weeks [ 36 , 37 ] . in these trials , pegifn--2b was dosed by weight ( 1.5 g / kg was fda approved ) and coupled with 800 mg of ribavirin ; pegifn--2a was given at a fixed dose of 180 g along with a weight - adjusted , higher dose of ribavirin ( 1.000 mg / day or 1.200 mg / day for patients weighing < 75 kg or > 75 kg , resp . ) . these trials demonstrated that higher svr rates could be achieved with the combination of pegifn- weekly plus oral ribavirin given twice daily than with the combination of ifn- given 3 times weekly plus ribavirin or than with pegifn- monotherapy . the issue of influence of ribavirin dose by body weight on the response rate was first addressed . in the pegifn--2b study , a post hoc analysis demonstrated that an svr of 61% was achieved in the subgroup whose daily dose of ribavirin exceeded 10.6 mg / kg . logistic regression analyses observed that the response rates generally increased as ribavirin dose increased up to about 13 mg / kg / day . some studies highlighted the potential importance of higher doses of ribavirin [ 38 , 39 ] . the first 4 weeks of weight - based ribavirin exposure ( > 13 mg / kg / day ) have been associated with the achievement of an rvr . in non - rvr patients , one post hoc analysis showed that providing and maintaining optimal dose of ribavirin within 12 weeks of treatment was pivotal for the attainment of a cevr . patients with a cevr in this study received a ribavirin dose of 16.3 mg / kg / day . moreover , a higher weight - based dose of ribavirin ( 15.2 mg / kilogram / day ) was associated with a lower relapse rate and higher svr rate . later , the optimal treatment duration and ribavirin dose were investigated in a multicenter rct in which all chc patients received pegifn--2a at a dose of 180 g , while patients in the four arms received either 24 or 48 weeks of ribavirin at a dose of 800 mg or at the higher , weight - based doses of 1.000 or 1.200 mg daily . in the subsequent registration trial , a high frequency of svrs occurred in patients with hcv-2 or hcv-3 , regardless of the regimen ( 79% to 84% ) , but optimal frequencies of svrs in hcv-1 ( 52% ) required longer duration and full - dose ribavirin , independent of the level of hcv rna . in patients with hcv-1 with a low viral load ( < 2 10 copies / ml , or 800.000 iu / ml ) , the svr was highest in those who had received the higher ribavirin dose and who were treated for 48 weeks ( 61% ) . this regimen was also optimal for patients with hcv-1 and a high viral load ( svr rate : 46% ) . in contrast , in patients with hcv-2 or hcv-3 , regardless of the pretreatment viral load , no differences were detected with the 4 treatment regimens . another single - arm , open - label , historical - control study of 24 weeks of treatment with pegifn--2b plus ribavirin limited to patients with hcv-2 or hcv-3 demonstrated that 24 weeks of treatment was sufficient in hcv-2- or hcv-3-infected patients , with an overall svr rate of 81% . this study supports the current recommendations that patients with hcv-1 require 48 weeks of pegifn- therapy with higher doses of ribavirin , while patients with hcv-2 or hcv-3 can be treated for only 24 weeks and with only 800 mg daily of ribavirin [ 7 , 45 ] . so far , there are 3 rcts to compare the rates of svr of these two pegifns . one rct showed no significant difference between the two available peginterferon - ribavirin regimens in patients infected with hcv genotype 1 . two rcts showed that svr rates were significantly greater in hcv patients treated with pegifn--2a than patients treated with pegifn--2b [ 12 , 47 , 48 ] . one recent meta - analysis showed that peginterferon alpfa-2a significantly increased the number of patients who achieved a sustained virological response ( svr ) versus peginterferon alfa-2b ( 47% versus 41% ; risk ratio : 1.11 ; 95% confidence interval : 1.041.19 ; p = .004 ( eight trials ) ) . physicians should look specifically for contraindications to antiviral therapy and assess both therapeutic risk and benefit . ribavirin is contraindicated in pregnancy , necessitating strict precautions and contraception in women of childbearing age and their sexual partners and in hcv - infected men with female partners of childbearing age . flu - like side effects of ifn can be managed with acetaminophen or nonsteroidal anti - inflammatory drugs ; antidepressants and hypnotics can be used for depression and insomnia , respectively . for management of neutropenia , dose reduction suffices ; the addition of granulocyte colony - stimulating factor is generally not recommended , although it may be considered in individual cases of severe neutropenia . treatment with ribavirin should be avoided in patients with ischemic cardiovascular and cerebrovascular disease and in patients with renal insufficiency . patients with decompensated cirrhosis are at high risk of adverse events and relatively contraindicated to ifn - ribavirin . patients receiving combination therapy had an increased risk for requiring medication dose reduction ( rr = 2.44 ; 95% ci = 1.583.75 ) or discontinuation ( rr = 1.28 ; 95% ci = 1.071.52 ) compared with those receiving ifn monotherapy . the rates of ifn dose reduction and discontinuation were similar among subjects receiving pegifn and conventional ifn [ 11 , 12 ] . with the great progress in the management of chc , clinical factors have been identified as predictors for the efficacy of the ifn - based therapy . they could be divided into two major categories : baseline and on - treatment predictors ( table 2 ) . the pretreatment variable most strongly predictive of an svr is the presence of hcv-2 or hcv-3 infection , whether with conventional ifns or pegifns , alone or in combination with ribavirin [ 5 , 6 , 36 , 37 ] . on the basis of variations in the nucleotide sequence of hcv , six genotypes ( numbered 16 ) and more than 50 subtypes ( identified by lowercase letters , e.g. , 1a and 1b ) have been identified . why hcv-1 is harder to treat than other hcv genotypes is not yet fully understood . several studies demonstrated that there exists a genotype - specific difference of viral kinetics [ 23 , 53 ] . the turnover of hepatocytes infected with hcv-1 is slower than that of hepatocytes infected with other hcv genotypes after initiation of ifn - based therapy [ 53 , 54 ] , implying that hcv-1 might be more resistant to antiviral therapy . under the current recommendation , svr rates were 42% to 60% for hcv-1 infection with a 48-week pegifn - ribavirin treatment , compared with 76% to 95% for hcv-2 or hcv-3 infections with a 24-week regimen [ 23 , 36 , 37 , 43 , 44 , 55 , 56 ] . patients with hcv-4 , which is common in egypt , are intermediate in responsiveness to therapy between those infected with hcv-1 and hcv-2 or hcv-3 , and it is suggested that they should be treated for a full 48 weeks with full - dose ribavirin , like patients with hcv-1 [ 4 , 41 ] . there is insufficient experience to provide recommendations for the treatment of persons with hcv-5 and hcv-6 so far . experienced providers need to make treatment judgments on a case - by - case basis . since hcv genotype is the strongest predictor of responses to ifn - based therapy for chc , it should be determined in all hcv - infected persons prior to treatment to determine the duration of therapy and the likelihood of response . pretreatment hcv rna level , even less important than hcv genotype , is a predictor of sustained response in ifn - based therapy [ 5 , 6 , 11 , 37 , 57 ] . the impact of hcv rna level on the response to combination therapy was different between patients with different hcv genotype infections . high viral load ( with a cutoff value of 200.000 copies / ml , or 40.000 iu / ml ) influenced the response rate in patients with hcv-1 ( 41% versus 56% ) but not those in patients with hcv-2 or hcv-3 ( 74% versus 81% ) . under the circumstances of a determined hcv genotype for chc patients , testing hcv rna levels is beneficial and recommended for hcv-1 patients but seems variable for hcv-2 or hcv-3 patients . higher quasispecies complexity at baseline has been observed in nonresponders than in sustained virological responders . an increasing number of mutations within the carboxyl terminal region of the hcv nonstructural 5a protein , named the ifn - sensitivity - determining region ( isdr ) , were correlated with treatment response in hcv-1-infected patients . patients infected with the so - called mutant type , defined by four or more amino acid substitutions in the isdr , showed a more favorable response toward ifn - based therapy in japan and taiwan [ 60 , 61 ] . additionally , a high degree ( 6 ) of sequence variation in the variable region 3 ( v3 ) plus its upstream flanking region of ns5a ( amino acid 23342379 ) , referred to as ifn / rbv resistance - determining region ( irrdr ) , would be a useful marker for predicting svr , whereas a less diverse ( 5 ) irrdr sequence predicts non - svr . the presence of bridging fibrosis and cirrhosis has been reported as one of the most unfavorable predictors for ifn - based therapy [ 5 , 6 , 12 , 51 , 64 , 65 ] . patients with cirrhosis generally respond poorly to standard ifn monotherapy , with svr rates of 5% to 20% [ 6 , 32 ] . responses are improved when conventional ifns or pegifns are combined with ribavirin , resulting in svr rates of 33% to 44% [ 6 , 36 , 37 ] . a gender effect on response has been reported . female sex was a predictor of svr in studies of conventional ifn - based therapy , but not in the studies of pegifn - ribavirin [ 11 , 36 , 43 ] . younger patients ( < 40 years ) had higher svr rates with pegifn - ribavirin [ 36 , 37 , 43 ] . several studies have demonstrated that svr rates are lower in patients with coexistent insulin resistance and/or hepatic steatosis or steatohepatitis [ 67 , 68 ] . in hcv-1 patients treated with pegifn - ribavirin , a lower svr rate was observed in patients with insulin resistance ( homeostasis model of assessment , homa - ir > 2 ) compared to those without insulin resistance [ 69 , 70 ] . kg / m are more likely to be insulin - resistant , to have more advanced hepatic steatosis or steatohepatitis and fibrosis , and to experience a reduced response to combination therapy [ 71 , 72 ] . additionally , other possible mechanisms of the impact of obesity on the therapeutic response include the linear correlation of efficacy and body - weight - based doses of ribavirin ( 10.615 mg / kg / day ) . to encourage weight loss and exercise before treatment , which has been associated with a reduction in steatosis fibrosis scores , excessive alcohol use could reduce the likelihood of a response to therapy [ 74 , 75 ] . to increase the efficacy of antiviral therapy , it has been suggested that abstinence be recommended before and during treatment for chc . racial differences in response to efficacy of ifn exist and have been one of the host factors . a lower response rate to ifn monotherapy was observed among african - american patients compared with white patients [ 17 , 76 ] . a pool analysis of two clinical trials with ifn - ribavirin combination therapy demonstrated that svrs were highest among asians ( 61% ) , followed by whites ( 39% ) , hispanics ( 23% ) , and african - americans ( 14% ) . hispanics and african - americans were less likely to respond to pegifn--ribavirin compared to whites . in studies of taiwanese chc patients , the svr rate was 23.7% , 37.1% , and 63.6% for a 24-week treatment of 3 mus of ifn- 3 times weekly alone , 6 mus of 3 times weekly alone , and 3 mus of 3 times weekly plus ribavirin , respectively [ 65 , 79 ] . the svr rate of hcv-1b patients to 24-week pegifn--ribavirin was 48.9% to 65.8% and could be as high as 80% with a 48-week regimen in taiwan [ 79 , 80 ] . a relative lower body weight ( 6770 kg ) in asian patients compared to u.s . patients ( 7881 kg ) may also play an important role . the different ethnic response rates may reflect the important role of genetics . host genetic variations are probably involved in the efficacy of ifn - based therapies for chc . genetic polymorphisms of human leukocyte antigen , cc chemokine receptor 5 , cytotoxic t lymphocyte antigen-4 , interleukin-10 , low molecular mass polypeptide 7 , mxa , and transforming growth factor-1 have been reported to have significant associations with responsiveness [ 8289 ] . tnf--308 polymorphism was associated with svrs to ifn - ribavirin in patients with hcv-1b infection and a high viral load . these results reflect the important role of unique genetic predisposition , at least in part , in the response to ifn - based therapy for chc . recent advances in pharmacogenomics have demonstrated the potential applications of genetic single nucleotide polymorphism and expression patterns in determining treatment responsiveness in chc [ 91 , 92 ] . a recent candidate gene study showed that genetic variation in the il28b gene , which encodes ifn-3 , is associated with spontaneous hcv clearance . several genome - wide associated studies observed that il28b single nucleotide polymorphisms played an important role in the treatment outcome of pegifn - rbv for chc [ 9496 ] . a genome - wide association study in 2010 confirmed that il28b genetic variation was the strongest genetic predictor in both natural and treatment - induced control of hcv . the increasing evidence for the role of ifn-3 for both spontaneous and treatment - induced control of hcv infection opens new avenues for prognosis and treatment of hcv infection . individuals with hcv genotype 1 or 4 who carry the risk allele , particularly in homozygosis , will have a very low probability of natural or treatment - induced clearance . half of the ethnic differences in response to interferon and ribavirin combination therapy can be explained by genetic polymorphism of il28b . because of the presumably shared routes of transmission , approximately one - fourth to one - third of all persons infected with hiv are coinfected with hcv . patients with hiv - hcv coinfection have been shown to respond less favorably to antiviral therapy than patients infected with hcv alone [ 98 , 99 ] . several rcts recommended 48 weeks of pegifn - ribavirin for hcv , regardless of hcv genotype , in hcv - hiv coinfected patients [ 100 , 101 ] . dual infections of hcv and hepatitis b virus ( hbv ) are not uncommon and occur in up to 5% of the general population in hcv - endemic areas . combined chronic hepatitis b and c leads to more severe liver disease and an increased risk of hcv . although hbv - hcv dual infection was refractory to conventional ifn monotherapy , recent studies in taiwan have demonstrated that conventional ifn - ribavirin combination therapy was effective in hcv clearance among hcv - dominant , hbv / hcv dually infected patients [ 105 , 106 ] . recently , a large , open - label , comparative , multicenter study confirmed the efficacy of pegifn - ribavirin for patients with chronic hcv - hbv dual infection in taiwan . nonresponders are more resistant to retreatment with subsequent ifn - based therapy , compared to relapsers ( or = 3.912 ; 95% ci = 1.45910.49 ) . retreatment with pegifn - ribavirin could achieve an svr rate of 47% to 60% for relapsers and 18% to 23% for nonresponders , hcv rna levels generally fall in a biphasic manner . the first rapid phase of viral suppression , from a few hours after the first ifn- injection to the end of the first day , is related to an inhibition of viral replication by a direct , nonspecific action of ifn-. this early initial decline in hcv rna levels correlates poorly with the eventual response to ifn - based therapy [ 74 , 113 ] . the second , slower phase of viral suppression , beginning on day 2 and gradually leading to seroclearance of hcv rna , is possibly related to the gradual clearance of infected cells by the patient 's immune system , while hcv replication is efficiently inhibited . this phase , less influenced by the dosage of ifn and hcv genotype , exhibits a good response to pegifn and is an excellent marker of an svr to the treatment [ 36 , 54 , 74 ] . an rvr at week 4 could predict an svr to ifn - ribavirin with a high degree of accuracy in both hcv-1 and hcv-2 patients , with positive predictive values of 78% and 92% , respectively . recent studies have demonstrated that an rvr is the single best predictor of an svr to pegifn - ribavirin for hcv-1 [ 114 , 115 ] and hcv-2 or hcv-3 patients [ 23 , 55 , 56 , 116 ] . for hcv-1 or hcv-4 patients with lower baseline viral loads and an rvr , an abbreviated 24-week regimen could achieve a comparable svr rate with a standard 48-week regimen [ 115 , 117 , 118 ] . selected patients with rvr might have their treatment courses abbreviated to 16 weeks if they are infected with hcv-2 or hcv-3 [ 23 , 56 ] . but , the shortening of therapy duration for genotype 2/3 with rvr is still controversial . abbreviated regimens may be considered in patients with a low baseline viral load who achieve an rvr [ 120 , 121 ] . among patients with an evr , however , as a negative predictor , non - evr is even an more robust predictor . in cases without an evr , the non - evr is a significantly negative predictor in hcv-1 patients , but not in hcv-2 patients . thus it is recommended that hcv-1 patients who do not achieve an evr at week 12 should discontinue the therapy beyond 12 weeks [ 22 , 78 ] . recently , stratification of early virological response ( evr ) into complete evr ( cevr ) and partial evr ( pevr ) has been possible to further improve the prediction of an svr and may allow for optimizing treatment duration in non - rvr patients . studies for hcv-1 non - rvr patients have demonstrated that the current recommended 48 weeks of treatment could achieve high svr rates in patients with a cevr but could lower rates of svr in patients with a pevr [ 124 , 125 ] . the svr rates would be more than 90% if patients could reach a cevr with a standard regimen ( 48 weeks for hcv-1 or 24 weeks for hcv-2 ) . for non - rvr patients , hcv viral loads < 10 iu / ml at week 4 provided an early and accurate prediction of who would or would not achieve a cevr and following svr . in hcv-1-infected patients with a pevr , the svr rates were 10% and 21% only and the relapse rates were up to 83% and 63% in the 24-week and 48-week groups , respectively . the treatment responses were inadequate , even with a standard 48-week regimen in these patients [ 124 , 125 ] . based on these predictors associated with treatment efficacy , response - guided individualized therapy has become a major consideration for clinicians . it is desirable to expose chc patients to the lowest doses and shortest durations of treatment , to reduce the likelihood of adverse events and to minimize costs , without compromising treatment efficacy . on the other hand , some difficult - to - treat patients have to receive longer and/or higher dose therapy to achieve responses . to date , hcv genotype , baseline viral load as well as on - treatment virological responses will provide information for individualized therapy decisionmaking for chc patients in the near future [ 115 , 126 ] . people who have an rvr may have a chance to abbreviate their treatment courses to avoid unnecessary costs and preventable drug side effects . in patients without an rvr treated with standard of care , the svr rate would be more than 90% if cevr could be accomplished . in patients with only a pevr , it has been suggested to extend the treatment course to 72 weeks [ 124 , 125 , 127 ] or adhere to high - dose peginterferon plus ribavirin combination therapy . in the future , additional therapy other than interferon - based treatment , such as protease inhibitors , might be anticipated in those difficult - to - treat patients . one would like to be able to evaluate whether a treatment response is likely as early as possible so that individualized strategies can be made or altered earlier before or during the treatment course . < 10 iu / ml at week 4 provided an accurate prediction of cevr and svr in non - rvr patients . medical adherence is an important factor associated with response to ifn - ribavirin , especially among patients with hcv-1 infection . in a retrospective analysis of data collected in the large registration trials of ifn - ribavirin , svrs have been reported to be more likely in patients who had taken at least 80% of all projected ifn injections and at least 80% of all projected ribavirin for at least 80% of the anticipated duration of treatment . recent development of direct - acting antiviral agents , also named specifically targeted antiviral therapy for hepatitis c ( stat - c ) compounds , to treat hcv has focused predominantly on inhibitors of the viral enzymes ns3/4a protease and the rna - dependent rna polymerase ns5b [ 129 , 130 ] . the administration of hcv ns3/4a protease inhibitors to patients with chronic hcv infections has demonstrated that they have dramatic antiviral effects and that compounds acting via this mechanism are likely to form a key component of future anti - hcv therapy . newer data have demonstrated promise for 2 protease inhibitors , sch 503034 ( boceprevir ) and vx-950 ( telaprevir ) , both of which appear to be able to improve sustained response while shortening duration of therapy . telaprevir ( vx-950 ) , the hcv protease inhibitor , is in the most advanced phase of clinical development . a first case of sustained virological response ( svr ) achieved in a patient with chronic hepatitis c by monotherapy with telaprevir without interferon therapy was reported . owing to a low genetic barrier , resistant variants emerge within a few days when used in monotherapy , thereby decreasing its efficacy . consequently , telaprevir has been combined with pegylated - interferon and ribavirin in clinical trials . this triple combination is more effective but has a higher rate of adverse events ( notably rash ) than the standard of care , despite the shorter duration of therapy . results of the milestone studies prove 1 and 2 indicate that 12 weeks of telaprevir - based triple therapy is too short because of the high rate of relapse after treatment completion . however , 24 to 48 weeks of total therapy including 12 weeks of triple therapy with telaprevir in addition to standard treatment greatly improved svr rates in treatment - nave genotype 1 patients compared with the standard of care . prove 3 has shown that telaprevir is also highly effective in the treatment of prior nonresponders or relapsers infected with hcv genotype 1 [ 130 , 135 ] .

since 1986 , interferon - alfa ( ifn- ) monotherapy has been administered for patients with chronic hepatitis c ( chc ) . however , sustained response rate is only about 8% to 9% . subsequent introduction of ribavirin in combination with ifn- was a major breakthrough in the treatment of chc . sustained virological responses ( svrs ) rate is about 30% in hepatitis c virus genotype 1 ( hcv-1 ) patients , and is about 65% in hcv-2 or -3 patients . after 2000 , pegylated interferon ( pegifn ) much improved the rates of svr . presently , pegifn--ribavirin combination therapy has been current standard of care for patients infected with hcv . in patients with hcv-1 , treatment for 48 weeks is optimal , but 24 weeks of treatment is sufficient in hcv-2 or -3 infected patients . clinical factors have been identified as predictors for the efficacy of the ifn - based therapy . the baseline factor most strongly predictive of an svr is the presence of hcv-2 or -3 infections . rapid virological response ( rvr ) is the single best predictor of an svr to pegifn - ribavirin therapy . if patients ca n't achieve a rvr but achieve a complete early virological response ( cevr ) , treatment with current standard of care can provide more than 90% svr rate . hcv-1 patients who do not achieve an evr should discontinue the therapy . recent advances of protease inhibitor may contribute the development of a novel triple combination therapy .

1. Introduction 2. Approved Agents for Treatment of Hepatitis C 3. Assessment of Treatment Response for Hepatitis C 4. Evolution of IFN-Based Therapy for Chronic Hepatitis C 5. Factors Associated with Treatment Efficacy 6. Protease Inhibitors and IFN-Based Therapy

interferon - alfa ( ifn- ) monotherapy has been found with normalization of alanine aminotransferase ( alt ) levels in a few patients diagnosed as non - a , non - b hepatitis even before hepatitis c virus ( hcv ) was identified as the chief etiologic agent in this diagnosis . in 1989 , the first cases of successful treatment of documented chronic hepatitis c ( chc ) with ifn- monotherapy were reported , but relapse after the cessation of treatment was common [ 2 , 3 ] . now , pegifn--ribavirin combination treatment has been recommended for all patients infected with hcv . for patients infected with hcv genotype 1 ( hcv-1 ) , the recommended treatment duration is 48 weeks , whereas for patients infected with hcv-2 or hcv-3 , the recommended treatment duration is 24 weeks . there are two distinct but complementary mechanisms for the antiviral effects of ifn- : ( a ) induction of a non - virus - specific antiviral state in infected cells , resulting in direct inhibition of viral replication , and ( b ) immunomodulatory effects that enhance the host 's specific antiviral immune responses and may accelerate the death of infected cells . these two pegifns much improved the rates of svr in comparison with their nonpegylated counterparts [ 11 , 12 ] . among the suggested , but not proven , roles of ribavirin in the treatment of chc are an immunologic modulation through switching the t - cell phenotype from type 2 to type 1 ; inhibition of host inosine monophosphate dehydrogenase activity ; depletion of intracellular guanosine triphosphate pools ; induction of mutational catastrophe ; or a moderate , transient , early direct antiviral effect . additionally , ribavirin may act synergistically with ifn by upregulating the activity of double - stranded rna - activated protein kinase and enhances the action of interferon - alfa against hepatitis c virus . the main difference in t - cell reactivity of patients treated with ifn - ribavirin was a significant decrease of the expression of ifn- , whereas inf- expression was similar to that in patients receiving ifn monotherapy . they include the following : rapid virologic response ( rvr ) : pcr - seronegative of hcv rna at week 4 ; early virologic response ( evr ) : there are two kinds stratifications of evr : complete evr ( cevr ) : pcr - seronegative of hcv rna at week 12;partial evr ( pevr ) : decrease of hcv rna by > 2 log from baseline values at week 12 ; complete evr ( cevr ) : pcr - seronegative of hcv rna at week 12 ; partial evr ( pevr ) : decrease of hcv rna by > 2 log from baseline values at week 12 ; end - of - treatment virologic response ( etvr ) : pcr - seronegative of hcv rna at the end of therapy ; virologic breakthrough : hcv rna reappearance in serum while still on treatment ; sustained virologic response ( svr ) : pcr - seronegative of hcv rna 6 months after completing therapy ; virologic relapse : pcr - seronegative of hcv rna at the end of therapy , with return of circulating virus after completion of therapy ; nonresponders : persistently seropositive for hcv rna throughout treatment . initially , a 6-month course of 3 weekly injections of 3 mus of ifn- was approved for treatment of chc , and a biochemical response , defined as the normalization of alt levels , was assigned as the primary end point [ 2 , 3 ] . however , these initial responses to ifn- monotherapy are usually transient , and sustained response is documented in only about 8% to 9% of patients . the introduction of ribavirin in combination with ifn- was a major breakthrough in the treatment of chc . a meta - analysis in 1995 showed that the svr rate was significantly higher for ifn - ribavirin combination therapy than for ifn or ribavirin monotherapy ( odds ratio [ or ] : ifn - ribavirin versus ifn = 9.8 ; 95% confidence interval [ ci ] = l.950 ) . the overall svr rates for 24 and 48 weeks of therapy were 33% and 41% , respectively , for patients receiving ifn--2b - ribavirin , compared with svr rates of 6% at 24 weeks and 16% at 48 weeks ifn--2b monotherapy . svrs to 48 or 24 weeks of combination therapy occurred in 29% and 17% of hcv-1 patients , respectively , and in 65% and 66% of hcv-2 or hcv-3 patients . the two studies reinforced the importance of longer duration therapy for 48 weeks in patients with hcv-1 infection . similarly , svrs to 48 or 24 weeks of combination therapy occurred in 38% and 27% of patients with pretreatment hcv rna levels of greater than 2 10 copies / ml , respectively , but the svr rates were no different for those with lower levels ( 45% and 43% , resp . ) in 1999 , the easl international consensus conference on hepatitis c recommended that , for patients with chc who have not been previously treated , ( a ) standard therapy should consist of ifn- and ribavirin in combination for 24 weeks and that ( b ) treatment should be extended to 48 weeks in patients with both hcv-1 and hcv rna levels greater than 2 10 copies / ml . four rcts compared the efficacy and safety of once - weekly pegifn- monotherapy compared with ifn- monotherapy three times per week for the treatment of chronic hcv infection in treatment - nave patients [ 11 , 12 , 32 , 33 ] . the recommended dose of pegifn--2a monotherapy , administered fixed at 180 g / week for 48 weeks , achieved higher svr rates compared with ifn--2a monotherapy ( 30% to 39% versus 8% to 19% ) [ 12 , 32 , 33 ] ; the pegifn--2b monotherapy , administered according to body weight at 1.5 g / kg / week for 48 weeks , achieved an svr rate of 23% , compared to 12% with ifn--2b monotherapy . this study , however , showed that pegifn monotherapy was both well tolerated and effective in cirrhotic chc patients , with an svr rate of 30% . pegifn monotherapy has been recommended for patients with contraindications to ribavirin , such as those with renal insufficiency , hemoglobinopathies , and ischemic cardiovascular disease . the first 4 weeks of weight - based ribavirin exposure ( > 13 mg / kg / day ) have been associated with the achievement of an rvr . in non - rvr patients , one post hoc analysis showed that providing and maintaining optimal dose of ribavirin within 12 weeks of treatment was pivotal for the attainment of a cevr . later , the optimal treatment duration and ribavirin dose were investigated in a multicenter rct in which all chc patients received pegifn--2a at a dose of 180 g , while patients in the four arms received either 24 or 48 weeks of ribavirin at a dose of 800 mg or at the higher , weight - based doses of 1.000 or 1.200 mg daily . in the subsequent registration trial , a high frequency of svrs occurred in patients with hcv-2 or hcv-3 , regardless of the regimen ( 79% to 84% ) , but optimal frequencies of svrs in hcv-1 ( 52% ) required longer duration and full - dose ribavirin , independent of the level of hcv rna . in patients with hcv-1 with a low viral load ( < 2 10 copies / ml , or 800.000 iu / ml ) , the svr was highest in those who had received the higher ribavirin dose and who were treated for 48 weeks ( 61% ) . this regimen was also optimal for patients with hcv-1 and a high viral load ( svr rate : 46% ) . in contrast , in patients with hcv-2 or hcv-3 , regardless of the pretreatment viral load , no differences were detected with the 4 treatment regimens . another single - arm , open - label , historical - control study of 24 weeks of treatment with pegifn--2b plus ribavirin limited to patients with hcv-2 or hcv-3 demonstrated that 24 weeks of treatment was sufficient in hcv-2- or hcv-3-infected patients , with an overall svr rate of 81% . this study supports the current recommendations that patients with hcv-1 require 48 weeks of pegifn- therapy with higher doses of ribavirin , while patients with hcv-2 or hcv-3 can be treated for only 24 weeks and with only 800 mg daily of ribavirin [ 7 , 45 ] . so far , there are 3 rcts to compare the rates of svr of these two pegifns . one rct showed no significant difference between the two available peginterferon - ribavirin regimens in patients infected with hcv genotype 1 . one recent meta - analysis showed that peginterferon alpfa-2a significantly increased the number of patients who achieved a sustained virological response ( svr ) versus peginterferon alfa-2b ( 47% versus 41% ; risk ratio : 1.11 ; 95% confidence interval : 1.041.19 ; p = .004 ( eight trials ) ) . with the great progress in the management of chc , clinical factors have been identified as predictors for the efficacy of the ifn - based therapy . the pretreatment variable most strongly predictive of an svr is the presence of hcv-2 or hcv-3 infection , whether with conventional ifns or pegifns , alone or in combination with ribavirin [ 5 , 6 , 36 , 37 ] . the turnover of hepatocytes infected with hcv-1 is slower than that of hepatocytes infected with other hcv genotypes after initiation of ifn - based therapy [ 53 , 54 ] , implying that hcv-1 might be more resistant to antiviral therapy . patients with hcv-4 , which is common in egypt , are intermediate in responsiveness to therapy between those infected with hcv-1 and hcv-2 or hcv-3 , and it is suggested that they should be treated for a full 48 weeks with full - dose ribavirin , like patients with hcv-1 [ 4 , 41 ] . since hcv genotype is the strongest predictor of responses to ifn - based therapy for chc , it should be determined in all hcv - infected persons prior to treatment to determine the duration of therapy and the likelihood of response . pretreatment hcv rna level , even less important than hcv genotype , is a predictor of sustained response in ifn - based therapy [ 5 , 6 , 11 , 37 , 57 ] . high viral load ( with a cutoff value of 200.000 copies / ml , or 40.000 iu / ml ) influenced the response rate in patients with hcv-1 ( 41% versus 56% ) but not those in patients with hcv-2 or hcv-3 ( 74% versus 81% ) . under the circumstances of a determined hcv genotype for chc patients , testing hcv rna levels is beneficial and recommended for hcv-1 patients but seems variable for hcv-2 or hcv-3 patients . an increasing number of mutations within the carboxyl terminal region of the hcv nonstructural 5a protein , named the ifn - sensitivity - determining region ( isdr ) , were correlated with treatment response in hcv-1-infected patients . patients infected with the so - called mutant type , defined by four or more amino acid substitutions in the isdr , showed a more favorable response toward ifn - based therapy in japan and taiwan [ 60 , 61 ] . the presence of bridging fibrosis and cirrhosis has been reported as one of the most unfavorable predictors for ifn - based therapy [ 5 , 6 , 12 , 51 , 64 , 65 ] . female sex was a predictor of svr in studies of conventional ifn - based therapy , but not in the studies of pegifn - ribavirin [ 11 , 36 , 43 ] . in hcv-1 patients treated with pegifn - ribavirin , a lower svr rate was observed in patients with insulin resistance ( homeostasis model of assessment , homa - ir > 2 ) compared to those without insulin resistance [ 69 , 70 ] . to increase the efficacy of antiviral therapy , it has been suggested that abstinence be recommended before and during treatment for chc . a pool analysis of two clinical trials with ifn - ribavirin combination therapy demonstrated that svrs were highest among asians ( 61% ) , followed by whites ( 39% ) , hispanics ( 23% ) , and african - americans ( 14% ) . in studies of taiwanese chc patients , the svr rate was 23.7% , 37.1% , and 63.6% for a 24-week treatment of 3 mus of ifn- 3 times weekly alone , 6 mus of 3 times weekly alone , and 3 mus of 3 times weekly plus ribavirin , respectively [ 65 , 79 ] . host genetic variations are probably involved in the efficacy of ifn - based therapies for chc . tnf--308 polymorphism was associated with svrs to ifn - ribavirin in patients with hcv-1b infection and a high viral load . these results reflect the important role of unique genetic predisposition , at least in part , in the response to ifn - based therapy for chc . patients with hiv - hcv coinfection have been shown to respond less favorably to antiviral therapy than patients infected with hcv alone [ 98 , 99 ] . several rcts recommended 48 weeks of pegifn - ribavirin for hcv , regardless of hcv genotype , in hcv - hiv coinfected patients [ 100 , 101 ] . although hbv - hcv dual infection was refractory to conventional ifn monotherapy , recent studies in taiwan have demonstrated that conventional ifn - ribavirin combination therapy was effective in hcv clearance among hcv - dominant , hbv / hcv dually infected patients [ 105 , 106 ] . recently , a large , open - label , comparative , multicenter study confirmed the efficacy of pegifn - ribavirin for patients with chronic hcv - hbv dual infection in taiwan . nonresponders are more resistant to retreatment with subsequent ifn - based therapy , compared to relapsers ( or = 3.912 ; 95% ci = 1.45910.49 ) . retreatment with pegifn - ribavirin could achieve an svr rate of 47% to 60% for relapsers and 18% to 23% for nonresponders , hcv rna levels generally fall in a biphasic manner . this phase , less influenced by the dosage of ifn and hcv genotype , exhibits a good response to pegifn and is an excellent marker of an svr to the treatment [ 36 , 54 , 74 ] . an rvr at week 4 could predict an svr to ifn - ribavirin with a high degree of accuracy in both hcv-1 and hcv-2 patients , with positive predictive values of 78% and 92% , respectively . recent studies have demonstrated that an rvr is the single best predictor of an svr to pegifn - ribavirin for hcv-1 [ 114 , 115 ] and hcv-2 or hcv-3 patients [ 23 , 55 , 56 , 116 ] . selected patients with rvr might have their treatment courses abbreviated to 16 weeks if they are infected with hcv-2 or hcv-3 [ 23 , 56 ] . in cases without an evr , the non - evr is a significantly negative predictor in hcv-1 patients , but not in hcv-2 patients . thus it is recommended that hcv-1 patients who do not achieve an evr at week 12 should discontinue the therapy beyond 12 weeks [ 22 , 78 ] . recently , stratification of early virological response ( evr ) into complete evr ( cevr ) and partial evr ( pevr ) has been possible to further improve the prediction of an svr and may allow for optimizing treatment duration in non - rvr patients . studies for hcv-1 non - rvr patients have demonstrated that the current recommended 48 weeks of treatment could achieve high svr rates in patients with a cevr but could lower rates of svr in patients with a pevr [ 124 , 125 ] . the svr rates would be more than 90% if patients could reach a cevr with a standard regimen ( 48 weeks for hcv-1 or 24 weeks for hcv-2 ) . in patients without an rvr treated with standard of care , the svr rate would be more than 90% if cevr could be accomplished . in patients with only a pevr , it has been suggested to extend the treatment course to 72 weeks [ 124 , 125 , 127 ] or adhere to high - dose peginterferon plus ribavirin combination therapy . in the future , additional therapy other than interferon - based treatment , such as protease inhibitors , might be anticipated in those difficult - to - treat patients . medical adherence is an important factor associated with response to ifn - ribavirin , especially among patients with hcv-1 infection . in a retrospective analysis of data collected in the large registration trials of ifn - ribavirin , svrs have been reported to be more likely in patients who had taken at least 80% of all projected ifn injections and at least 80% of all projected ribavirin for at least 80% of the anticipated duration of treatment . recent development of direct - acting antiviral agents , also named specifically targeted antiviral therapy for hepatitis c ( stat - c ) compounds , to treat hcv has focused predominantly on inhibitors of the viral enzymes ns3/4a protease and the rna - dependent rna polymerase ns5b [ 129 , 130 ] . a first case of sustained virological response ( svr ) achieved in a patient with chronic hepatitis c by monotherapy with telaprevir without interferon therapy was reported . results of the milestone studies prove 1 and 2 indicate that 12 weeks of telaprevir - based triple therapy is too short because of the high rate of relapse after treatment completion . however , 24 to 48 weeks of total therapy including 12 weeks of triple therapy with telaprevir in addition to standard treatment greatly improved svr rates in treatment - nave genotype 1 patients compared with the standard of care . prove 3 has shown that telaprevir is also highly effective in the treatment of prior nonresponders or relapsers infected with hcv genotype 1 [ 130 , 135 ] .

human immunodeficiency virus type 1 ( hiv-1 ) , the causative agent of acquired immune deficiency syndrome ( aids ) , remains a significant challenge to global health . since its initial identification in 1983 , hiv-1 infection has reached the status of a pandemic . in 2009 alone , there were approximately 2.7 million new infections and about 2.0 million deaths from aids related causes . currently there is no cure for hiv-1 infection . while progression of the disease can be controlled by highly active antiretroviral therapy ( haart ) , a combination of drugs designed to inhibit different stages in the virus life cycle , the complexity of the haart regimen , and the ability of the virus to evolve resistance suggest that alternative drug targets for hiv-1 treatment and prophylaxis are needed . one potentially attractive target for pharmacological interference in the hiv-1 life cycle is the virus requirement for a programmed 1 ribosomal frameshift ( 1 prf ) in order to express its enzymes . ribosomal frameshifting is a recoding mechanism common among viruses with polycistronic ( multiple open reading frames , or orfs , in a single gene ) genomes . it allows viruses to translate polypeptides in different orfs by avoiding the stop codon(s ) present in the single mrna transcript . in hiv-1 , gag , the precusor of the viral structural proteins , is produced via normal translational rules , while pol , the precursor of the viral enzymes , is synthesized as a fused gag - pol polyprotein via 1 prf . this occurs with a frequency of 510% of ribosomes translating the full - length viral mrna . a critical molar ratio of gag - pol to gag protein is required for hiv-1 replication and infectivity ; alterations to this ratio have been shown to be detrimental . 1 prf in hiv-1 is controlled by two cis acting mrna elements : a heptameric slippery sequence ( u uuu uua ) , with the 0 frame indicated by spaces and where the frameshift actually occurs , and a downstream two - stem helix immediately following the slippery sequence , also known as the frameshift stimulatory signal ( hiv-1 fss , figure 1 ) . while several mechanisms have been proposed to account for the frameshift , it is currently hypothesized that this event results from an incomplete translocation for a limited number of ribosomes , due to resistance of the fss to unwinding . modification of the slippery site or stimulatory sequence ( either via natural variation or laboratory mutations ) in ways affecting frameshifting efficiency translates to a decrease in viral replication . these and other results have led several groups to propose 1 prf as a potential target for developing antiretroviral agents for hiv-1 . ( left ) proposed secondary structure of the hiv-1 fss , supported by nmr structural studies . ( right ) htlv-2 fss stem - loop sequence used as a specificity control in this work . note that when the slippery sequence occupies the decoding site of the ribosome , the lower stem is unwound and it is the upper stem - loop that acts as the effective frameshift stimulatory signal . nmr structural analyses indicate that the hiv-1 fss rna consists of a g - c rich upper stem - loop structure , separated from a flexible lower stem by a gga trinucleotide bulge ( figure 1 ) . the bulge produces a roughly 60 bend between the upper and lower stems . this stability is believed to play a vital role in the stimulation of the frameshift , since the ribosome must unwind the stem during translation . the highly structured acaa tetraloop is uncommon among tetraloops but is conserved among all hiv-1 group m subtypes except the uncommon h and j subtypes . likewise , the heptameric slippery sequence is conserved across all hiv-1 group m subtypes . shape analysis of the intact hiv genome suggests a more complex structure for the fss rna , although the upper stem - loop is retained . since other viruses also rely on frameshifting , targeting frameshift - regulating structures may have general utility beyond the context of hiv . for example , human t - cell leukemia virus type 2 ( htlv-2 ) uses two 1 prf events similar to hiv-1 in order to synthesize fused gag - pro and gag - pro - pol precursor proteins . the rna responsible for the 1 prf essential for expression of gag - pro in htlv-2 also consists of two cis - acting rna elements , a heptanucleotide ( aaaaaac ) slippery site and a stem - loop shown in figure 1 . this htlv-2 fss served as a sequence specificity control in the experiments we describe herein . the first attempted use of a synthetic molecule to alter hiv-1 frameshifting and thereby influence viral replication was reported by green and co - workers in 1998 . the authors showed that 1,4-bis[n-(3-n , n - dimethylpropyl)amidino]benzene tetrahydrochloride , a bis guanidinium - containing compound termed , was able to stimulate 1 frameshifting , alter the gag - pol : gag ratio , inhibit hiv-1 replication in cem cells ( a lymphocytic cell line ) , and interfere with the formation of viral particles in chronically infected ch-1 cells ( a cos cell line stably transfected with hiv - gpt , an hiv derivative in which the e. coli gpt gene replaces hiv env(28 ) ) . increases in reverse transcriptase ( rt ) following treatment with 1.5 mm rg501 were also observed , as would be expected for an increase in gag - pol production . recently , the butcher group confirmed that this compound indeed binds the hiv-1 fss rna with weak affinity ( kd 360 m ) , and they carried out nmr structure analysis , indicating that rg501 binds in the major groove of the upper stem - loop . unfortunately , as noted by the authors , rg501 is a relatively nonselective binder and interacts with other rnas . it stimulates frameshifting in viruses with different fss , and likely that also interacts with the ribosome.rg501 is also toxic , a likely result of its lack of selectivity . moreover , its interference with hiv replication begins at concentrations below those observed to affect frameshifting . other compounds such as guanidinoneomycin , idarubicin , and doxorubicin have been shown to bind the hiv-1 fss . doxorubicin was found to bind with a kd of 2.8 m , decreased frameshifting in a rabbit reticulocyte assay , and also significantly reduced overall translation . a screen of an arg - rich peptide library revealed a sequence able to significantly reduce frameshifting , but this displayed no selectivity for the hiv-1 fss relative to other frameshift - stimulating constructs and likely also interacts with the ribosome . thus , as far as we are aware , there are no reported examples of synthetic molecules able to alter hiv-1 frameshifting and interfere with viral infectivity via selective , high - affinity binding to the fss rna . building on our laboratory s longstanding interest in understanding the factors that drive affinity and sequence selectivity in small molecule recognition of rna , we previously reported the use of an 11,325-member resin - bound dynamic combinatorial library ( designed based on the structure of dna - binding , bisintercalating peptide antibiotics ) to identify a compound ( 1 ) able to bind the hiv-1 fss upper stem - loop with moderate affinity ( kd = 4.1 2.4 m immobilized on an surface plasmon resonance ( spr ) chip via one of its amine groups ; kd = 350 110 nm in solution as measured by fluorescence ) and good selectivity . subsequent efforts revealed that replacement of the disulfide bridge with an olefin ( to produce compounds 2 and 3 ) could be accomplished without any reduction in affinity . these studies also indicated that affinity was essentially abolished if the -surface area of the molecule was reduced , while the peptidic portion of 1 was required for sequence - selective binding . while the binding ability of 2 and 3 was intriguing , subsequent preliminary experiments indicated the compounds were unable to inhibit virus in a pseudotyped hiv-1 assay ( data not shown ) . given these results , we hypothesized that further increases in affinity were essential . we anticipated that increasing the surface area of 1 could represent a viable strategy for enhancing the affinity for the hiv-1 fss without significant reductions in selectivity . in particular , incorporation of a benzo[g]quinoline moiety to produce 4 and 5 was viewed as attractive . this hypothesis was supported in part by parallel efforts in our laboratory on the design and synthesis of compounds targeting cug repeat rna in which incorporation of a benzo[g]quinoline was found to enhance affinity and to provide compounds with activity in vivo in a mouse model of type 1 myotonic dystrophy . synthesis of 4 and 5 proceeded via cross - metathesis of half - structure 6 , by analogy to our previous work . we also synthesized 7 and 8 ( one - armed benzo[g]quinoline - bearing structures ) , as well as compounds 9 and 10 , to test the effect of sequential removal of the putative intercalators . compound 11 was synthesized in order to ascertain the effectiveness of the olefin bioisostere in improving cellular availability and bioactivity relative to an easily reduced disulfide . synthesized compounds were first analyzed for binding to the hiv-1 fss by surface plasmon resonance ( spr ) . this technique allows the equilibrium constant ( kd ) and kinetic rate constants ( kon , koff ) to be determined in a label - free format . a 5-biotin labeled hiv-1 fss rna upper stem - loop ( sequence as in figure 1 ) was immobilized on a streptavidin - functionalized sensor chip . compound solutions in hbs buffer ( 0.01 m hepes , 0.150 m nacl , ph = 7.4 ) were flowed over the rna , and the reference - subtracted sensorgrams were recorded . the associative and dissociative phases of the experimental sensorgrams for at least five different concentrations were globally fit to a 1:1 langmuir equation to obtain the binding constants . under these conditions , benzo[g]quinoline - containing compounds 4 and 5 bound the hiv-1 fss with affinities ( kd ) of 89 nm and 102 nm , respectively , in each case representing an approximately 50-fold increased affinity over 2 and 3 ( table 1 ) . binding by benzo[g]quinoline containing monomer 6 was 200-fold weaker relative to that of the dimers 4 and 5 . also resulted in an approximately 200-fold decrease in affinity for 7 and a 30-fold decrease for 8 , while removal of both benzo[g]quinoline groups completely abrogated binding between 10 and the hiv-1 fss rna , consistent with our prior results , indicating the importance of the heterocyclic group for affinity . disulfide linked compound 11 bound the target rna with a kd of 0.741 m , which is 7-fold weaker relative to the olefin analogs 4 and 5 . no binding was observed between the 2-ethyl benzo[g]quinoline carboxylic acid and the hiv-1fss by spr . confirming their anticipated selectivity for the hiv-1 fss , compounds 410 showed no binding to the htlv-2 fss by spr at concentrations up to 3 m ( supporting information p s12 ) . conditions : ( a ) hbs buffer ( 0.01 m hepes , 0.150 m nacl , ph = 7.4 ) ; ( b ) 20 mm hepes , 150 mm nacl , 5 mm mgcl2 , and 0.005% tween-20 . the dissociation phase for spr traces obtained at the highest concentrations for compounds 4 and 5 were not well fit by a single - exponential function . to control for the possibility of compound aggregation interfering with the measurement , we therefore reexamined these compounds using a running buffer containing detergent ( 20 mm hepes , 150 mm nacl , 5 mm mgcl2 , and 0.005% tween-20 ) . this had only a marginal impact on the fitted kinetic parameters ( table 1 ) , suggesting aggregation is not a significant complicating factor in the assay . an spr chip with a very low density of rna provided similar dissociation constants ( supporting information ) . to confirm thermodynamic dissociation constants measured by spr with a fully solution - phase method , and to further assess sequence selectivity , saturable quenching of benzo[g]quinoline fluorescence was observed as a function of added unlabeled rna . dissociation constants obtained in this manner ( table 2 ) for 4 and 5 binding the hiv-1 fss rna are in general agreement with those obtained via spr . by fluorescence we observe a 5-fold selectivity for 4 binding the fss rna vs total yeast trna , while selectivity for fss rna vs fss dna was 4-fold . the selectivity of 5 for fss rna vs trna was somewhat lower ( 2.14-fold ) . curiously , unlike 4 , 5 had no selectivity for fss rna vs fss dna . thus , as 2 and 3 were previously found to have no measurable affinity for trna , we conclude that the increase in affinity obtained on replacement of the quinoline moieties of 2 and 3 with benzo[g]quinoline does come with a cost of some decrease in selectivity . this result is in contrast to what was observed in a related series of compounds optimized for binding triplet repeat rna sequences relevant to type 1 myotonic dystrophy . it is likely that these differences have a structural foundation , and further experiments will be needed to explore this hypothesis . reported error is the standard deviation on each measurement taken in triplicate with 1 min spacing between measurements . before analyzing the effect of compounds on hiv-1 frameshifting in cells , we verified that they were capable of crossing cell membranes , and not toxic at reasonable concentrations . human embryonic kidney cells ( hek 293 ft ) were treated with 4 , 5 , and 11 at concentrations of 50 , 100 , 200 , and 500 m for 12 h. cells were then washed using standard methods to ensure that cell staining was not simply due to surface capture . the inherent fluorescence of the benzo[g]quinoline chromophore in these compounds allowed direct visualization of cell penetration via fluorescence microscopy ( figure 2 shows results for 50 m treatment ; additional images are provided in the supporting information ) , confirming that cells internalized all three compounds . compounds did not appear to localize in any specific subcellular structures but were visible throughout the cytoplasm and nucleus . . bright - field ( gray ) and false - color fluorescence images of compound 4 , 5 , and 11 incubated with hek 293 ft cells at a concentration of 50 m . compounds were incubated with hek 293 ft cells cultured in dmem for 24 h , after which a 10:1 media to wst-1 cell proliferation reagent mixture replaced the growth media for 2 h. absorbance was then measured at 450 and 690 nm . disulfide linked compound 11 proved toxic at concentrations above 10 m , while no significant toxicity for compounds 4 and 5 occurred at concentrations below 60 m ( figure 3 ) . the higher toxicity observed for 11 relative to 4 and 5 is not surprising and likely reflects the lability of the disulfide bond in the reducing environment of the cell . toxicity of hiv-1 fss ligands in hek 293 ft cells , assessed via wst-1 cell proliferation assay . results are shown for exposure to 4 ( blue triangles ) , 5 ( green triangles ) , or 11 ( red circles ) , in comparison to addition of buffer alone ( black squares ) . compounds were next evaluated for their ability to alter hiv-1 fss - dependent frameshifting , using a dual - luciferase reporter assay . in this system , the fss sequence has been inserted between renilla ( rluc ) and firefly ( fluc ) luciferase genes . several constructs were employed . in the pdualhiv(1 ) construct , fluc is in the 1 reading frame relative to rluc , while , in the pdualhiv(0 ) construct , fluc is in the 0 reading frame relative to rluc . in both cases , fluc is expressed only as a fused rluc - fluc protein , but in the case of pdualhiv(1 ) , a 1 frameshift is required . to ascertain if compound - dependent effects on frameshifting are specific to hiv-1 , we also carried out an analogous 1 prf assay using constructs in which the htlv-2 frameshift site was inserted between rluc and fluc genes , such that fluc is only synthesized by a 1prf event [ pdualhtlv-2 ( 1 ) ] . hek 293 ft cells were transiently transfected separately with pdualhiv(1 ) and pdualhiv(0 ) plasmids . the frameshift efficiency ( defined as the ratio of firefly to renilla luciferase activities ) in the pdualhiv(1 ) transfected cells was measured in the presence of varying concentrations of compounds ( 050 m ) . a statistically significant ( p < 0.005 ) dose - dependent increase in fluc to rluc ratios , > 50% at 50 m , was observed following treatment with 4 or 5 ( figure 4 ) . control compound 10 , which showed no measurable binding to the hiv-1 fss by spr , had no significant effect on 1 prf in pdualhiv(1 ) transfected cells under similar conditions . in hek 293 ft cells transfected with the pdualhiv(0 ) plasmids , the fused renilla - firefly luciferase protein is synthesized by conventional translation rules , i.e. without ribosomal frameshifting . therefore , to confirm that the observed effect in pdualhiv(1 ) cells is a direct effect of compound on frameshift - dependent translation , pdualhiv(0 ) transfected cells were treated analogously with compounds 4 and 5 . no change in fluc to rluc ratio relative to untreated pdualhiv(0 ) transfected hek 293 ft cells was observed , suggesting that 4 and 5 specifically stimulate 1 frameshift translation in cells . disulfide linked 11 stimulated hiv-1 frameshifting by approximately 25% ( supporting information ) ; this lower effect compared to the cases of 4 and 5 is consistent with its lower affinity to the hiv-1 fss and likely lability in the cell . treatment of hek 293 ft transfected with the pdualhtlv-2 ( 1 ) construct with compounds 4 , 5 , or 11 resulted in no change in fluc / rluc ratio compared to the case of untreated cells . this is consistent with in vitro spr results in which no binding was observed between these compounds and the htlv-2 fss rna . compounds 4 and 5 increase frameshifting ( > 50% ) in a dual - luciferase assay incorporating the hiv-1 fss but have no effect on frameshifting in analogous assay incorporating the htlv-2 fss . relative frameshift efficiency in hek 293 ft cells treated with 4 , 5 or control peptide 10 after transfection with pdualhiv ( 1 ) or pdualhtlv-2 ( 1 ) . the frameshift efficiency was calculated as the ratio of fluc to rluc in pdualhiv(1 ) or pdualhtlv-2 ( 1 ) transiently transfected cells . this ratio was arbitrarily set to 100% for plasmid - transfected cells , but not exposed to compounds . error is sem on three replicates for each concentration . in order for the observed 1 frameshift effect of compounds to be significant , it was important that it correlate to an equivalent reduction in viral replication . to assess this , we analyzed the effect of compounds in a single - round infection with pseudotyped hiv in hek 293 t producer cells and tzm - bl target cells ( a hela cell line ) . the wild type hiv proviral vector ( pdhiv3-gfp ) codes for all hiv-1nl43 genes except nef ( which is replaced with gfp ) and env , thus preserving gag and pol , and the frameshift required for production of the gag - pol polyprotein . however , we observed a statistically significant ( p < 0.001 ) and concentration - dependent decrease in infectivity of the pseudotyped hiv-1 virions when producer cells were treated with 4 and 5 ( figure 5 ) . the decrease in infectivity was > 90% at a concentration of 20 m 4 , and > 90% at a concentration of 40 m for 5 . the core peptide alone ( 10 ) showed a modest decrease in infectivity at a concentration of 40 m , likely via nonspecific effects , given its lack of affinity for the fss . the protease inhibitor indinavir was tested in parallel to calibrate the assay ; its ec50 was found to be 14.8 nm , in line with previously reported values . treatment of viral producer cells with compounds 4 and 5 yields a strong inhibition of the infectivity of pseudotyped hiv-1 virions into tzm - bl reporter cells . infectivity is measured as relative luminescence from the stably expressed firefly luciferase expressed via the hiv ltr promoter in the tzm - bl cell line . error bars indicate standard deviation on the mean ( n = 3 ) . while virus titer was determined in these experiments using an elisa assay to normalize viral load into target cells , a compound - dependent decrease in viral production was also readily observable via fluorescence microscopy , as the pseudotyped hiv carries gfp as a marker ( figure 6 ) . phase - contrast images of these cells showed no morphological changes , consistent with wst-1 results ( supporting information ) . the concentration range required for a decrease in viral infectivity by 4 and 5 was similar to the range at which a significant increase in 1 frameshift was observed in hek 293 ft cells , supporting the hypothesis that these compounds exert their antiviral activity primarily by altering 1 prf . in order to provide further support for this hypothesis , viral particles were isolated from supernatants from these experiments by spinning through a 20% sucrose cushion , and probed via western blot for the presence of reverse transcriptase ( rt ) . as rt is produced only as part of the gag - pol fusion , increasing amounts of rt relative to capsid protein p24 ( a structural protein produced as part of gag ) would be required given an increase in frameshifting . indeed , that is what is observed as a function of added 4 or 5 ( figure 7 ) . the amount of rt in unprocessed gag - pol ( p160 ) relative to mature rt ( p66 + p51 ) also increased significantly ; this may indicate that treatment with compounds 4 and 5 also inhibits pol processing . however , the pattern of p160 cleavage intermediates observed on treatment with indinavir is significantly different , indicating that 4 and 5 likely do not directly interact with viral protease ( supporting information p s19 ) . compound - dependent changes in viral production may be observed directly via fluorescence microscopy . proviral expression of gfp is reduced in the presence of compounds 4 and 5 compared to 10 and the untreated control ( 40 mm concentration shown ) . images were acquired 24 h after treatment prior to harvesting viral particles for tzm - bl single - cycle infectivity . equal loads of viral particles ( measured by p24 elisa ) were isolated from media of viral producer cells by spinning through a 20% sucrose cushion at 100,000 g . the viral particles were western blotted for rt ( abcam ) and p24 ( nih aids reagent program , cat # 3537 ) . densitometry of nonsaturated bands was used to calculate ratios for p160/(p66+p51 ) ( listed below the rt blot ) and the ratio of total ( all bands ) rt / p24 are ( listed below the p24 blot ) . designing small molecules that target specific rna sequences and elicit a desired rna mediated biological response constitutes one of the signature challenges in chemical biology . in this paper , we have demonstrated that a moderate - affinity hit compound for the hiv-1 fss rna , obtained from a resin - bound dynamic combinatorial library screen , can be transformed into high - affinity binders . an intriguing observation from our initial screen was the finding that a symmetrical compound was selected , despite the target rna being nonsymmetrical . this work confirms that both putative intercalators are required for high - affinity binding , as compounds 7 and 8 had only weak affinity for the fss . structural analysis will be essential to fully understand the binding mode of 4 and 5 , but the recognition of a nonsymmetrical rna binding site by a symmetrical , dimeric molecule is not unprecedented ; for example , arya and colleagues have described the use of neomycin dimers for recognition of hiv tar rna , and the hergenrother group designed deoxystreptamine dimers that bound nonsymmetrical rna loops . likewise , structural information should prove useful for understanding the significantly higher selectivity of 4 for fss rna over fss dna and trna relative to 5 . these enhanced compounds are able to alter frameshifting in a dual luciferase assay in hek 293 ft cells and strongly inhibit viral infectivity in a pseudotyped hiv assay . relative to previously studied compounds targeting frameshifting in hiv , compounds 4 and 5 produce a roughly equivalent increase in rt at a 37.5-fold lower concentration than rg501 . as such , compounds 4 and 5 represent promising leads for further study , as well as interesting tools to further investigate the mechanism of 1 prf . since the pseudotyped hiv used in this assay is only capable of one round of replication , we anticipate that stronger effects will be observed with wild - type hiv in a spreading infection assay . of course , further enhancements in affinity and selectivity would likely improve activity as well . as discussed above , previous high - throughput screening efforts targeting frameshift - modulating compounds in which bicistronic reporters provided the assay readout largely yielded compounds acting on the ribosome . given this observation , it has been suggested by others that assays focused on direct binding to the fss rna could be advantageous , as such compounds could potentially increase frameshifting by inhibiting unwinding of the upper stem - loop . the results presented herein support this hypothesis . in the context of hiv biology , our results with compounds 4 and 5 indicate that an increase in frameshifting and a concomitant increase in the gag - pol / gag ratio decreases viral fitness . this is consistent with the work of mak and colleagues , who increased the gag - pol / gag ratio via cotransfection . commercially available reagents were obtained from sigma - aldrich chemical co. ( st . louis , mo ) , tci america ( portland , or ) , fisher scientific , emd chemicals ( gibbstown , nj ) , advanced chemtech ( louisville , ky ) , and alfa aesar , and were used without further purification unless otherwise noted . water used for reactions and aqueous workup was glass - distilled from a deionized water feed . reaction progress was monitored by analytical thin - layer chromatography ( tlc ) using em silica gel 60 f-254 precoated glass plates ( 0.25 mm ) . compounds were visualized on the tlc plates with a uv lamp ( dual wavelength ; = 254 nm , = 360 nm ) . synthesized compounds were purified using flash column chromatography on em silica gel 60 ( 230400 ) mesh or alternatively via preparative reversed phase hplc . cells were cultured in dulbecco s modified eagle s medium ( dmem ) , supplemented with 10% fbs and 1% penicillin1% streptomycin . premixed wst-1 cell proliferation reagent was purchased from clontech , and luminescence assays were carried out using a promega dual - luciferase assay kit following manufacture s instructions . h nmr spectra were recorded at 25 c on either a bruker avance 400 ( 400 mhz ) or bruker avance 500 ( 500 mhz ) instrument and processed using mestrenova nmr processing software . chemical shifts ( ) are reported in parts per million ( ppm ) downfield from tetramethylsilane and referenced to the residual protium signal in the nmr solvents . data are reported as follows : chemical shift , multiplicity ( s = singlet , d = doublet , t = triplet , m = multiplet , and q = quartet ) , coupling constant ( j ) in hertz ( hz ) , and integration . c spectra were recorded at 25 c on a bruker avance 500 instrument operating at 126 mhz . chemical shifts ( ) are reported in ppm downfield from tetramethylsilane and referenced ( except in d2o ) to the primary carbon resonance in the nmr solvent . high - resolution mass spectra ( hrms ) were acquired at the university of buffalo chemistry department mass spectrometry facility , buffalo , ny . compounds were synthesized following procedures analogous to those previously reported . all compounds were produced in > 95% purity , as determined by analytical hplc . briefly , 4 and 5 were synthesized from olefin precursor monomer 6 , which was assembled on wang resin by standard fmoc solid phase peptide synthesis ( spps ) methods . one half of the resin - bound monomer was cleaved using 50% tfa in dcm with 1% tes , and was used as solution phase partner in an olefin metathesis reaction employing grubbs second generation catalyst . the olefin products ( isomer ratio of z / e = 2:3 ) were isolated using reverse phase hplc on a c18 column ( waters , xbridge prep c18 5 m obd , 19 mm 250 mm ) using gradient elution from 5 to 100% acetonitrile/0.1% tfa in water/0.1% tfa . olefin geometries were assigned by comparing the olefin proton chemical shifts of the e - olefin downfield to the z - isomer and also by the infrared spectra of the compounds as described previously . compounds 7 and 8 were synthesized by a slight modification to the procedures described above . the tripeptide ( phe - pro - allylgly ) was first assembled using fmoc peptide coupling chemistry on wang resin . 2-ethyl benzo[g]quinoline carboxylic acid was coupled to half the material and cleaved with tfa . this was then employed as the solution phase component in olefin cross - metathesis with the remaining bead - bound portion of fmoc - protected tripeptide . surface plasmon resonance ( spr ) experiments were performed on a biacore - x instrument ( biacore , inc . , uppsala , sweden ) on a cm5 sensor chip . approximately 2000 ru of streptavidin ( rockland immunochemicals ) was immobilized in both flow cells using edc / nhs chemistry . 5-biotinylated - rna ( either 500 nm 5-biotin - hiv-1 fss or 500 nm 5-biotin - htlv-2 fss rna sequences , obtained commercially from integrated dna technologies inc . ) was captured onto the streptavidin surface in one flow cell to a density of approximately 3001200 ru . the streptavidin surface in the second flow cell was then blocked with biotin solution and served as a reference cell to correct for any nonspecific binding . binding constant measurements were carried out for each compound once on a low density ( approximately 300 ru ) chip and once on a high - density ( approximately 1200 ru ) chip in order to ensure binding was not influenced by rna density . kinetic binding experiments were carried out by flowing various concentrations of compound in ( a ) hbs buffer ( 0.01 m hepes , 0.150 m nacl , ph = 7.4 ) at a 60 l per min flow rate or ( b ) 20 mm hepes , 150 mm nacl , 5 mm mgcl2 , and 0.005% tween-20 at a 50 l per min flow rate over the captured rna sequence . where necessary , a 20 s , 0.5 or 1 m aqueous nacl injection was sufficient for regeneration ( compounds displaying fast off - rates did not require regeneration of the chip between injections ) . binding constants were obtained by global fit ( conditions ( a ) ) of association and dissociation phases of at least five referenced - subtracted and blank - corrected sensorgrams to a 1:1 langmuir binding equation , or via individual fits ( conditions ( b ) using biaevaluation software ) . fluorescence titrations were carried out in 20 mm hepes , 150 mm nacl , using a cary eclipse spectrofluorometer . all titrations started at a volume of 500 l with the compound at 1 m . rna was then titrated in from a high - concentration stock ( 10 m for hiv-1 fss rna ; 40 m for hiv-1 fss dna and trna ) in 110 l increments . after rna was added , the solution was thoroughly mixed in the cuvette via pipetting and allowed to stand for 10 min to reach equilibrium . each measurement was taken three times with a 1 min waiting period between scans to confirm equilibrium was reached . hek 293 ft cells grown to 80% confluence at 37 c and 5% co2 were exposed to compounds at a concentration of 50 m for 12 h in a 96-well tissue culture plate . after removal of the culture media [ dmem containing 10% fbs , 1% penicillin - streptomycin ( gibco ) ] , the cells were washed twice with pbs to remove excess and surface - bound compounds . cells were then imaged while in buffer under a fluorescence microscope ( olympus ix70 ) in the 96-well plate using 358-nm excitation and 460-nm emission filters . hek 293 ft cells were plated in a 96-well tissue culture plate in dmem ( 10% fetal bovine serum , 1% penicillin - streptomycin ) and allowed to grow to 80% confluence at 37 c under co2 . varying compound concentrations ( up to 0.5 mm ) and control ( identical volumes of sterile h2o ) in triplicate were incubated with cells for 24 h at 37 c . ten microliters of premix wst-1 cell proliferation reagent ( clontech ) was added to each well including blanks ( dmem ) , followed by a 2 h incubation at 37 c . hek 293 ft cells were plated in 96-well plates at densities of 1.5 10 cells / well 6 h before transfection in dmem ( 10% fetal bovine serum , 1% penicillin - streptomycin ) . cells were transiently transfected in separate wells with 0.2 g of plasmid dna ( pdualhiv(0 ) , pdualhiv(1 ) , pdualhtlv-2(0 ) , or pdualhtlv-2(1 ) ) , using lipofectamine 2000 transfection reagent ( invitrogen ) and following the manufacturer s protocol . five hours after transfection , various compound concentrations ( 050 m ) were added directly to the cells in triplicate and incubated for 36 h at 37 c . the culture media was gently aspirated , washed with 1 pbs , and lysed with 200 l 1 passive lysis buffer . rluc and fluc activities were measured with 5 l of cell lysate and 25 l of luciferase reagent using the dual - luciferase assay system ( promega ) in the same wells . fluc and rluc luminescence values were measured on a modulus microplate reader ( turner biosystems ) . relative frameshift efficiencies were calculated by comparing the fluc / rluc luminescence ratio of cells treated with compounds to untreated cells . the antiviral activity of 4 , 5 , and 10 was measured by single - round infectivity assay with pseudotyped hiv-1 using hek293 t producer cells . the hiv-1 proviral vector ( pdhiv3-gfp ) codes for all hiv-1nl43 genes except nef ( replaced with gfp ) and env , thus preserving gag and pol , and the frameshift required for production of the gag - pol polyprotein . a single - round infectivity assay was conducted by transient transfection of the viral vector with vsv - g coat protein vector at a ratio of 1:0.5 using fugene hd ( promega ) . the virus producer cells were dosed with compounds four hours after transfection , and viral particles were harvested from the media 24 h after transfecting by filtering through a 0.45-m syringe filter . the infections were performed using tzm - bl reporter cells that contain stably integrated firefly luciferase that is driven by the hiv - ltr promoter . triplicate infections in 96-well plates at 10,000 cells / well with 500 pg p24/well proceeded for 48 h before the addition of steadyglo reagent ( promega ) to each well for 30 min . luminescence was measured as a quantitative metric for changes in viral infectivity in the presence of compound .

the life cycle of the human immunodeficiency virus type 1 ( hiv-1 ) has an absolute requirement for ribosomal frameshifting during protein translation in order to produce the polyprotein precursor of the viral enzymes . while an rna stem - loop structure ( the hiv-1 frameshift stimulating signal , or hiv-1 fss ) controls the frameshift efficiency and has been hypothesized as an attractive therapeutic target , developing compounds that selectively bind this rna and interfere with hiv-1 replication has proven challenging . building on our prior discovery of a hit molecule able to bind this stem - loop , we now report the development of compounds displaying high affinity for the hiv-1 fss . these compounds are able to enhance frameshifting more than 50% in a dual - luciferase assay in human embryonic kidney cells , and they strongly inhibit the infectivity of pseudotyped hiv-1 virions .

Introduction Results and Discussion Discussion and Conclusions Experimental Section

human immunodeficiency virus type 1 ( hiv-1 ) , the causative agent of acquired immune deficiency syndrome ( aids ) , remains a significant challenge to global health . while progression of the disease can be controlled by highly active antiretroviral therapy ( haart ) , a combination of drugs designed to inhibit different stages in the virus life cycle , the complexity of the haart regimen , and the ability of the virus to evolve resistance suggest that alternative drug targets for hiv-1 treatment and prophylaxis are needed . one potentially attractive target for pharmacological interference in the hiv-1 life cycle is the virus requirement for a programmed 1 ribosomal frameshift ( 1 prf ) in order to express its enzymes . ribosomal frameshifting is a recoding mechanism common among viruses with polycistronic ( multiple open reading frames , or orfs , in a single gene ) genomes . in hiv-1 , gag , the precusor of the viral structural proteins , is produced via normal translational rules , while pol , the precursor of the viral enzymes , is synthesized as a fused gag - pol polyprotein via 1 prf . 1 prf in hiv-1 is controlled by two cis acting mrna elements : a heptameric slippery sequence ( u uuu uua ) , with the 0 frame indicated by spaces and where the frameshift actually occurs , and a downstream two - stem helix immediately following the slippery sequence , also known as the frameshift stimulatory signal ( hiv-1 fss , figure 1 ) . while several mechanisms have been proposed to account for the frameshift , it is currently hypothesized that this event results from an incomplete translocation for a limited number of ribosomes , due to resistance of the fss to unwinding . ( left ) proposed secondary structure of the hiv-1 fss , supported by nmr structural studies . ( right ) htlv-2 fss stem - loop sequence used as a specificity control in this work . note that when the slippery sequence occupies the decoding site of the ribosome , the lower stem is unwound and it is the upper stem - loop that acts as the effective frameshift stimulatory signal . nmr structural analyses indicate that the hiv-1 fss rna consists of a g - c rich upper stem - loop structure , separated from a flexible lower stem by a gga trinucleotide bulge ( figure 1 ) . this stability is believed to play a vital role in the stimulation of the frameshift , since the ribosome must unwind the stem during translation . shape analysis of the intact hiv genome suggests a more complex structure for the fss rna , although the upper stem - loop is retained . for example , human t - cell leukemia virus type 2 ( htlv-2 ) uses two 1 prf events similar to hiv-1 in order to synthesize fused gag - pro and gag - pro - pol precursor proteins . the rna responsible for the 1 prf essential for expression of gag - pro in htlv-2 also consists of two cis - acting rna elements , a heptanucleotide ( aaaaaac ) slippery site and a stem - loop shown in figure 1 . the authors showed that 1,4-bis[n-(3-n , n - dimethylpropyl)amidino]benzene tetrahydrochloride , a bis guanidinium - containing compound termed , was able to stimulate 1 frameshifting , alter the gag - pol : gag ratio , inhibit hiv-1 replication in cem cells ( a lymphocytic cell line ) , and interfere with the formation of viral particles in chronically infected ch-1 cells ( a cos cell line stably transfected with hiv - gpt , an hiv derivative in which the e. coli gpt gene replaces hiv env(28 ) ) . recently , the butcher group confirmed that this compound indeed binds the hiv-1 fss rna with weak affinity ( kd 360 m ) , and they carried out nmr structure analysis , indicating that rg501 binds in the major groove of the upper stem - loop . it stimulates frameshifting in viruses with different fss , and likely that also interacts with the ribosome.rg501 is also toxic , a likely result of its lack of selectivity . other compounds such as guanidinoneomycin , idarubicin , and doxorubicin have been shown to bind the hiv-1 fss . doxorubicin was found to bind with a kd of 2.8 m , decreased frameshifting in a rabbit reticulocyte assay , and also significantly reduced overall translation . a screen of an arg - rich peptide library revealed a sequence able to significantly reduce frameshifting , but this displayed no selectivity for the hiv-1 fss relative to other frameshift - stimulating constructs and likely also interacts with the ribosome . thus , as far as we are aware , there are no reported examples of synthetic molecules able to alter hiv-1 frameshifting and interfere with viral infectivity via selective , high - affinity binding to the fss rna . building on our laboratory s longstanding interest in understanding the factors that drive affinity and sequence selectivity in small molecule recognition of rna , we previously reported the use of an 11,325-member resin - bound dynamic combinatorial library ( designed based on the structure of dna - binding , bisintercalating peptide antibiotics ) to identify a compound ( 1 ) able to bind the hiv-1 fss upper stem - loop with moderate affinity ( kd = 4.1 2.4 m immobilized on an surface plasmon resonance ( spr ) chip via one of its amine groups ; kd = 350 110 nm in solution as measured by fluorescence ) and good selectivity . subsequent efforts revealed that replacement of the disulfide bridge with an olefin ( to produce compounds 2 and 3 ) could be accomplished without any reduction in affinity . while the binding ability of 2 and 3 was intriguing , subsequent preliminary experiments indicated the compounds were unable to inhibit virus in a pseudotyped hiv-1 assay ( data not shown ) . we anticipated that increasing the surface area of 1 could represent a viable strategy for enhancing the affinity for the hiv-1 fss without significant reductions in selectivity . in particular , incorporation of a benzo[g]quinoline moiety to produce 4 and 5 was viewed as attractive . this hypothesis was supported in part by parallel efforts in our laboratory on the design and synthesis of compounds targeting cug repeat rna in which incorporation of a benzo[g]quinoline was found to enhance affinity and to provide compounds with activity in vivo in a mouse model of type 1 myotonic dystrophy . compound 11 was synthesized in order to ascertain the effectiveness of the olefin bioisostere in improving cellular availability and bioactivity relative to an easily reduced disulfide . synthesized compounds were first analyzed for binding to the hiv-1 fss by surface plasmon resonance ( spr ) . a 5-biotin labeled hiv-1 fss rna upper stem - loop ( sequence as in figure 1 ) was immobilized on a streptavidin - functionalized sensor chip . under these conditions , benzo[g]quinoline - containing compounds 4 and 5 bound the hiv-1 fss with affinities ( kd ) of 89 nm and 102 nm , respectively , in each case representing an approximately 50-fold increased affinity over 2 and 3 ( table 1 ) . also resulted in an approximately 200-fold decrease in affinity for 7 and a 30-fold decrease for 8 , while removal of both benzo[g]quinoline groups completely abrogated binding between 10 and the hiv-1 fss rna , consistent with our prior results , indicating the importance of the heterocyclic group for affinity . confirming their anticipated selectivity for the hiv-1 fss , compounds 410 showed no binding to the htlv-2 fss by spr at concentrations up to 3 m ( supporting information p s12 ) . to control for the possibility of compound aggregation interfering with the measurement , we therefore reexamined these compounds using a running buffer containing detergent ( 20 mm hepes , 150 mm nacl , 5 mm mgcl2 , and 0.005% tween-20 ) . dissociation constants obtained in this manner ( table 2 ) for 4 and 5 binding the hiv-1 fss rna are in general agreement with those obtained via spr . thus , as 2 and 3 were previously found to have no measurable affinity for trna , we conclude that the increase in affinity obtained on replacement of the quinoline moieties of 2 and 3 with benzo[g]quinoline does come with a cost of some decrease in selectivity . this result is in contrast to what was observed in a related series of compounds optimized for binding triplet repeat rna sequences relevant to type 1 myotonic dystrophy . before analyzing the effect of compounds on hiv-1 frameshifting in cells , we verified that they were capable of crossing cell membranes , and not toxic at reasonable concentrations . human embryonic kidney cells ( hek 293 ft ) were treated with 4 , 5 , and 11 at concentrations of 50 , 100 , 200 , and 500 m for 12 h. cells were then washed using standard methods to ensure that cell staining was not simply due to surface capture . the inherent fluorescence of the benzo[g]quinoline chromophore in these compounds allowed direct visualization of cell penetration via fluorescence microscopy ( figure 2 shows results for 50 m treatment ; additional images are provided in the supporting information ) , confirming that cells internalized all three compounds . bright - field ( gray ) and false - color fluorescence images of compound 4 , 5 , and 11 incubated with hek 293 ft cells at a concentration of 50 m . toxicity of hiv-1 fss ligands in hek 293 ft cells , assessed via wst-1 cell proliferation assay . compounds were next evaluated for their ability to alter hiv-1 fss - dependent frameshifting , using a dual - luciferase reporter assay . the frameshift efficiency ( defined as the ratio of firefly to renilla luciferase activities ) in the pdualhiv(1 ) transfected cells was measured in the presence of varying concentrations of compounds ( 050 m ) . control compound 10 , which showed no measurable binding to the hiv-1 fss by spr , had no significant effect on 1 prf in pdualhiv(1 ) transfected cells under similar conditions . no change in fluc to rluc ratio relative to untreated pdualhiv(0 ) transfected hek 293 ft cells was observed , suggesting that 4 and 5 specifically stimulate 1 frameshift translation in cells . disulfide linked 11 stimulated hiv-1 frameshifting by approximately 25% ( supporting information ) ; this lower effect compared to the cases of 4 and 5 is consistent with its lower affinity to the hiv-1 fss and likely lability in the cell . treatment of hek 293 ft transfected with the pdualhtlv-2 ( 1 ) construct with compounds 4 , 5 , or 11 resulted in no change in fluc / rluc ratio compared to the case of untreated cells . compounds 4 and 5 increase frameshifting ( > 50% ) in a dual - luciferase assay incorporating the hiv-1 fss but have no effect on frameshifting in analogous assay incorporating the htlv-2 fss . the frameshift efficiency was calculated as the ratio of fluc to rluc in pdualhiv(1 ) or pdualhtlv-2 ( 1 ) transiently transfected cells . in order for the observed 1 frameshift effect of compounds to be significant , it was important that it correlate to an equivalent reduction in viral replication . to assess this , we analyzed the effect of compounds in a single - round infection with pseudotyped hiv in hek 293 t producer cells and tzm - bl target cells ( a hela cell line ) . the wild type hiv proviral vector ( pdhiv3-gfp ) codes for all hiv-1nl43 genes except nef ( which is replaced with gfp ) and env , thus preserving gag and pol , and the frameshift required for production of the gag - pol polyprotein . however , we observed a statistically significant ( p < 0.001 ) and concentration - dependent decrease in infectivity of the pseudotyped hiv-1 virions when producer cells were treated with 4 and 5 ( figure 5 ) . the core peptide alone ( 10 ) showed a modest decrease in infectivity at a concentration of 40 m , likely via nonspecific effects , given its lack of affinity for the fss . treatment of viral producer cells with compounds 4 and 5 yields a strong inhibition of the infectivity of pseudotyped hiv-1 virions into tzm - bl reporter cells . the concentration range required for a decrease in viral infectivity by 4 and 5 was similar to the range at which a significant increase in 1 frameshift was observed in hek 293 ft cells , supporting the hypothesis that these compounds exert their antiviral activity primarily by altering 1 prf . in order to provide further support for this hypothesis , viral particles were isolated from supernatants from these experiments by spinning through a 20% sucrose cushion , and probed via western blot for the presence of reverse transcriptase ( rt ) . in this paper , we have demonstrated that a moderate - affinity hit compound for the hiv-1 fss rna , obtained from a resin - bound dynamic combinatorial library screen , can be transformed into high - affinity binders . this work confirms that both putative intercalators are required for high - affinity binding , as compounds 7 and 8 had only weak affinity for the fss . structural analysis will be essential to fully understand the binding mode of 4 and 5 , but the recognition of a nonsymmetrical rna binding site by a symmetrical , dimeric molecule is not unprecedented ; for example , arya and colleagues have described the use of neomycin dimers for recognition of hiv tar rna , and the hergenrother group designed deoxystreptamine dimers that bound nonsymmetrical rna loops . these enhanced compounds are able to alter frameshifting in a dual luciferase assay in hek 293 ft cells and strongly inhibit viral infectivity in a pseudotyped hiv assay . since the pseudotyped hiv used in this assay is only capable of one round of replication , we anticipate that stronger effects will be observed with wild - type hiv in a spreading infection assay . given this observation , it has been suggested by others that assays focused on direct binding to the fss rna could be advantageous , as such compounds could potentially increase frameshifting by inhibiting unwinding of the upper stem - loop . louis , mo ) , tci america ( portland , or ) , fisher scientific , emd chemicals ( gibbstown , nj ) , advanced chemtech ( louisville , ky ) , and alfa aesar , and were used without further purification unless otherwise noted . premixed wst-1 cell proliferation reagent was purchased from clontech , and luminescence assays were carried out using a promega dual - luciferase assay kit following manufacture s instructions . one half of the resin - bound monomer was cleaved using 50% tfa in dcm with 1% tes , and was used as solution phase partner in an olefin metathesis reaction employing grubbs second generation catalyst . binding constant measurements were carried out for each compound once on a low density ( approximately 300 ru ) chip and once on a high - density ( approximately 1200 ru ) chip in order to ensure binding was not influenced by rna density . where necessary , a 20 s , 0.5 or 1 m aqueous nacl injection was sufficient for regeneration ( compounds displaying fast off - rates did not require regeneration of the chip between injections ) . cells were transiently transfected in separate wells with 0.2 g of plasmid dna ( pdualhiv(0 ) , pdualhiv(1 ) , pdualhtlv-2(0 ) , or pdualhtlv-2(1 ) ) , using lipofectamine 2000 transfection reagent ( invitrogen ) and following the manufacturer s protocol . rluc and fluc activities were measured with 5 l of cell lysate and 25 l of luciferase reagent using the dual - luciferase assay system ( promega ) in the same wells . the antiviral activity of 4 , 5 , and 10 was measured by single - round infectivity assay with pseudotyped hiv-1 using hek293 t producer cells . the hiv-1 proviral vector ( pdhiv3-gfp ) codes for all hiv-1nl43 genes except nef ( replaced with gfp ) and env , thus preserving gag and pol , and the frameshift required for production of the gag - pol polyprotein . a single - round infectivity assay was conducted by transient transfection of the viral vector with vsv - g coat protein vector at a ratio of 1:0.5 using fugene hd ( promega ) .

search cvd is an ancillary study to the search for diabetes in youth study , conducted in colorado and ohio . search is a multicenter study that conducts population - based ascertainment of nongestational cases of physician - diagnosed diabetes in youth aged < 20 years at diagnosis ( 13 ) . a total of 406 youth who had a physician diagnosis of type 1 diabetes were registered with search , were residents of colorado or ohio , were aged 1126 years during 20092011 , and had duration of diabetes of at least 5 years were enrolled in the search cvd study . during the same time period , a total of 204 frequency - matched ( by age , sex , and race / ethnicity ) youth without diabetes ( control subjects ) were also recruited in the study . because all search cases arose from health care provider offices , we recruited control youth from primary care offices in the same geographic areas . within clinical sites , control recruitment sampled youth based on the distribution of age , sex , and racial / ethnic background of case subjects . for defined periods of time , participating primary care offices provided an initial study brochure , and patients and their parent or guardian were asked to complete a one - page information form and an indication of permission for study staff to contact them regarding participation in the study . the study staff contacted those interested in learning more about the study and recruited participants accordingly . control participants were confirmed to be nondiabetic based on fasting glucose levels < 126 mg / dl ( 14 ) . the study was reviewed and approved by the local institutional review boards that had jurisdiction over the local study population , and all participants provided signed informed consent or assent . youth with diabetes were asked to withhold their diabetes medications , including short - acting insulin , on the morning of the visit until after the blood draw was complete . all participants were asked to refrain from any strenuous exercise , smoking , or caffeinated drinks 12 h prior to the visit . race / ethnicity was categorized as non - hispanic white ( nhw ) and race / ethnicity other than nhw , including hispanic , african american , and asian / pacific islander racial / ethnic groups . participants completed standardized questionnaires including medical history , medication inventory , smoking status , physical activity , daily insulin dose , family history of diabetes , and cvd . cigarette smoking was defined as having smoked cigarettes on 1 of the 30 days preceding the survey . participants were asked the average number of days in a typical week that they participated in physical activity for at least 20 min that made them sweat or breathe hard and were then categorized as physically inactive ( 02 days / week ) or physically active ( 37 days / week ) ( 15 ) . height was measured in centimeters using a stadiometer and weight in kilograms using a standardized weighing machine . bmi was calculated as weight in kilograms divided by the square of height in meters , and age- and sex - specific bmi z scores were derived based on the centers for disease control and prevention national standards ( 16 ) . waist circumference was measured to the nearest 0.1 cm with the national health and nutrition examination survey ( nhanes ) protocol ( 17 ) . resting systolic blood pressure ( sbp ) and diastolic blood pressure ( dbp ) were measured three times , using aneroid sphygmomanometer , while the subjects were seated for at least 5 min , and the average of the three measurements was taken . laboratory samples were obtained under conditions of metabolic stability , defined , for subjects with type 1 diabetes , as no episodes of diabetic ketoacidosis during the previous month . a fasting blood draw was conducted for the assessment of the metabolic parameters ( a1c , ldl cholesterol , hdl , and triglyceride levels ) . blood specimens were processed locally and shipped to a central laboratory ( northwest lipid metabolism and diabetes research laboratories , seattle , wa ) . urinary albumin was measured from overnight timed urine samples by radioimmunoassay and was expressed as albumin excretion rate ( aer ) . aer between 20 and 200 g / min was defined as microalbuminuria , while 200 g / min was defined as macroalbuminuria . high - performance liquid chromatography ( tosoh bioscience , san francisco , ca ) was used to measure a1c . measurements of triglyceride and hdl cholesterol were performed enzymatically on a hitachi 917 autoanalyzer ( roche molecular biochemicals diagnostics , indianapolis , in ) . ldl cholesterol was calculated by the friedewald equation for individuals with triglyceride concentration < 400 mg / dl and the beta quantification procedure for those with triglycerides of 400 mg / dl . all measurements of hrv were conducted in the morning between 7 and 11 a.m. in a room with a stable room temperature while the participant was lying in the resting supine position for 10 min and breathing at a normal pace , using the sphygmocor device ( atcor medical ) . the device takes into account the normal heart beats , ignoring the ectopic beats , to derive the statistical parameters of the normal r - r intervals ( nn intervals ) of the ecg and computes several time and frequency domain hrv indices . the time domain indices of hrv used in these analyses were the sd of the nn intervals ( sdnn ) and the root mean square differences of successive nn intervals ( rmssd ) . the frequency domain indices of hrv that were measured were the normalized hf ( high frequency ) power , lf ( low frequency ) power , and the lf - to - hf ratio . the sphgmocor device derives the normalized lf and hf power by expressing them as a fraction of the total power [ lf n.u . = lf/(total power [ tp ] very low frequency [ vlf ] ) 100 and hf n.u . sdnn is a measure of overall hrv , so lower sdnn levels indicate reduced overall hrv ( 18 ) . rmssd and hf power represent the parasympathetic component of the hrv ( 19 ) , and thus parasympathetic loss is quantified by the reduction in the rmssd and hf power . the lf power is indicative of the sympathetic control of the cardiac function , and an increased lf - to - hf ratio denotes the increased sympathovagal balance ( 20 ) . combined parasympathetic and sympathetic loss is indicated by a reduction in all the above hrv parameters including the lf - to - hf ratio . statistical analyses were performed using sas for windows ( version 9.2 ; sas institute , cary , nc ) . sdnn , rmssd , triglyceride , and aer were log transformed to better meet model assumptions ( e.g. , homogeneity of variance ) . t tests and tests were used to test for differences in continuous and categorical variables between youth with and without type 1 diabetes , respectively . ancova was used to assess the relationship between diabetes status ( type 1 diabetic vs. control subjects ) and several hrv parameters , independent of demographic , anthropometric , and traditional cvd risk factors . for a better understanding of the influence of hyperglycemia on hrv abnormalities , youth with type 1 diabetes were categorized according to their glycemic control as optimal ( a1c < 7.5% ) and suboptimal ( a1c 7.5% ) , based on american diabetes association ( ada ) recommendations ( 21 ) , and each category was compared with the referent control group . youth with diabetes were asked to withhold their diabetes medications , including short - acting insulin , on the morning of the visit until after the blood draw was complete . all participants were asked to refrain from any strenuous exercise , smoking , or caffeinated drinks 12 h prior to the visit . race / ethnicity was self - reported using 2000 u.s . census based questions . for these analyses , race / ethnicity was categorized as non - hispanic white ( nhw ) and race / ethnicity other than nhw , including hispanic , african american , and asian / pacific islander racial / ethnic groups . participants completed standardized questionnaires including medical history , medication inventory , smoking status , physical activity , daily insulin dose , family history of diabetes , and cvd . cigarette smoking was defined as having smoked cigarettes on 1 of the 30 days preceding the survey . participants were asked the average number of days in a typical week that they participated in physical activity for at least 20 min that made them sweat or breathe hard and were then categorized as physically inactive ( 02 days / week ) or physically active ( 37 days / week ) ( 15 ) . height was measured in centimeters using a stadiometer and weight in kilograms using a standardized weighing machine . bmi was calculated as weight in kilograms divided by the square of height in meters , and age- and sex - specific bmi z scores were derived based on the centers for disease control and prevention national standards ( 16 ) . waist circumference was measured to the nearest 0.1 cm with the national health and nutrition examination survey ( nhanes ) protocol ( 17 ) . resting systolic blood pressure ( sbp ) and diastolic blood pressure ( dbp ) were measured three times , using aneroid sphygmomanometer , while the subjects were seated for at least 5 min , and the average of the three measurements was taken . laboratory samples were obtained under conditions of metabolic stability , defined , for subjects with type 1 diabetes , as no episodes of diabetic ketoacidosis during the previous month . a fasting blood draw was conducted for the assessment of the metabolic parameters ( a1c , ldl cholesterol , hdl , and triglyceride levels ) . blood specimens were processed locally and shipped to a central laboratory ( northwest lipid metabolism and diabetes research laboratories , seattle , wa ) . urinary albumin was measured from overnight timed urine samples by radioimmunoassay and was expressed as albumin excretion rate ( aer ) . aer between 20 and 200 g / min was defined as microalbuminuria , while 200 g / min was defined as macroalbuminuria . high - performance liquid chromatography ( tosoh bioscience , san francisco , ca ) was used to measure a1c . measurements of triglyceride and hdl cholesterol were performed enzymatically on a hitachi 917 autoanalyzer ( roche molecular biochemicals diagnostics , indianapolis , in ) . ldl cholesterol was calculated by the friedewald equation for individuals with triglyceride concentration < 400 mg / dl and the beta quantification procedure for those with triglycerides of 400 mg / dl . all measurements of hrv were conducted in the morning between 7 and 11 a.m. in a room with a stable room temperature while the participant was lying in the resting supine position for 10 min and breathing at a normal pace , using the sphygmocor device ( atcor medical ) . the device takes into account the normal heart beats , ignoring the ectopic beats , to derive the statistical parameters of the normal r - r intervals ( nn intervals ) of the ecg and computes several time and frequency domain hrv indices . the time domain indices of hrv used in these analyses were the sd of the nn intervals ( sdnn ) and the root mean square differences of successive nn intervals ( rmssd ) . the frequency domain indices of hrv that were measured were the normalized hf ( high frequency ) power , lf ( low frequency ) power , and the lf - to - hf ratio . the sphgmocor device derives the normalized lf and hf power by expressing them as a fraction of the total power [ lf n.u . = lf/(total power [ tp ] very low frequency [ vlf ] ) 100 and hf n.u . sdnn is a measure of overall hrv , so lower sdnn levels indicate reduced overall hrv ( 18 ) . rmssd and hf power represent the parasympathetic component of the hrv ( 19 ) , and thus parasympathetic loss is quantified by the reduction in the rmssd and hf power . the lf power is indicative of the sympathetic control of the cardiac function , and an increased lf - to - hf ratio denotes the increased sympathovagal balance ( 20 ) . combined parasympathetic and sympathetic loss is indicated by a reduction in all the above hrv parameters including the lf - to - hf ratio . statistical analyses were performed using sas for windows ( version 9.2 ; sas institute , cary , nc ) . sdnn , rmssd , triglyceride , and aer were log transformed to better meet model assumptions ( e.g. , homogeneity of variance ) . t tests and tests were used to test for differences in continuous and categorical variables between youth with and without type 1 diabetes , respectively . ancova was used to assess the relationship between diabetes status ( type 1 diabetic vs. control subjects ) and several hrv parameters , independent of demographic , anthropometric , and traditional cvd risk factors . for a better understanding of the influence of hyperglycemia on hrv abnormalities , youth with type 1 diabetes were categorized according to their glycemic control as optimal ( a1c < 7.5% ) and suboptimal ( a1c 7.5% ) , based on american diabetes association ( ada ) recommendations ( 21 ) , and each category was compared with the referent control group . a total of 530 youth , 354 with and 176 without type 1 diabetes , with complete hrv measurements were included in these analyses . table 1 shows the characteristics of the study population by diabetes status . there were no significant differences in mean age or sex distribution among youth with and without diabetes , although youth with diabetes were more likely to be nhw than control subjects ( 87 vs. 75% , p = 0.0005 ) . bmi , waist circumference , and sbp were similar among youth with and without type 1 diabetes . however , youth with type 1 diabetes had a worse metabolic profile with higher a1c ( p < 0.0001 ) and ldl cholesterol ( p = 0.03 ) levels and a higher prevalence of microalbuminuria ( 9 vs. 2.7% , p = 0.006 ) . dbp and heart rate were also significantly higher among youth with type 1 diabetes compared with their healthy counterparts ( p = 0.04 and < 0.0001 , respectively ) . youth with type 1 diabetes had significantly worse hrv parameters with lower sdnn , rmssd , and hf and higher lf and lf - to - hf ratio ( all p < 0.05 ) compared with youth without diabetes . characteristics of youth with and without type 1 diabetes participating in search cvd table 2 displays the results of the multiple linear regression analyses exploring the association between diabetes status and hrv parameters , independent of other demographic , anthropometric , and traditional cvd risk factors . all hrv variables were altered among youth with type 1 diabetes compared with control subjects , independent of age , sex , race / ethnicity , and traditional cvd risk factors , suggesting a role for diabetes - related hyperglycemia in mediating these abnormalities . associations between hrv parameters and type 1 diabetes status from multiple linear regression analysis sdnn , a marker of overall hrv , was 10 ms lower among youth with type 1 diabetes compared with control subjects ( p = 0.003 ) . other variables independently associated with lower sdnn were older age ( p = 0.05 ) , female sex ( p = 0.0007 ) , elevated ldl cholesterol ( p = 0.05 ) , triglyceride levels ( p = 0.03 ) , and presence of microalbuminuria ( p = 0.02 ) . rmssd was 13.5 ms lower in youth with type 1 diabetes versus control subjects ( p = 0.001 ) . other variables associated with lower rmssd , independent of diabetes status , were older age ( p = 0.02 ) and higher ldl cholesterol ( p = 0.01 ) . increasing age , female sex , and race / ethnicity other than nhw were also significantly associated with lower hf power . lf power , a surrogate for sympathetic dysfunction , was higher among youth with type 1 diabetes compared with healthy control subjects by 5.2 n.u . the lf - to - hf ratio was higher by 0.2 units among youth with type 1 diabetes compared with control subjects ( p = 0.005 ) . table 3 further explores the role of hyperglycemia by comparing adjusted mean levels of various hrv parameters in youth with type 1 diabetes with optimal ( a1c < 7.5% ) and suboptimal ( a1c 7.5% ) glycemic control versus those observed among healthy youth after adjustment for demographic , anthropometric , and traditional cvd risk factors . youth with type 1 diabetes and suboptimal glycemic control had significantly lower sdnn , rmssd , and hf power and higher lf power compared with the youth without diabetes . differences in hrv parameters between youth with type 1 diabetes and optimal glycemic control and youth without diabetes were substantially reduced and were not statistically significant . differences ( 95% ci ) in adjusted mean hrv parameters between youth with type 1 diabetes with optimal ( a1c < 7.5% ) and suboptimal ( a1c 7.5% ) glycemic control compared with control youth sdnn , a marker of overall hrv , was 10 ms lower among youth with type 1 diabetes compared with control subjects ( p = 0.003 ) . other variables independently associated with lower sdnn were older age ( p = 0.05 ) , female sex ( p = 0.0007 ) , elevated ldl cholesterol ( p = 0.05 ) , triglyceride levels ( p = 0.03 ) , and presence of microalbuminuria ( p = 0.02 ) . rmssd was 13.5 ms lower in youth with type 1 diabetes versus control subjects ( p = 0.001 ) . other variables associated with lower rmssd , independent of diabetes status , were older age ( p = 0.02 ) and higher ldl cholesterol ( p = 0.01 ) . increasing age , female sex , and race / ethnicity other than nhw were also significantly associated with lower hf power . lf power , a surrogate for sympathetic dysfunction , was higher among youth with type 1 diabetes compared with healthy control subjects by 5.2 n.u . the lf - to - hf ratio was higher by 0.2 units among youth with type 1 diabetes compared with control subjects ( p = 0.005 ) . table 3 further explores the role of hyperglycemia by comparing adjusted mean levels of various hrv parameters in youth with type 1 diabetes with optimal ( a1c < 7.5% ) and suboptimal ( a1c 7.5% ) glycemic control versus those observed among healthy youth after adjustment for demographic , anthropometric , and traditional cvd risk factors . youth with type 1 diabetes and suboptimal glycemic control had significantly lower sdnn , rmssd , and hf power and higher lf power compared with the youth without diabetes . differences in hrv parameters between youth with type 1 diabetes and optimal glycemic control and youth without diabetes were substantially reduced and were not statistically significant . differences ( 95% ci ) in adjusted mean hrv parameters between youth with type 1 diabetes with optimal ( a1c < 7.5% ) and suboptimal ( a1c 7.5% ) glycemic control compared with control youth we found evidence of reduced overall hrv , including a pattern of parasympathetic loss with sympathetic overdrive , among youth and young adults with type 1 diabetes , independent of traditional cvd risk factors . our findings suggest an important role for hyperglycemia in mediating these abnormalities in a contemporary cohort of diverse youth with an average duration of diabetes of ~10 years . older age , female sex , elevated ldl cholesterol and triglyceride levels , and microalbuminuria were also associated with reduced hrv , independent of diabetes status . although reduced hrv has been associated with an increased risk of arrhythmia , sudden death , and silent myocardial infarction in adults ( 1 ) , it is difficult to directly quantify the clinical importance of the observed reduction in overall hrv among youth with type 1 diabetes seen in our study . in a recent metanalysis of 21 studies of nearly 3,489 post myocardial infarction patients , individuals with sdnn < 70 ms had four times greater risk of death compared with those with sdnn > 70 ms ( 22 ) . the youth with suboptimal glycemic control in our study also had an adjusted mean value of sdnn < 70 ms , suggesting that their hrv impairment is likely to be clinically important . our finding of a specific pattern of early can in youth with type 1 diabetes is consistent with previous studies in other young - adult populations ( 23,24 ) . we also found that altered hrv parameters among youth with type 1 diabetes compared with control subjects were independent of traditional cvd risk factors , suggesting an important role for hyperglycemia in the pathogenesis of can . hyperglycemia induces abnormal signaling of the autonomic neurons via accumulation of advanced glycation end products , activation of polyol pathway , and ischemic atrophy of the autonomic nerve fibers innervating cardiac and vascular tissue ( 25 ) . both the parasympathetic and sympathetic divisions of the autonomic nervous system are typically affected in can , with parasympathetic impairment preceding the sympathetic dysfunction ( 1 ) . our data suggest that contemporary youth with type 1 diabetes with an average disease duration of 10 years already display early signs of can characterized by overall reduced hrv and vagal impairment with sympathetic override . however , it has been shown that the shift in cardiac sympathovagal balance from parasympathetic to sympathetic control over heart rhythm may lead to increased cardiovascular morbidity and mortality in people with diabetes ( 4 ) . therefore , the ada recommends that persons with type 1 diabetes be screened for can starting 5 years after diagnosis with the goal of detecting early abnormalities that may be reversible ( 26 ) . the role of improved glycemic control in attenuating the risk of microvascular and macrovascular complications of diabetes has been documented by landmark studies such as the diabetes control and complications trial ( dcct ) and uk prospective diabetes study ( ukpds ) ( 27,28 ) . a beneficial effect of tight glycemic control on early can has been shown in young adults with type 1 diabetes ( 27,28 ) . burger et al . ( 29 ) demonstrated an improvement in hrv parameters 1 year after an intensive therapy among 10 patients with early can but not among the 13 patients with advanced can , thus suggesting that early can is amenable to improved glycemic control . long - term poor glycemic control has been identified as a major factor in the development and progression of diabetic can ( 30 ) . in our study , nearly 78% of youth with diabetes had a1c levels > 7.5% , indicating an urgent need for efforts focused at improving glycemic control even among contemporary cohorts of youth with type 1 diabetes in developed countries such as the u.s . , which may , in turn , result in reduction in subclinical cardiovascular abnormalities . we found that sdnn was 7 ms lower for each 10-year increase in age , while females had on average 12 ms lower sdnn compared with males . the reduced hrv among female participants in our study is in agreement with that shown by the pittsburgh epidemiology of diabetes complications study ( 31 ) . similar sex and age influences on the hrv have also been demonstrated in healthy adults ( 34 ) , although the factors responsible for these differences are not clear . similar to our findings , higher triglyceride and lower hdl cholesterol have been associated with can among adults with type 1 diabetes in the eurodiab study ( 4 ) . previous studies have demonstrated a significant relationship between can and decline in renal function in individuals with diabetes ( 35 ) . we found that sddn was 11.3 ms lower in youth with microalbuminuria compared with those with normal urinary albumin excretion levels . first , the cross - sectional nature of the study limits our ability to evaluate the temporal trend in the development and progression of can among youth with type 1 diabetes . we therefore intend to longitudinally follow this cohort to better understand the progression of can over time . second , and related , while our results strongly suggest a role for diabetes - related hyperglycemia in mediating the hrv abnormalities , the design of the study did not permit us to directly quantify this effect . finally , the hrv measures used in our study are derived from a 10-min recording of the baseline ecg . while this is a relatively short length of recording , it is considered standard practice for clinical and research purposes and it is advocated by the task force of the european society of cardiology and the north american society of pacing and electrophysiology ( 36 ) , as opposed to the hrv measures derived from the 24-h holter recordings . the major strengths of our study are the relatively large and diverse sample of contemporary youth with type 1 diabetes and the simple noninvasive , bedside assessment of multiple measures of hrv that have been validated as surrogate markers of the autonomic function in several human studies ( 37,38 ) . in summary , we found evidence of reduced hrv among youth with type 1 diabetes with a relatively short duration of diabetes compared with healthy control subjects . the specific pattern identified suggests an early and potentially reversible can stage , characterized by parasympathetic loss with sympathetic overdrive . these abnormalities were independent of traditional cvd risk factors , suggesting a major role for diabetes - related hyperglycemia . improved glycemic control may be beneficial in slowing the progression or reversing the signs of early can among youth with type 1 diabetes .

objectivethis study compared heart rate variability ( hrv ) parameters in youth with and without type 1 diabetes and explored potential contributors of altered hrv.research design and methodshrv parameters were measured among 354 youth with type 1 diabetes ( mean age 18.8 years , diabetes duration 9.8 years , and mean a1c 8.9% ) and 176 youth without diabetes ( mean age 19.2 years ) participating in the search cvd study . multiple linear regression was used to assess the relationship between diabetes status and hrv parameters , adjusting for covariates.resultscompared with control subjects , youth with type 1 diabetes had reduced overall hrv ( 10.09 ms lower sd of nn intervals [ sdnn ] ) and markers of parasympathetic loss ( 13.5 ms reduced root mean square successive difference of nn intervals [ rmssd ] and 5.2 normalized units ( n.u . ) reduced high frequency [ hf ] power ) with sympathetic override ( 5.2 n.u . increased low frequency [ lf ] power ) , independent of demographic , anthropometric , and traditional cardiovascular risk factors . older age , female sex , higher ldl cholesterol and triglyceride levels , and presence of microalbuminuria were independently associated with lower hrv but did not account for the observed differences between youth with and without diabetes . youth with type 1 diabetes and a1c levels 7.5% had significantly worse hrv parameters than control subjects ; however , in youth with optimal glycemic control ( a1c < 7.5% ) , hrv parameters did not differ significantly from control subjects.conclusionsyouth with type 1 diabetes have signs of early cardiac autonomic neuropathy : reduced overall hrv and parasympathetic loss with sympathetic override . the main driver of these subclinical abnormalities appears to be hyperglycemia .

RESEARCH DESIGN AND METHODS Anthropometric and metabolic measurements Assessment of HRV Statistical analyses RESULTS Reduced overall HRV Markers of parasympathetic loss Markers of sympathetic override CONCLUSIONS

search cvd is an ancillary study to the search for diabetes in youth study , conducted in colorado and ohio . search is a multicenter study that conducts population - based ascertainment of nongestational cases of physician - diagnosed diabetes in youth aged < 20 years at diagnosis ( 13 ) . a total of 406 youth who had a physician diagnosis of type 1 diabetes were registered with search , were residents of colorado or ohio , were aged 1126 years during 20092011 , and had duration of diabetes of at least 5 years were enrolled in the search cvd study . during the same time period , a total of 204 frequency - matched ( by age , sex , and race / ethnicity ) youth without diabetes ( control subjects ) were also recruited in the study . because all search cases arose from health care provider offices , we recruited control youth from primary care offices in the same geographic areas . within clinical sites , control recruitment sampled youth based on the distribution of age , sex , and racial / ethnic background of case subjects . for defined periods of time , participating primary care offices provided an initial study brochure , and patients and their parent or guardian were asked to complete a one - page information form and an indication of permission for study staff to contact them regarding participation in the study . the study was reviewed and approved by the local institutional review boards that had jurisdiction over the local study population , and all participants provided signed informed consent or assent . youth with diabetes were asked to withhold their diabetes medications , including short - acting insulin , on the morning of the visit until after the blood draw was complete . race / ethnicity was categorized as non - hispanic white ( nhw ) and race / ethnicity other than nhw , including hispanic , african american , and asian / pacific islander racial / ethnic groups . participants completed standardized questionnaires including medical history , medication inventory , smoking status , physical activity , daily insulin dose , family history of diabetes , and cvd . bmi was calculated as weight in kilograms divided by the square of height in meters , and age- and sex - specific bmi z scores were derived based on the centers for disease control and prevention national standards ( 16 ) . resting systolic blood pressure ( sbp ) and diastolic blood pressure ( dbp ) were measured three times , using aneroid sphygmomanometer , while the subjects were seated for at least 5 min , and the average of the three measurements was taken . laboratory samples were obtained under conditions of metabolic stability , defined , for subjects with type 1 diabetes , as no episodes of diabetic ketoacidosis during the previous month . a fasting blood draw was conducted for the assessment of the metabolic parameters ( a1c , ldl cholesterol , hdl , and triglyceride levels ) . high - performance liquid chromatography ( tosoh bioscience , san francisco , ca ) was used to measure a1c . ldl cholesterol was calculated by the friedewald equation for individuals with triglyceride concentration < 400 mg / dl and the beta quantification procedure for those with triglycerides of 400 mg / dl . all measurements of hrv were conducted in the morning between 7 and 11 a.m. in a room with a stable room temperature while the participant was lying in the resting supine position for 10 min and breathing at a normal pace , using the sphygmocor device ( atcor medical ) . the time domain indices of hrv used in these analyses were the sd of the nn intervals ( sdnn ) and the root mean square differences of successive nn intervals ( rmssd ) . the frequency domain indices of hrv that were measured were the normalized hf ( high frequency ) power , lf ( low frequency ) power , and the lf - to - hf ratio . the sphgmocor device derives the normalized lf and hf power by expressing them as a fraction of the total power [ lf n.u . = lf/(total power [ tp ] very low frequency [ vlf ] ) 100 and hf n.u . sdnn is a measure of overall hrv , so lower sdnn levels indicate reduced overall hrv ( 18 ) . rmssd and hf power represent the parasympathetic component of the hrv ( 19 ) , and thus parasympathetic loss is quantified by the reduction in the rmssd and hf power . the lf power is indicative of the sympathetic control of the cardiac function , and an increased lf - to - hf ratio denotes the increased sympathovagal balance ( 20 ) . sdnn , rmssd , triglyceride , and aer were log transformed to better meet model assumptions ( e.g. t tests and tests were used to test for differences in continuous and categorical variables between youth with and without type 1 diabetes , respectively . ancova was used to assess the relationship between diabetes status ( type 1 diabetic vs. control subjects ) and several hrv parameters , independent of demographic , anthropometric , and traditional cvd risk factors . for a better understanding of the influence of hyperglycemia on hrv abnormalities , youth with type 1 diabetes were categorized according to their glycemic control as optimal ( a1c < 7.5% ) and suboptimal ( a1c 7.5% ) , based on american diabetes association ( ada ) recommendations ( 21 ) , and each category was compared with the referent control group . for these analyses , race / ethnicity was categorized as non - hispanic white ( nhw ) and race / ethnicity other than nhw , including hispanic , african american , and asian / pacific islander racial / ethnic groups . participants completed standardized questionnaires including medical history , medication inventory , smoking status , physical activity , daily insulin dose , family history of diabetes , and cvd . bmi was calculated as weight in kilograms divided by the square of height in meters , and age- and sex - specific bmi z scores were derived based on the centers for disease control and prevention national standards ( 16 ) . resting systolic blood pressure ( sbp ) and diastolic blood pressure ( dbp ) were measured three times , using aneroid sphygmomanometer , while the subjects were seated for at least 5 min , and the average of the three measurements was taken . laboratory samples were obtained under conditions of metabolic stability , defined , for subjects with type 1 diabetes , as no episodes of diabetic ketoacidosis during the previous month . a fasting blood draw was conducted for the assessment of the metabolic parameters ( a1c , ldl cholesterol , hdl , and triglyceride levels ) . high - performance liquid chromatography ( tosoh bioscience , san francisco , ca ) was used to measure a1c . measurements of triglyceride and hdl cholesterol were performed enzymatically on a hitachi 917 autoanalyzer ( roche molecular biochemicals diagnostics , indianapolis , in ) . ldl cholesterol was calculated by the friedewald equation for individuals with triglyceride concentration < 400 mg / dl and the beta quantification procedure for those with triglycerides of 400 mg / dl . the time domain indices of hrv used in these analyses were the sd of the nn intervals ( sdnn ) and the root mean square differences of successive nn intervals ( rmssd ) . the frequency domain indices of hrv that were measured were the normalized hf ( high frequency ) power , lf ( low frequency ) power , and the lf - to - hf ratio . the sphgmocor device derives the normalized lf and hf power by expressing them as a fraction of the total power [ lf n.u . = lf/(total power [ tp ] very low frequency [ vlf ] ) 100 and hf n.u . sdnn is a measure of overall hrv , so lower sdnn levels indicate reduced overall hrv ( 18 ) . rmssd and hf power represent the parasympathetic component of the hrv ( 19 ) , and thus parasympathetic loss is quantified by the reduction in the rmssd and hf power . t tests and tests were used to test for differences in continuous and categorical variables between youth with and without type 1 diabetes , respectively . ancova was used to assess the relationship between diabetes status ( type 1 diabetic vs. control subjects ) and several hrv parameters , independent of demographic , anthropometric , and traditional cvd risk factors . for a better understanding of the influence of hyperglycemia on hrv abnormalities , youth with type 1 diabetes were categorized according to their glycemic control as optimal ( a1c < 7.5% ) and suboptimal ( a1c 7.5% ) , based on american diabetes association ( ada ) recommendations ( 21 ) , and each category was compared with the referent control group . a total of 530 youth , 354 with and 176 without type 1 diabetes , with complete hrv measurements were included in these analyses . there were no significant differences in mean age or sex distribution among youth with and without diabetes , although youth with diabetes were more likely to be nhw than control subjects ( 87 vs. 75% , p = 0.0005 ) . bmi , waist circumference , and sbp were similar among youth with and without type 1 diabetes . however , youth with type 1 diabetes had a worse metabolic profile with higher a1c ( p < 0.0001 ) and ldl cholesterol ( p = 0.03 ) levels and a higher prevalence of microalbuminuria ( 9 vs. 2.7% , p = 0.006 ) . dbp and heart rate were also significantly higher among youth with type 1 diabetes compared with their healthy counterparts ( p = 0.04 and < 0.0001 , respectively ) . youth with type 1 diabetes had significantly worse hrv parameters with lower sdnn , rmssd , and hf and higher lf and lf - to - hf ratio ( all p < 0.05 ) compared with youth without diabetes . characteristics of youth with and without type 1 diabetes participating in search cvd table 2 displays the results of the multiple linear regression analyses exploring the association between diabetes status and hrv parameters , independent of other demographic , anthropometric , and traditional cvd risk factors . all hrv variables were altered among youth with type 1 diabetes compared with control subjects , independent of age , sex , race / ethnicity , and traditional cvd risk factors , suggesting a role for diabetes - related hyperglycemia in mediating these abnormalities . associations between hrv parameters and type 1 diabetes status from multiple linear regression analysis sdnn , a marker of overall hrv , was 10 ms lower among youth with type 1 diabetes compared with control subjects ( p = 0.003 ) . other variables independently associated with lower sdnn were older age ( p = 0.05 ) , female sex ( p = 0.0007 ) , elevated ldl cholesterol ( p = 0.05 ) , triglyceride levels ( p = 0.03 ) , and presence of microalbuminuria ( p = 0.02 ) . rmssd was 13.5 ms lower in youth with type 1 diabetes versus control subjects ( p = 0.001 ) . other variables associated with lower rmssd , independent of diabetes status , were older age ( p = 0.02 ) and higher ldl cholesterol ( p = 0.01 ) . increasing age , female sex , and race / ethnicity other than nhw were also significantly associated with lower hf power . lf power , a surrogate for sympathetic dysfunction , was higher among youth with type 1 diabetes compared with healthy control subjects by 5.2 n.u . the lf - to - hf ratio was higher by 0.2 units among youth with type 1 diabetes compared with control subjects ( p = 0.005 ) . table 3 further explores the role of hyperglycemia by comparing adjusted mean levels of various hrv parameters in youth with type 1 diabetes with optimal ( a1c < 7.5% ) and suboptimal ( a1c 7.5% ) glycemic control versus those observed among healthy youth after adjustment for demographic , anthropometric , and traditional cvd risk factors . youth with type 1 diabetes and suboptimal glycemic control had significantly lower sdnn , rmssd , and hf power and higher lf power compared with the youth without diabetes . differences in hrv parameters between youth with type 1 diabetes and optimal glycemic control and youth without diabetes were substantially reduced and were not statistically significant . differences ( 95% ci ) in adjusted mean hrv parameters between youth with type 1 diabetes with optimal ( a1c < 7.5% ) and suboptimal ( a1c 7.5% ) glycemic control compared with control youth sdnn , a marker of overall hrv , was 10 ms lower among youth with type 1 diabetes compared with control subjects ( p = 0.003 ) . other variables independently associated with lower sdnn were older age ( p = 0.05 ) , female sex ( p = 0.0007 ) , elevated ldl cholesterol ( p = 0.05 ) , triglyceride levels ( p = 0.03 ) , and presence of microalbuminuria ( p = 0.02 ) . rmssd was 13.5 ms lower in youth with type 1 diabetes versus control subjects ( p = 0.001 ) . other variables associated with lower rmssd , independent of diabetes status , were older age ( p = 0.02 ) and higher ldl cholesterol ( p = 0.01 ) . increasing age , female sex , and race / ethnicity other than nhw were also significantly associated with lower hf power . lf power , a surrogate for sympathetic dysfunction , was higher among youth with type 1 diabetes compared with healthy control subjects by 5.2 n.u . the lf - to - hf ratio was higher by 0.2 units among youth with type 1 diabetes compared with control subjects ( p = 0.005 ) . table 3 further explores the role of hyperglycemia by comparing adjusted mean levels of various hrv parameters in youth with type 1 diabetes with optimal ( a1c < 7.5% ) and suboptimal ( a1c 7.5% ) glycemic control versus those observed among healthy youth after adjustment for demographic , anthropometric , and traditional cvd risk factors . youth with type 1 diabetes and suboptimal glycemic control had significantly lower sdnn , rmssd , and hf power and higher lf power compared with the youth without diabetes . differences in hrv parameters between youth with type 1 diabetes and optimal glycemic control and youth without diabetes were substantially reduced and were not statistically significant . differences ( 95% ci ) in adjusted mean hrv parameters between youth with type 1 diabetes with optimal ( a1c < 7.5% ) and suboptimal ( a1c 7.5% ) glycemic control compared with control youth we found evidence of reduced overall hrv , including a pattern of parasympathetic loss with sympathetic overdrive , among youth and young adults with type 1 diabetes , independent of traditional cvd risk factors . our findings suggest an important role for hyperglycemia in mediating these abnormalities in a contemporary cohort of diverse youth with an average duration of diabetes of ~10 years . older age , female sex , elevated ldl cholesterol and triglyceride levels , and microalbuminuria were also associated with reduced hrv , independent of diabetes status . although reduced hrv has been associated with an increased risk of arrhythmia , sudden death , and silent myocardial infarction in adults ( 1 ) , it is difficult to directly quantify the clinical importance of the observed reduction in overall hrv among youth with type 1 diabetes seen in our study . the youth with suboptimal glycemic control in our study also had an adjusted mean value of sdnn < 70 ms , suggesting that their hrv impairment is likely to be clinically important . our finding of a specific pattern of early can in youth with type 1 diabetes is consistent with previous studies in other young - adult populations ( 23,24 ) . we also found that altered hrv parameters among youth with type 1 diabetes compared with control subjects were independent of traditional cvd risk factors , suggesting an important role for hyperglycemia in the pathogenesis of can . our data suggest that contemporary youth with type 1 diabetes with an average disease duration of 10 years already display early signs of can characterized by overall reduced hrv and vagal impairment with sympathetic override . however , it has been shown that the shift in cardiac sympathovagal balance from parasympathetic to sympathetic control over heart rhythm may lead to increased cardiovascular morbidity and mortality in people with diabetes ( 4 ) . therefore , the ada recommends that persons with type 1 diabetes be screened for can starting 5 years after diagnosis with the goal of detecting early abnormalities that may be reversible ( 26 ) . the role of improved glycemic control in attenuating the risk of microvascular and macrovascular complications of diabetes has been documented by landmark studies such as the diabetes control and complications trial ( dcct ) and uk prospective diabetes study ( ukpds ) ( 27,28 ) . a beneficial effect of tight glycemic control on early can has been shown in young adults with type 1 diabetes ( 27,28 ) . ( 29 ) demonstrated an improvement in hrv parameters 1 year after an intensive therapy among 10 patients with early can but not among the 13 patients with advanced can , thus suggesting that early can is amenable to improved glycemic control . long - term poor glycemic control has been identified as a major factor in the development and progression of diabetic can ( 30 ) . in our study , nearly 78% of youth with diabetes had a1c levels > 7.5% , indicating an urgent need for efforts focused at improving glycemic control even among contemporary cohorts of youth with type 1 diabetes in developed countries such as the u.s . , which may , in turn , result in reduction in subclinical cardiovascular abnormalities . we found that sdnn was 7 ms lower for each 10-year increase in age , while females had on average 12 ms lower sdnn compared with males . similar sex and age influences on the hrv have also been demonstrated in healthy adults ( 34 ) , although the factors responsible for these differences are not clear . similar to our findings , higher triglyceride and lower hdl cholesterol have been associated with can among adults with type 1 diabetes in the eurodiab study ( 4 ) . previous studies have demonstrated a significant relationship between can and decline in renal function in individuals with diabetes ( 35 ) . we found that sddn was 11.3 ms lower in youth with microalbuminuria compared with those with normal urinary albumin excretion levels . first , the cross - sectional nature of the study limits our ability to evaluate the temporal trend in the development and progression of can among youth with type 1 diabetes . second , and related , while our results strongly suggest a role for diabetes - related hyperglycemia in mediating the hrv abnormalities , the design of the study did not permit us to directly quantify this effect . the major strengths of our study are the relatively large and diverse sample of contemporary youth with type 1 diabetes and the simple noninvasive , bedside assessment of multiple measures of hrv that have been validated as surrogate markers of the autonomic function in several human studies ( 37,38 ) . in summary , we found evidence of reduced hrv among youth with type 1 diabetes with a relatively short duration of diabetes compared with healthy control subjects . the specific pattern identified suggests an early and potentially reversible can stage , characterized by parasympathetic loss with sympathetic overdrive . these abnormalities were independent of traditional cvd risk factors , suggesting a major role for diabetes - related hyperglycemia . improved glycemic control may be beneficial in slowing the progression or reversing the signs of early can among youth with type 1 diabetes .

the management of cancer patients has been revolutionized in the last few decades by advances in targeted therapies , which , unlike conventional chemotherapy , enable the precise targeting of cancer cells giving rise to the concept of precision medicine . the term targeted therapy encompasses a wide spectrum of drug classes including inhibitors of signal transduction ( receptor tyrosine kinases ) , hormonal agents , inhibitors of angiogenesis , dna damage modulators and modulators of immune system , to name a few . of these targeted therapies , immune modulators or immune - checkpoint inhibitors ( icis ) have garnered significant attention in the last 5 years due to their increasing spectrum of activity in oncology and have , as such , radically changed the therapeutic landscape of advanced solid and hematologic malignancies . to date , four different icis have been approved by the food and drug administration ( fda ) for various malignancies since 2011 ( 1 ) . the use of icis is expected to increase significantly in the near future with several new single and combination therapies under active research in phase i and ii trials . owing to their unique anti - cancer mechanism , icis are associated with unusual response patterns and adverse event profiles on imaging , which are currently under investigation . as icis become the standard of care for an increasing number of cancers and prolong life expectancy of patients with metastatic disease , radiologists , as an integral part of the multidisciplinary oncologic patient care , need be familiar with the mechanistic background of these anticancer agents , their immune - related tumor response patterns , immune - related adverse event ( irae ) profiles , to contribute to clinical decision making . in this article , we will review the molecular bases of anti - cancer immunotherapeutic agents , their clinical application in solid and hematologic malignancies , the immune - related patterns of response observed on imaging studies and the imaging features of iraes . using the immune system to treat diseases dates back to 1796 , when edward jenner produced the first vaccine involving immunization with cowpox to prevent smallpox . it was only in the late 1980s that immunotherapy has been used to treat cancer , when rosenberg ( 2 ) and colleagues reported a low tumor regression rate ( 2.63.3% ) in 1205 patients with metastatic cancer who underwent different types of immunotherapy . since then , cancer immunotherapy , which utilizes the immune system to kill cancer cells , has become an established modality for cancer care . several agents have been used over the years , targeting the immune systems at different levels and in different ways . cytokine il-2 has shown objective response in patients with melanoma and renal cell carcinoma ( rcc ) ( 3 ) . vaccine sipuleucel - t has been fda approved in april 2010 for treatment of metastatic prostate cancer ( 4 ) . patient s own t - cells have been engineered with chimeric antigen receptors to recognize and fight leukemias and lymphomas ( 5 ) . more recently , icis , which act unleashing the immune system to fight the cancer , have gained fda approval for treatment of different types of solid and hematologic malignancies ( 6 ) . immune - checkpoint inhibitors act by inhibiting crucial regulatory steps in the immune system , promoting the activation and proliferation of t - cells to induce tumor infiltration and regression . based on the checkpoint target , three main types of icis have been studied so far and gained fda approval : cytotoxic t - lymphocyte antigen-4 ( ctla-4 ) antibodies and programmed cell death ( pd-1 ) and programmed cell death ligand ( pd - l1 and pd - l2 ) antibodies . ctla-4 , discovered in 1987 , is a negative regulator of t cell activation , inhibiting cd4 + and cd8 + cell activation , triggered by the antigen presenting cells ( 789 ) . ipilimumab ( yervoy ; bristol - myers squibb , new york , ny , usa ) , a ctla4 inhibitor , was the first ici , to gain approval by the u.s . fda in 2011 , based on two pivotal randomized trials ( 1 ) , which have shown improved overall survival in patients with advanced melanoma . it has also been approved as adjuvant therapy for high - risk melanoma , as an alternative to interferon . ipilimumab is administered intravenously every 3 weeks for a total of 4 doses , called the induction regimen . additional ctla-4 inhibiting monoclonal antibodies have been investigated in other solid malignancies , such as tremelimumab for malignant mesothelioma ( 101112 ) . pd-1 , pd - l1 and pd - l2 are transmembrane inhibitory proteins expressed on t cells , b cells and natural killer ( nk ) cells , binding to pd - ligand 1 and pd - ligand 2 on multiple tissue types and hematopoietic cells , respectively ( 13 ) . nivolumab , pemrolizumab and atezolizumab have been fda approved and are increasingly used in multiple cancer types ( 141516 ) . nivolumab , igg4 subclass pd-1 inhibitor , is fda approved for metastatic melanoma , advanced non - small cell lung cancer ( nsclc ) ( 17 ) , rcc ( 18 ) and hodgkin s disease ( 1920212223242526 ) . pembrolizumab is fda approved for metastatic melanoma and advanced nsclc and atezolizumab has been recently fda approved for the treatment of urothelial bladder cancer ( 27 ) . in addition , these agents and other antibodies targeting pd-1 or pd - l1 are under investigation in a broad spectrum of malignancies , such as mismatch - repair deficient colorectal carcinoma , non - hodgkin s lymphoma and cancers of the head and neck . other potential targets for checkpoint inhibition have been studied , including t - cell immunoglobulin and mucin domain 3 , lymphocyte activation gene 3 , b and t - cell lymphocyte attenuator , and v - domain ig suppressor of t - cell activation . in addition , multiple clinical trials investigating combination regimen of checkpoint inhibitors are underway . blocking the ctla-4 and the pd-1/pd - l1 pathways , in fact , has been proved to have synergistic effect ( 2829 ) . the combination ipilimumab and nivolumab has demonstrated a significant higher response rate than ipilumamb alone in metastatic melanoma and has already been fda approved for the treatment of unresectable or metastatic melanoma across braf status ( 2829 ) . clinical trials combining ipilimumab and nivolumab are underway in nsclc , epithelial ovarian / fallopian tube carcinoma , hodgkin s lymphoma , rcc and soft tissue sarcoma ( 2829 ) . cancer immunotherapy is associated with a variety of new imaging features , including unconventional response patterns and new spectrum of drug - related toxicities . the patterns of response seen with icis differ in many ways from the ones seen with conventional cytotoxic agents and molecular targeted therapies . treatment response to immune - modulatory agents , in fact , can be seen long after the start of treatment ; furthermore , increased and new lesions soon after start of treatment do not necessarily represent worsening of disease , as they do with conventional therapies . the immuno - modulation elicits an inflammatory reaction , which can cause initial increase in tumor size , giving the appearance of pseudoprogression , before leading to response or stability of disease ( 30313233 ) . new lesions can also be seen in the context of the initial inflammatory reaction , likely representing undetected lesions at baseline , increasing in size during the initial immune reaction . the pseudoprogression pattern of response is supported by a case study of an ipilimumab - treated patient which showed increased tumor burden on imaging after 12 weeks of therapy , while histologic exam demonstrated that the increased in size was due to inflammatory response caused by t - cell infiltration rather than tumor cell proliferation ( 30 ) . in this setting of unconventional response , the application of conventional tumor response criteria , such as response evaluation criteria for solid tumors ( recist ) and world health organiziation ( who ) criterion , where new lesions and increase tumor size suggest progression of disease , would underestimate the benefit of therapy and lead to premature discontinuation of treatment . hence , to assess this atypical pattern of response , in 2009 , a committee of 200 oncologists , immunotherapists , and regulatory experts proposed a new immune - related response criteria ( irrc ) ( 34353637 ) . immune - related response criteria revisits the definition of progression of disease , preventing new measurable or non - measurable lesions from defining progression and incorporates new measurable lesions into the tumor burden . radiologic aspects of immune - related tumor response criteria and patterns of iraes in patients undergoing ipilimumab therapy ( 3133 ) . maintaining the concept of measurable and non - measurable lesions and the bidimensional measurement according to who criterion , irrc proposes a new immune - related ( ir ) response assessment , as follow : 1 ) immune - related complete response : complete response is defined as disappearance of all measureable and non - measurable lesions , with no new lesions . complete response must be confirmed by a subsequent , consecutive assessment at least four weeks apart . 2 ) immune - related partial response : partial response is defined as decrease in the total tumor burden of 50 percent or more from the baseline , confirmed by a subsequent scan at least four weeks later . increase in size of some of the lesions and/or appearance of new lesions , as long as the total tumor burden meets the response criterion of 50 percent decrease , are allowed . 3 ) immune - related stable disease : stability of the tumor burden , not meeting the criteria for partial , complete response or for progressive disease . 4 ) immune - related progressive disease : progression of disease is defined as 25 percent increase in the tumor burden from the baseline or nadir study . a short - term follow - up imaging study , at least four weeks apart , while irrc accurately revisits the parameter defining the response assessment , it presents some limitations and scope for areas for improvement . for example , a major pitfall of the original irrc is the use of bidimensional measurements according to who criterion , defined as the product of the longest diameter and the longest perpendicular diameter ( ld lpd ) . prior studies have in fact demonstrated that bidimensional measurements are not suitable for capturing small changes of tumor burden , as they are subject to a higher variability than unidimensional measurements ( 38394041 ) . furthermore , since multiple prior trials have used recist , which is based on unidimensional measurements , the use of bidimensional measurements in irrc makes it difficult to directly compare results ( 4243 ) . tumor density , volume , metabolic activities and other functional information , should also be incorporated in the criterion to evaluate immune - related response and are under investigation ( 3644 ) . the field of atypical response with icis , as single agents or in combination therapy , has to be further explored ( 45464748 ) . a recent study by hodi et al . ( 49 ) evaluated atypical response patterns using irrc in comparison with recist1.1 in 327 patients with melanoma during treatment with pembrolizumab , the largest retrospective study to date , evaluating immune - related responses in patients on pd-1 inhibitor therapy . the study found that , based on survival analysis , conventional recist underestimates the benefit of pembrolizumab in approximately 15% of patients in comparison with irrc . the article provides important insights for future directions toward an irrecist 1.1 , which , maintaining the modifications introduced by irrc , will propose a common denominator with conventional recist , for allowing accurate comparison of the immune - related response ( 39 ) . in the clinical practice of a radiology reading room of a tertiary cancer center , the non - conventional patterns of response elicited by immunomodulatory agents , translate into three main categories , which radiologists should be aware of : 1 ) initial progression followed by response or stability : increase in tumor burden and/or presence of new lesions at the first follow up imaging study after start of immunomodulatory agent should not be reported as progression of disease . in fact , this could represent initial transient flare up of tumor burden , followed by improvement or stabilization of disease . patients with this type of response pattern are usually asymptomatic during the flare ; conversely , patients with true progression will show clinical symptoms . in the absence of clear symptomatic progression , a short - term follow up scan ( at least 4 weeks ) should be suggested in the radiology report , to avoid considering failure of immunotherapy . 2 ) prolonged stable disease followed by response : stability of the tumor burden can be seen for a long period of time after initiation of icis , before seeing response , which could become apparent only after the induction period of 4 doses adminsistered 3 weeks apart . practically , after the induction period of 12 weeks , is the time a radiologist should carefully look for changes in the tumor burden . 3 ) long period of stability : although there is no visible decrease in size of the lesions , even long period of stability can be clinically significant . in the clinical practice , reporting stability of an otherwise incurable and aggressive disease after multiple imaging studies can be clinically relevant . hence , the time of initiation of treatment should be known to the radiologist , and the stability over a long period of time should be emphasized in the radiology report . immune - checkpoint inhibitors can cause a variety of iraes , either symptomatic or clinically silent , most of them associated with radiology manifestations . while both clinical and radiographic characterization of iraes is still ongoing , several these include enterocolitis , hepatitis , thyroid disorders , hypophysitis , pancreatitis , pneumonitis and dermatitis ( 50 ) . additional imaging findings , such as retroperitoneal stranding , sarcoid - like distributed lymphadenopathy , myositis and arthritis have also been attributed to the proinflammatory state elicited by icis . iraes are quite frequent as the body s innate control against autoimmunity is down - regulated by icis ( 50 ) , more so with ctla-4 inhibitors , which involves the early activation of t - cells , less with pd-1 and pd - l1 antibodies , involved in the later phases of the activation . with early detection , iraes can be successfully managed delaying or discontinuing the therapy , based on the severity of the event , and high dose corticosteroids . hence , prompt recognition of drug - related toxicity by the radiologist is crucial for clinical decisionmaking . pneumonitis : pneumonitis is a serious , potentially life - threatening iraes , which resulted in three deaths in a phase i trial . pneumonitis as new consolidative or ground - glass opacities has been reported in up to 5% of patients receiving immune checkpoint inhibition ( fig . 1 ; please also see fig . 4 m , p regarding pet findings ) ( 50 ) . although imaging features have yet to be described , initial reports of ici - related pneumonitis indicate a spectrum of radiographic manifestations of the entity . three cases of pneumonitis associated with the use of anti - pd-1 antibodies in patients with melanoma have been reported with two main radiologic patterns : an acute respiratory distress syndrome - like pattern , with diffuse ground - glass and reticular opacities , consolidation associated with traction bronchiectasis , decreased lung volumes and pleural effusion and a non - specific interstitial pneumonia ( nsip)-pattern , with peripheral ground - glass and reticular opacities , predominantly at the lung bases ( 5152 ) . a case study of two patients reports anti - pd-1 pneumonitis in advanced nsclc patients treated with nivolumab ( 51 ) . ct of both patients demonstrated ground - glass , reticular opacities and rapidly evolving consolidations in a peripheral distribution , in a radiographic pattern of cryptogenic organizing pneumonia . nivolumab was stopped and treatment with high dose corticosteroids was initiated , with improvement of respiratory symptoms and radiographic findings . after steroid taper , one patient , not on any active treatment , developed another episode of pneumonitis ( pneumonitis flare ) and had to be restarted on corticosteroids with subsequent clinical and radiologic improvement , further indicating the complicated nature of this irae ( fig . immune - related colitis is the most commonly reported irae , and can be observed even in asymptomatic patients . it is most commonly associated with ipilimumab and usually occurs between 510 weeks of initiation of therapy ( fig . two distinct ct appearances have been described : diffuse bowel wall thickening , with or without fluid filled distended colon , with mesenteric vessel engorgement , or segmental bowel wall thickening and pericolonic stranding , often superimposed to diverticulitis , so called segmental colitis associated with diverticulosis ( 54 ) . it is important to differentiate between the two patterns as their management differs : after stopping the drug , diffuse colitis is treated with high dose steroids , segmental colitis is treated with steroids and antibiotics . hepatitis : immune - related hepatitis usually appears between 614 weeks of initiation of therapy . imaging findings can be subtle and a high index of suspicion should be maintained in patients with systemic symptoms and altered liver function tests ( lfts ) . classic imaging appearance of acute hepatitis is seen in such patients , as reported in a study of 6 melanoma patients with ipilimumab - associated hepatitis ( 55 ) . conversely , imaging of asymptomatic patients , with mildly increased lfts can be normal ( 5556 ) . histologically , ipilimumab - associated hepatitis has been described as hepatocyte injury , with acute hepatitis pattern or as bile duct injury ( 56 ) . endocrine iraes are quite common and usually appear between 720 weeks of initiation of therapy . they might cause serious and life - changing symptoms due to permanent damage of the hypophysis or the thyroid glands . they have to be promptly recognized by the radiologist , as symptoms can resolve with adequate hormone replacement , allowing the patients to continue a therapy they are benefiting from . thyroid disorders are the most common clinically reported endocrine iraes , particularly in women on anti - pd-1 inhibitors and can clinically manifests with hypothyroidism , most commonly , followed by hyperthyroidisim , whereas thyroiditis has not yet been reported ( 5057 ) . imaging features have been described as new enlargement of the thyroid gland , enhancement and diffuse fludeoxyglucose ( fdg ) uptake on pet / ct , as reported by a study on ipilimumab related adverse event ( 50 ) . hypophysitis is more commonly associated with ipilimumab , is rare with pd-1 and pd - l1 inhibitor monotherapies , and has a median appearance at 11 weeks , but can be seen as early as 4 weeks ( fig . 4n ) ( 58 ) . clinical picture and imaging findings resemble those seen with primary lymphocytic hypophysitis , with avid enhancement of the hypophysis on contrast enhanced ct , t1 precontrast and t2 hypointensity and homogeneous enhancement on mri ( 57 ) . additional rare immune - related endocrinopathies have been described and include ipilimumab graves ophthalmopathy and anti pd-1 type i diabetes mellitus , which has been reported in only 4 patients to date , but imaging features have yet to be described ( fig . awareness that these findings might reflect inflammatory response rather than new sites of disease is crucial for the radiologist . examples include new sarcoid - like distributed lymph nodes ( bilateral hilar and mediastinal ) , which have been reported in up to 5% of patients on icis treatment ( 5059606162 ) . although the imaging appearance of these nodes can be indistinguishable from nodal metastases , the distribution resembling typical sarcoidosis , in absence of infection and occurring in the setting of response at other sites , favor sarcoid - like reaction and ace - level and tissue diagnosis can support the diagnosis of irae ( 50 ) . treatment should not be discontinued as these nodes are likely a flare phenomenon at initiation of therapy and might regress spontaneously . new abdominal lymphadenopathy , stranding in perirenal or retroperitoneal fat and focal muscle abnormalities have also been observed and are also likely a marker of lymphocytic infiltration , indicating treatment effect , rather than new sites of disease . new focal intramuscular enhancement seen on contrast - enhanced ct or new intramuscular tracer uptake on pet / ct has been described as drug - related myositis and should be reported . an interesting relationship between tumor response and iraes has been described : radiologic manifestations of iraes have been associated with improved tumor response and disease control ( 53 ) . a study of 119 patients with metastatic melanoma on ipilimumab therapy showed iraes in 17% of patients , who were more likely to show response and disease control ( 55% ) ( 63 ) . for example , ipilimumab and nivolumab therapy for the treatment of metastatic melanoma , as it demonstrates increase antitumor activity , shows increased incidence of drug - associated adverse events . the combination of pembrolizumab and ipilimumab has also been associated with increased adverse events , the thyroid gland being most affected organ , manifesting as hypothyroidism . this interesting relationship between response and toxicities , however , need to be further investigated with a rigorous study design controlling for important factors such as time of therapy duration . cancer immunotherapy is associated with a variety of new imaging features , including unconventional response patterns and new spectrum of drug - related toxicities . the patterns of response seen with icis differ in many ways from the ones seen with conventional cytotoxic agents and molecular targeted therapies . treatment response to immune - modulatory agents , in fact , can be seen long after the start of treatment ; furthermore , increased and new lesions soon after start of treatment do not necessarily represent worsening of disease , as they do with conventional therapies . the immuno - modulation elicits an inflammatory reaction , which can cause initial increase in tumor size , giving the appearance of pseudoprogression , before leading to response or stability of disease ( 30313233 ) . new lesions can also be seen in the context of the initial inflammatory reaction , likely representing undetected lesions at baseline , increasing in size during the initial immune reaction . the pseudoprogression pattern of response is supported by a case study of an ipilimumab - treated patient which showed increased tumor burden on imaging after 12 weeks of therapy , while histologic exam demonstrated that the increased in size was due to inflammatory response caused by t - cell infiltration rather than tumor cell proliferation ( 30 ) . in this setting of unconventional response , the application of conventional tumor response criteria , such as response evaluation criteria for solid tumors ( recist ) and world health organiziation ( who ) criterion , where new lesions and increase tumor size suggest progression of disease , would underestimate the benefit of therapy and lead to premature discontinuation of treatment . hence , to assess this atypical pattern of response , in 2009 , a committee of 200 oncologists , immunotherapists , and regulatory experts proposed a new immune - related response criteria ( irrc ) ( 34353637 ) . immune - related response criteria revisits the definition of progression of disease , preventing new measurable or non - measurable lesions from defining progression and incorporates new measurable lesions into the tumor burden . radiologic aspects of immune - related tumor response criteria and patterns of iraes in patients undergoing ipilimumab therapy ( 3133 ) . maintaining the concept of measurable and non - measurable lesions and the bidimensional measurement according to who criterion , irrc proposes a new immune - related ( ir ) response assessment , as follow : 1 ) immune - related complete response : complete response is defined as disappearance of all measureable and non - measurable lesions , with no new lesions . complete response must be confirmed by a subsequent , consecutive assessment at least four weeks apart . 2 ) immune - related partial response : partial response is defined as decrease in the total tumor burden of 50 percent or more from the baseline , confirmed by a subsequent scan at least four weeks later . increase in size of some of the lesions and/or appearance of new lesions , as long as the total tumor burden meets the response criterion of 50 percent decrease , are allowed . 3 ) immune - related stable disease : stability of the tumor burden , not meeting the criteria for partial , complete response or for progressive disease . 4 ) immune - related progressive disease : progression of disease is defined as 25 percent increase in the tumor burden from the baseline or nadir study . a short - term follow - up imaging study , at least four weeks apart , while irrc accurately revisits the parameter defining the response assessment , it presents some limitations and scope for areas for improvement . for example , a major pitfall of the original irrc is the use of bidimensional measurements according to who criterion , defined as the product of the longest diameter and the longest perpendicular diameter ( ld lpd ) . prior studies have in fact demonstrated that bidimensional measurements are not suitable for capturing small changes of tumor burden , as they are subject to a higher variability than unidimensional measurements ( 38394041 ) . furthermore , since multiple prior trials have used recist , which is based on unidimensional measurements , the use of bidimensional measurements in irrc makes it difficult to directly compare results ( 4243 ) . tumor density , volume , metabolic activities and other functional information , should also be incorporated in the criterion to evaluate immune - related response and are under investigation ( 3644 ) . the field of atypical response with icis , as single agents or in combination therapy , has to be further explored ( 45464748 ) . a recent study by hodi et al . ( 49 ) evaluated atypical response patterns using irrc in comparison with recist1.1 in 327 patients with melanoma during treatment with pembrolizumab , the largest retrospective study to date , evaluating immune - related responses in patients on pd-1 inhibitor therapy . the study found that , based on survival analysis , conventional recist underestimates the benefit of pembrolizumab in approximately 15% of patients in comparison with irrc . the article provides important insights for future directions toward an irrecist 1.1 , which , maintaining the modifications introduced by irrc , will propose a common denominator with conventional recist , for allowing accurate comparison of the immune - related response ( 39 ) . in the clinical practice of a radiology reading room of a tertiary cancer center , the non - conventional patterns of response elicited by immunomodulatory agents , translate into three main categories , which radiologists should be aware of : 1 ) initial progression followed by response or stability : increase in tumor burden and/or presence of new lesions at the first follow up imaging study after start of immunomodulatory agent should not be reported as progression of disease . in fact , this could represent initial transient flare up of tumor burden , followed by improvement or stabilization of disease . patients with this type of response pattern are usually asymptomatic during the flare ; conversely , patients with true progression will show clinical symptoms . in the absence of clear symptomatic progression , a short - term follow up scan ( at least 4 weeks ) should be suggested in the radiology report , to avoid considering failure of immunotherapy . 2 ) prolonged stable disease followed by response : stability of the tumor burden can be seen for a long period of time after initiation of icis , before seeing response , which could become apparent only after the induction period of 4 doses adminsistered 3 weeks apart . practically , after the induction period of 12 weeks , is the time a radiologist should carefully look for changes in the tumor burden . 3 ) long period of stability : although there is no visible decrease in size of the lesions , even long period of stability can be clinically significant . in the clinical practice , reporting stability of an otherwise incurable and aggressive disease after multiple imaging studies can be clinically relevant . hence , the time of initiation of treatment should be known to the radiologist , and the stability over a long period of time should be emphasized in the radiology report . immune - checkpoint inhibitors can cause a variety of iraes , either symptomatic or clinically silent , most of them associated with radiology manifestations . while both clinical and radiographic characterization of iraes is still ongoing , several these include enterocolitis , hepatitis , thyroid disorders , hypophysitis , pancreatitis , pneumonitis and dermatitis ( 50 ) . additional imaging findings , such as retroperitoneal stranding , sarcoid - like distributed lymphadenopathy , myositis and arthritis have also been attributed to the proinflammatory state elicited by icis . iraes are quite frequent as the body s innate control against autoimmunity is down - regulated by icis ( 50 ) , more so with ctla-4 inhibitors , which involves the early activation of t - cells , less with pd-1 and pd - l1 antibodies , involved in the later phases of the activation . with early detection , iraes can be successfully managed delaying or discontinuing the therapy , based on the severity of the event , and high dose corticosteroids . hence , prompt recognition of drug - related toxicity by the radiologist is crucial for clinical decisionmaking . pneumonitis : pneumonitis is a serious , potentially life - threatening iraes , which resulted in three deaths in a phase i trial . pneumonitis as new consolidative or ground - glass opacities has been reported in up to 5% of patients receiving immune checkpoint inhibition ( fig . 1 ; please also see fig . 4 m , p regarding pet findings ) ( 50 ) . although imaging features have yet to be described , initial reports of ici - related pneumonitis indicate a spectrum of radiographic manifestations of the entity . three cases of pneumonitis associated with the use of anti - pd-1 antibodies in patients with melanoma have been reported with two main radiologic patterns : an acute respiratory distress syndrome - like pattern , with diffuse ground - glass and reticular opacities , consolidation associated with traction bronchiectasis , decreased lung volumes and pleural effusion and a non - specific interstitial pneumonia ( nsip)-pattern , with peripheral ground - glass and reticular opacities , predominantly at the lung bases ( 5152 ) . a case study of two patients reports anti - pd-1 pneumonitis in advanced nsclc patients treated with nivolumab ( 51 ) . ct of both patients demonstrated ground - glass , reticular opacities and rapidly evolving consolidations in a peripheral distribution , in a radiographic pattern of cryptogenic organizing pneumonia . nivolumab was stopped and treatment with high dose corticosteroids was initiated , with improvement of respiratory symptoms and radiographic findings . after steroid taper , one patient , not on any active treatment , developed another episode of pneumonitis ( pneumonitis flare ) and had to be restarted on corticosteroids with subsequent clinical and radiologic improvement , further indicating the complicated nature of this irae ( fig . immune - related colitis is the most commonly reported irae , and can be observed even in asymptomatic patients . it is most commonly associated with ipilimumab and usually occurs between 510 weeks of initiation of therapy ( fig . two distinct ct appearances have been described : diffuse bowel wall thickening , with or without fluid filled distended colon , with mesenteric vessel engorgement , or segmental bowel wall thickening and pericolonic stranding , often superimposed to diverticulitis , so called segmental colitis associated with diverticulosis ( 54 ) . it is important to differentiate between the two patterns as their management differs : after stopping the drug , diffuse colitis is treated with high dose steroids , segmental colitis is treated with steroids and antibiotics . hepatitis : immune - related hepatitis usually appears between 614 weeks of initiation of therapy . imaging findings can be subtle and a high index of suspicion should be maintained in patients with systemic symptoms and altered liver function tests ( lfts ) . classic imaging appearance of acute hepatitis is seen in such patients , as reported in a study of 6 melanoma patients with ipilimumab - associated hepatitis ( 55 ) . conversely , imaging of asymptomatic patients , with mildly increased lfts can be normal ( 5556 ) . histologically , ipilimumab - associated hepatitis has been described as hepatocyte injury , with acute hepatitis pattern or as bile duct injury ( 56 ) . endocrine iraes are quite common and usually appear between 720 weeks of initiation of therapy . they might cause serious and life - changing symptoms due to permanent damage of the hypophysis or the thyroid glands . they have to be promptly recognized by the radiologist , as symptoms can resolve with adequate hormone replacement , allowing the patients to continue a therapy they are benefiting from . thyroid disorders are the most common clinically reported endocrine iraes , particularly in women on anti - pd-1 inhibitors and can clinically manifests with hypothyroidism , most commonly , followed by hyperthyroidisim , whereas thyroiditis has not yet been reported ( 5057 ) . imaging features have been described as new enlargement of the thyroid gland , enhancement and diffuse fludeoxyglucose ( fdg ) uptake on pet / ct , as reported by a study on ipilimumab related adverse event ( 50 ) . hypophysitis is more commonly associated with ipilimumab , is rare with pd-1 and pd - l1 inhibitor monotherapies , and has a median appearance at 11 weeks , but can be seen as early as 4 weeks ( fig . 4n ) ( 58 ) . clinical picture and imaging findings resemble those seen with primary lymphocytic hypophysitis , with avid enhancement of the hypophysis on contrast enhanced ct , t1 precontrast and t2 hypointensity and homogeneous enhancement on mri ( 57 ) . additional rare immune - related endocrinopathies have been described and include ipilimumab graves ophthalmopathy and anti pd-1 type i diabetes mellitus , which has been reported in only 4 patients to date , but imaging features have yet to be described ( fig . awareness that these findings might reflect inflammatory response rather than new sites of disease is crucial for the radiologist . examples include new sarcoid - like distributed lymph nodes ( bilateral hilar and mediastinal ) , which have been reported in up to 5% of patients on icis treatment ( 5059606162 ) . although the imaging appearance of these nodes can be indistinguishable from nodal metastases , the distribution resembling typical sarcoidosis , in absence of infection and occurring in the setting of response at other sites , favor sarcoid - like reaction and ace - level and tissue diagnosis can support the diagnosis of irae ( 50 ) . treatment should not be discontinued as these nodes are likely a flare phenomenon at initiation of therapy and might regress spontaneously . new abdominal lymphadenopathy , stranding in perirenal or retroperitoneal fat and focal muscle abnormalities have also been observed and are also likely a marker of lymphocytic infiltration , indicating treatment effect , rather than new sites of disease . new focal intramuscular enhancement seen on contrast - enhanced ct or new intramuscular tracer uptake on pet / ct has been described as drug - related myositis and should be reported . an interesting relationship between tumor response and iraes has been described : radiologic manifestations of iraes have been associated with improved tumor response and disease control ( 53 ) . a study of 119 patients with metastatic melanoma on ipilimumab therapy showed iraes in 17% of patients , who were more likely to show response and disease control ( 55% ) ( 63 ) . for example , ipilimumab and nivolumab therapy for the treatment of metastatic melanoma , as it demonstrates increase antitumor activity , shows increased incidence of drug - associated adverse events . the combination of pembrolizumab and ipilimumab has also been associated with increased adverse events , the thyroid gland being most affected organ , manifesting as hypothyroidism . this interesting relationship between response and toxicities , however , need to be further investigated with a rigorous study design controlling for important factors such as time of therapy duration . immune - checkpoint inhibitors consist of a new paradigm of anti - cancer drugs approved for treatment of a variety of solid and hematologic malignancies . with the increasing use of icis in a growing number of tumor types , awareness of the immune - related patterns of response , the clinical and imaging manifestations of iraes is key for radiologists , as they become increasingly involved in and key contributors of cancer patient care .

over the past five years immune - checkpoint inhibitors have dramatically changed the therapeutic landscape of advanced solid and hematologic malignancies . the currently approved immune - checkpoint inhibitors include antibodies to cytotoxic t - lymphocyte antigen-4 , programmed cell death ( pd-1 ) , and programmed cell death ligand ( pd - l1 and pd - l2 ) . response to immune - checkpoint inhibitors is evaluated on imaging using the immune - related response criteria . activation of immune system results in a unique toxicity profile termed immune - related adverse events . this article will review the molecular mechanism , clinical applications , imaging of immune - related response patterns and adverse events associated with immune - checkpoint inhibitors .

INTRODUCTION Immunotherapy Immune-Checkpoint Inhibitors: Mechanism of Action Immune-Related Imaging Features Immune-Related Response and Immune Response Criteria Immune-Related Response in the Radiology Reading Room Immune-Related Adverse Events CONCLUSION

the term targeted therapy encompasses a wide spectrum of drug classes including inhibitors of signal transduction ( receptor tyrosine kinases ) , hormonal agents , inhibitors of angiogenesis , dna damage modulators and modulators of immune system , to name a few . of these targeted therapies , immune modulators or immune - checkpoint inhibitors ( icis ) have garnered significant attention in the last 5 years due to their increasing spectrum of activity in oncology and have , as such , radically changed the therapeutic landscape of advanced solid and hematologic malignancies . owing to their unique anti - cancer mechanism , icis are associated with unusual response patterns and adverse event profiles on imaging , which are currently under investigation . as icis become the standard of care for an increasing number of cancers and prolong life expectancy of patients with metastatic disease , radiologists , as an integral part of the multidisciplinary oncologic patient care , need be familiar with the mechanistic background of these anticancer agents , their immune - related tumor response patterns , immune - related adverse event ( irae ) profiles , to contribute to clinical decision making . in this article , we will review the molecular bases of anti - cancer immunotherapeutic agents , their clinical application in solid and hematologic malignancies , the immune - related patterns of response observed on imaging studies and the imaging features of iraes . using the immune system to treat diseases dates back to 1796 , when edward jenner produced the first vaccine involving immunization with cowpox to prevent smallpox . since then , cancer immunotherapy , which utilizes the immune system to kill cancer cells , has become an established modality for cancer care . several agents have been used over the years , targeting the immune systems at different levels and in different ways . patient s own t - cells have been engineered with chimeric antigen receptors to recognize and fight leukemias and lymphomas ( 5 ) . more recently , icis , which act unleashing the immune system to fight the cancer , have gained fda approval for treatment of different types of solid and hematologic malignancies ( 6 ) . immune - checkpoint inhibitors act by inhibiting crucial regulatory steps in the immune system , promoting the activation and proliferation of t - cells to induce tumor infiltration and regression . based on the checkpoint target , three main types of icis have been studied so far and gained fda approval : cytotoxic t - lymphocyte antigen-4 ( ctla-4 ) antibodies and programmed cell death ( pd-1 ) and programmed cell death ligand ( pd - l1 and pd - l2 ) antibodies . ipilimumab ( yervoy ; bristol - myers squibb , new york , ny , usa ) , a ctla4 inhibitor , was the first ici , to gain approval by the u.s . fda in 2011 , based on two pivotal randomized trials ( 1 ) , which have shown improved overall survival in patients with advanced melanoma . pd-1 , pd - l1 and pd - l2 are transmembrane inhibitory proteins expressed on t cells , b cells and natural killer ( nk ) cells , binding to pd - ligand 1 and pd - ligand 2 on multiple tissue types and hematopoietic cells , respectively ( 13 ) . nivolumab , igg4 subclass pd-1 inhibitor , is fda approved for metastatic melanoma , advanced non - small cell lung cancer ( nsclc ) ( 17 ) , rcc ( 18 ) and hodgkin s disease ( 1920212223242526 ) . in addition , these agents and other antibodies targeting pd-1 or pd - l1 are under investigation in a broad spectrum of malignancies , such as mismatch - repair deficient colorectal carcinoma , non - hodgkin s lymphoma and cancers of the head and neck . other potential targets for checkpoint inhibition have been studied , including t - cell immunoglobulin and mucin domain 3 , lymphocyte activation gene 3 , b and t - cell lymphocyte attenuator , and v - domain ig suppressor of t - cell activation . blocking the ctla-4 and the pd-1/pd - l1 pathways , in fact , has been proved to have synergistic effect ( 2829 ) . cancer immunotherapy is associated with a variety of new imaging features , including unconventional response patterns and new spectrum of drug - related toxicities . treatment response to immune - modulatory agents , in fact , can be seen long after the start of treatment ; furthermore , increased and new lesions soon after start of treatment do not necessarily represent worsening of disease , as they do with conventional therapies . the pseudoprogression pattern of response is supported by a case study of an ipilimumab - treated patient which showed increased tumor burden on imaging after 12 weeks of therapy , while histologic exam demonstrated that the increased in size was due to inflammatory response caused by t - cell infiltration rather than tumor cell proliferation ( 30 ) . in this setting of unconventional response , the application of conventional tumor response criteria , such as response evaluation criteria for solid tumors ( recist ) and world health organiziation ( who ) criterion , where new lesions and increase tumor size suggest progression of disease , would underestimate the benefit of therapy and lead to premature discontinuation of treatment . hence , to assess this atypical pattern of response , in 2009 , a committee of 200 oncologists , immunotherapists , and regulatory experts proposed a new immune - related response criteria ( irrc ) ( 34353637 ) . immune - related response criteria revisits the definition of progression of disease , preventing new measurable or non - measurable lesions from defining progression and incorporates new measurable lesions into the tumor burden . radiologic aspects of immune - related tumor response criteria and patterns of iraes in patients undergoing ipilimumab therapy ( 3133 ) . maintaining the concept of measurable and non - measurable lesions and the bidimensional measurement according to who criterion , irrc proposes a new immune - related ( ir ) response assessment , as follow : 1 ) immune - related complete response : complete response is defined as disappearance of all measureable and non - measurable lesions , with no new lesions . 2 ) immune - related partial response : partial response is defined as decrease in the total tumor burden of 50 percent or more from the baseline , confirmed by a subsequent scan at least four weeks later . 3 ) immune - related stable disease : stability of the tumor burden , not meeting the criteria for partial , complete response or for progressive disease . 4 ) immune - related progressive disease : progression of disease is defined as 25 percent increase in the tumor burden from the baseline or nadir study . tumor density , volume , metabolic activities and other functional information , should also be incorporated in the criterion to evaluate immune - related response and are under investigation ( 3644 ) . ( 49 ) evaluated atypical response patterns using irrc in comparison with recist1.1 in 327 patients with melanoma during treatment with pembrolizumab , the largest retrospective study to date , evaluating immune - related responses in patients on pd-1 inhibitor therapy . the article provides important insights for future directions toward an irrecist 1.1 , which , maintaining the modifications introduced by irrc , will propose a common denominator with conventional recist , for allowing accurate comparison of the immune - related response ( 39 ) . hence , the time of initiation of treatment should be known to the radiologist , and the stability over a long period of time should be emphasized in the radiology report . immune - checkpoint inhibitors can cause a variety of iraes , either symptomatic or clinically silent , most of them associated with radiology manifestations . iraes are quite frequent as the body s innate control against autoimmunity is down - regulated by icis ( 50 ) , more so with ctla-4 inhibitors , which involves the early activation of t - cells , less with pd-1 and pd - l1 antibodies , involved in the later phases of the activation . with early detection , iraes can be successfully managed delaying or discontinuing the therapy , based on the severity of the event , and high dose corticosteroids . hence , prompt recognition of drug - related toxicity by the radiologist is crucial for clinical decisionmaking . pneumonitis : pneumonitis is a serious , potentially life - threatening iraes , which resulted in three deaths in a phase i trial . three cases of pneumonitis associated with the use of anti - pd-1 antibodies in patients with melanoma have been reported with two main radiologic patterns : an acute respiratory distress syndrome - like pattern , with diffuse ground - glass and reticular opacities , consolidation associated with traction bronchiectasis , decreased lung volumes and pleural effusion and a non - specific interstitial pneumonia ( nsip)-pattern , with peripheral ground - glass and reticular opacities , predominantly at the lung bases ( 5152 ) . ct of both patients demonstrated ground - glass , reticular opacities and rapidly evolving consolidations in a peripheral distribution , in a radiographic pattern of cryptogenic organizing pneumonia . immune - related colitis is the most commonly reported irae , and can be observed even in asymptomatic patients . it is most commonly associated with ipilimumab and usually occurs between 510 weeks of initiation of therapy ( fig . two distinct ct appearances have been described : diffuse bowel wall thickening , with or without fluid filled distended colon , with mesenteric vessel engorgement , or segmental bowel wall thickening and pericolonic stranding , often superimposed to diverticulitis , so called segmental colitis associated with diverticulosis ( 54 ) . hepatitis : immune - related hepatitis usually appears between 614 weeks of initiation of therapy . conversely , imaging of asymptomatic patients , with mildly increased lfts can be normal ( 5556 ) . imaging features have been described as new enlargement of the thyroid gland , enhancement and diffuse fludeoxyglucose ( fdg ) uptake on pet / ct , as reported by a study on ipilimumab related adverse event ( 50 ) . hypophysitis is more commonly associated with ipilimumab , is rare with pd-1 and pd - l1 inhibitor monotherapies , and has a median appearance at 11 weeks , but can be seen as early as 4 weeks ( fig . additional rare immune - related endocrinopathies have been described and include ipilimumab graves ophthalmopathy and anti pd-1 type i diabetes mellitus , which has been reported in only 4 patients to date , but imaging features have yet to be described ( fig . examples include new sarcoid - like distributed lymph nodes ( bilateral hilar and mediastinal ) , which have been reported in up to 5% of patients on icis treatment ( 5059606162 ) . new focal intramuscular enhancement seen on contrast - enhanced ct or new intramuscular tracer uptake on pet / ct has been described as drug - related myositis and should be reported . an interesting relationship between tumor response and iraes has been described : radiologic manifestations of iraes have been associated with improved tumor response and disease control ( 53 ) . for example , ipilimumab and nivolumab therapy for the treatment of metastatic melanoma , as it demonstrates increase antitumor activity , shows increased incidence of drug - associated adverse events . the combination of pembrolizumab and ipilimumab has also been associated with increased adverse events , the thyroid gland being most affected organ , manifesting as hypothyroidism . cancer immunotherapy is associated with a variety of new imaging features , including unconventional response patterns and new spectrum of drug - related toxicities . treatment response to immune - modulatory agents , in fact , can be seen long after the start of treatment ; furthermore , increased and new lesions soon after start of treatment do not necessarily represent worsening of disease , as they do with conventional therapies . the pseudoprogression pattern of response is supported by a case study of an ipilimumab - treated patient which showed increased tumor burden on imaging after 12 weeks of therapy , while histologic exam demonstrated that the increased in size was due to inflammatory response caused by t - cell infiltration rather than tumor cell proliferation ( 30 ) . in this setting of unconventional response , the application of conventional tumor response criteria , such as response evaluation criteria for solid tumors ( recist ) and world health organiziation ( who ) criterion , where new lesions and increase tumor size suggest progression of disease , would underestimate the benefit of therapy and lead to premature discontinuation of treatment . hence , to assess this atypical pattern of response , in 2009 , a committee of 200 oncologists , immunotherapists , and regulatory experts proposed a new immune - related response criteria ( irrc ) ( 34353637 ) . immune - related response criteria revisits the definition of progression of disease , preventing new measurable or non - measurable lesions from defining progression and incorporates new measurable lesions into the tumor burden . radiologic aspects of immune - related tumor response criteria and patterns of iraes in patients undergoing ipilimumab therapy ( 3133 ) . maintaining the concept of measurable and non - measurable lesions and the bidimensional measurement according to who criterion , irrc proposes a new immune - related ( ir ) response assessment , as follow : 1 ) immune - related complete response : complete response is defined as disappearance of all measureable and non - measurable lesions , with no new lesions . 2 ) immune - related partial response : partial response is defined as decrease in the total tumor burden of 50 percent or more from the baseline , confirmed by a subsequent scan at least four weeks later . 3 ) immune - related stable disease : stability of the tumor burden , not meeting the criteria for partial , complete response or for progressive disease . 4 ) immune - related progressive disease : progression of disease is defined as 25 percent increase in the tumor burden from the baseline or nadir study . tumor density , volume , metabolic activities and other functional information , should also be incorporated in the criterion to evaluate immune - related response and are under investigation ( 3644 ) . ( 49 ) evaluated atypical response patterns using irrc in comparison with recist1.1 in 327 patients with melanoma during treatment with pembrolizumab , the largest retrospective study to date , evaluating immune - related responses in patients on pd-1 inhibitor therapy . the article provides important insights for future directions toward an irrecist 1.1 , which , maintaining the modifications introduced by irrc , will propose a common denominator with conventional recist , for allowing accurate comparison of the immune - related response ( 39 ) . hence , the time of initiation of treatment should be known to the radiologist , and the stability over a long period of time should be emphasized in the radiology report . immune - checkpoint inhibitors can cause a variety of iraes , either symptomatic or clinically silent , most of them associated with radiology manifestations . iraes are quite frequent as the body s innate control against autoimmunity is down - regulated by icis ( 50 ) , more so with ctla-4 inhibitors , which involves the early activation of t - cells , less with pd-1 and pd - l1 antibodies , involved in the later phases of the activation . with early detection , iraes can be successfully managed delaying or discontinuing the therapy , based on the severity of the event , and high dose corticosteroids . hence , prompt recognition of drug - related toxicity by the radiologist is crucial for clinical decisionmaking . pneumonitis : pneumonitis is a serious , potentially life - threatening iraes , which resulted in three deaths in a phase i trial . although imaging features have yet to be described , initial reports of ici - related pneumonitis indicate a spectrum of radiographic manifestations of the entity . three cases of pneumonitis associated with the use of anti - pd-1 antibodies in patients with melanoma have been reported with two main radiologic patterns : an acute respiratory distress syndrome - like pattern , with diffuse ground - glass and reticular opacities , consolidation associated with traction bronchiectasis , decreased lung volumes and pleural effusion and a non - specific interstitial pneumonia ( nsip)-pattern , with peripheral ground - glass and reticular opacities , predominantly at the lung bases ( 5152 ) . ct of both patients demonstrated ground - glass , reticular opacities and rapidly evolving consolidations in a peripheral distribution , in a radiographic pattern of cryptogenic organizing pneumonia . immune - related colitis is the most commonly reported irae , and can be observed even in asymptomatic patients . it is most commonly associated with ipilimumab and usually occurs between 510 weeks of initiation of therapy ( fig . two distinct ct appearances have been described : diffuse bowel wall thickening , with or without fluid filled distended colon , with mesenteric vessel engorgement , or segmental bowel wall thickening and pericolonic stranding , often superimposed to diverticulitis , so called segmental colitis associated with diverticulosis ( 54 ) . hepatitis : immune - related hepatitis usually appears between 614 weeks of initiation of therapy . classic imaging appearance of acute hepatitis is seen in such patients , as reported in a study of 6 melanoma patients with ipilimumab - associated hepatitis ( 55 ) . conversely , imaging of asymptomatic patients , with mildly increased lfts can be normal ( 5556 ) . imaging features have been described as new enlargement of the thyroid gland , enhancement and diffuse fludeoxyglucose ( fdg ) uptake on pet / ct , as reported by a study on ipilimumab related adverse event ( 50 ) . hypophysitis is more commonly associated with ipilimumab , is rare with pd-1 and pd - l1 inhibitor monotherapies , and has a median appearance at 11 weeks , but can be seen as early as 4 weeks ( fig . additional rare immune - related endocrinopathies have been described and include ipilimumab graves ophthalmopathy and anti pd-1 type i diabetes mellitus , which has been reported in only 4 patients to date , but imaging features have yet to be described ( fig . examples include new sarcoid - like distributed lymph nodes ( bilateral hilar and mediastinal ) , which have been reported in up to 5% of patients on icis treatment ( 5059606162 ) . new focal intramuscular enhancement seen on contrast - enhanced ct or new intramuscular tracer uptake on pet / ct has been described as drug - related myositis and should be reported . an interesting relationship between tumor response and iraes has been described : radiologic manifestations of iraes have been associated with improved tumor response and disease control ( 53 ) . for example , ipilimumab and nivolumab therapy for the treatment of metastatic melanoma , as it demonstrates increase antitumor activity , shows increased incidence of drug - associated adverse events . the combination of pembrolizumab and ipilimumab has also been associated with increased adverse events , the thyroid gland being most affected organ , manifesting as hypothyroidism . immune - checkpoint inhibitors consist of a new paradigm of anti - cancer drugs approved for treatment of a variety of solid and hematologic malignancies . with the increasing use of icis in a growing number of tumor types , awareness of the immune - related patterns of response , the clinical and imaging manifestations of iraes is key for radiologists , as they become increasingly involved in and key contributors of cancer patient care .

cloning , expression , and purification of recombinant sbi fragments dna sequences encoding several sbi fragments were amplified by pcr using s. aureus strain mu50 genomic dna as template and cloned into a pet - based vector with a tev - cleavable n - terminal hexahistidine tag ( 27 ) , as described previously ( 23 , 24 ) . cells were grown at 37 c to an a600 value of 0.6 when they were induced by addition of isopropyl-1-thio--d - galactopyranoside ( melford ) to 0.2 mm and cultured for a further 4 h. n - labeled sbi - iii and n - labeled and n / c - labeled sbi - iv for nmr spectroscopy were produced by expression in minimal medium with nh4cl and nh4cl / c6-d - glucose ( spectra stable isotopes ) as the sole nitrogen and nitrogen / carbon sources . following cell harvest by centrifugation and lysis by sonication , the lysate was centrifuged at 40,000 g for 15 min , and the supernatant filtered through a 0.45-m filter . sbi fragments were purified by nickel ion - chelating chromatography using a 1-ml histrap column attached to ankta purifier ( ge healthcare ) . the column was then washed with binding buffer , and the bound proteins were eluted using a 0.051.0 m imidazole gradient . for nmr data acquisition , the n - terminal hexahistidine tag was removed by incubation with tev protease ( invitrogen ) , and the hexahistidine tag - free sbi fragment was then separated from tev protease ( itself his - tagged ) by a second passage through a histrap column . following tev removal of the hexahistidine tag , the sbi proteins contained n - terminal residues gam from the expression vector . nmr sample generation and nmr spectroscopy of sbi - iii and sbi - iv purified sbi - iii and sbi - iv were exchanged into 5 mm mes , 100 mm nacl , 1 mm edta , 1 mm benzamidine , ph 5.5 . all nmr data were acquired at 16 c on a varian unity inova spectrometer operating at a nominal proton frequency of 600 mhz , using a triple resonance 5-mm probe equipped with z - axis - pulsed - field gradients . nmr data were processed using the nmrpipe / nmrdraw software suite ( 28 ) and analyzed using ccpn analysis ( 29 ) . sequence - specific backbone resonance assignments of sbi - iv were made using three - dimensional hncacb , cbca(co)nh , hnco , hn(ca)co , hnha , and hbha(cbcaco)nh data sets . side chain resonance assignments of sbi - iv were made using ccc - tocsy - nnh , hcc - tocsy - nnh , hcch tocsy , and n - edited tocsy ( 12.1 , 12.1 , 15.6 , and 50 ms mixing times ) . sbi - iv noe distance restraints were obtained by analysis of n - noesy hsqc ( 50 , 100 , and 150 ms mixing times ) and c - noesy hsqc ( 70 and 175 ms mixing times ) spectra . distances involving methyl groups , aromatic ring protons , and non - stereospecifically assigned methylene protons were represented as a ( r ) sum ( 31 ) . backbone dihedral angles and were predicted from c , c , c , h , and backbone n chemical shifts using talos ( 32 ) . the dihedral angles were restrained to talos - predicted values 30 or 40 , and dihedral angles were restrained to talos - predicted values 50. hydrogen bond distance restraints were established from noe patterns , and the proximities of donor and acceptor groups in structures calculated using noe - derived distance restraints . for hydrogen bond restraints , the nh - o distance was assigned lower and upper distance bounds of 1.5 and 2.5 , and the n - o distance assigned lower and upper distance bounds of 2.5 and 3.5 . structures were calculated using the python interface of xplor - nih 2.18 ( 33 , 34 ) , using simulated annealing starting from random extended structures . ensembles of nmr structures were analyzed for violated restraints using the vmd - xplor visualization package ( 35 ) . the structure determination was carried out iteratively whereby consistently violated restraints were reassigned , wherever possible , using existing structures or removed until a consistent set of constraints was obtained with few violations in the ensemble . the ensemble of structures was further refined with xplor - nih standard refinement protocols using the final set of restraints . site - directed mutagenesis of sbi - iv sbi - iv r231a and n238a single mutants and r231a - n238a double mutant were made using the quikchange site - directed mutagenesis kit ( stratagene ) . the following oligonucleotide primers ( mwg biotech ag ) were used to make the sbi - iv r231a and n238a mutations : 5-caa ttg aaa aca gag ctt tag cac aac gag aag-3 ( forward primer ) , 5-ctt ctc gtt gtg cta aag ctc tgt ttt caa ttg-3 ( reverse primer ) , 5-gca caa cga gaa gtt gcc aaa gca cct atg gat gta aaa gag-3 ( forward primer ) , 5-ctc ttt tac atc cat agg tgc ttt ggc aac ttc tcg ttg tgc-3 ( reverse primer ) . the mutant proteins were expressed and purified as described above for wild - type sbi - iv . isothermal titration calorimetry experiments were done at 25 c using a vp - itc calorimeter ( microcal ) in a very similar way to that described for efb - c3 measurements ( 12 ) . sbi - iv , sbi - iii - iv , and c3dg were exchanged into 20 mm tris , 150 mm nacl , ph 8.0 . for each interaction , reciprocal experiments were done , one with c3dg in the cell and sbi - iv or sbi - iii - iv in the syringe and the other with sbi - iv or sbi - iii - iv in the cell and c3dg in the syringe . the concentration of protein in the cell ranged between 10.4 and 19 m , and the concentration of protein in the syringe ranged between 156 and 203 m . a 1-l injection of protein into the cell was followed by multiple injections of 5 l at 4-min intervals , and the evolved heat was measured . purified sbi - i - ii , sbi - ii , sbi - iii , sbi - iv , sbi - iv r231a , sbi - iv n238a , and sbi - iv r231a - n238a were each covalently coupled to a 1-ml nhs - activated sepharose high performance column ( ge healthcare ) according to the manufacturer 's instructions . after equilibration with phosphate - buffered saline ( pbs ) , 5 ml of human serum ( cambrex ) was applied to the column . columns were washed with pbs , and bound proteins were eluted with a 01 m nacl gradient over 10 ml . size exclusion chromatography , with a superdex-200 gel filtration column ( ge healthcare ) , was used for further purification of the bound fragments . surface plasmon resonance experiments were performed on a biacore x ( biacore , piscataway , nj ) instrument at 25 c in hepes - buffered saline ( 10 mm hepes , ph 7.2 , 3 mm edta , 150 mm nacl , 0.005% ( v / v ) surfactant p-20 ( biacore ) ) . 800 resonance units ( ru ) of c3dg were immobilized on a cm-5 sensor chip ( biacore ) by standard amino coupling chemistry following the manufacturer 's protocol , with a reference channel being sham - activated and deactivated . wild - type or mutant sbi - iv analytes were injected at a flow rate of 20 l / min for 60 s at the concentrations indicated in fig . the sensor chip was regenerated between successive analyte injections with a 60-s pulse of 2 m nacl , which brought the sensorgram signal back to baseline . complement assays the wielisa total complement system screen ( wieslab ) , described by seelen et al . ( 37 ) , was used to measure inhibition of the alternative complement pathway by a range of concentrations of sbi - iv , sbi - iv r231a , sbi - iv n238a , and sbi - iv r231-n238a , using a procedure similar to that described previously ( 23 ) . briefly , wild - type or mutant sbi - iv was added to a particular concentration in human serum , and assayed for complement activity after 30 min of preincubation at 37 c . the assay was completed in duplicate , according to the manufacturer 's instructions , and included a blank , a positive control ( human serum from healthy individuals ) , and a negative control ( heat - inactivated serum ) . inhibition of complement activation was quantified from absorbance at 405 nm using the calculation ( sample - negative control)/(positive control - negative control ) 100% . sbi - iv structure determination a semi - automated procedure for iterative noe assignment was used to determine the structure of sbi - iv . the final structures were generated using 1116 noe - derived distance restraints ( comprising 320 intraresidue , 621 sequential and medium range , and 175 long range noes where long range means between amino acids five or more apart in the sequence ) , 78 hydrogen bond restraints and 138 and dihedral angle restraints ( 68 and 70 ) ( table 1 ) . the ensemble of 20 simulated annealing structures selected from 50 calculations on the basis of lowest energy , and the average structure , are shown in fig . the ensemble of structures has a backbone r.m.s.d . from the mean of 0.29 and an r.m.s.d . a ramachandran plot of the structures with procheck - nmr ( 36 ) indicates that 100% of the residues ( excluding gly and pro residues ) lie in the most favored or additionally allowed regions ( table 1 ) . table 1structural statistics on the nmr - derived structures of sbi - iv total number of noe restraints 1116 intraresidue 320 sequential / med . range ( i to i + 1 4 ) 621 long range 175 number of dihedral angle restraints 138 number of hydrogen bond restraints 78 r.m.s.d . for backbone atoms 0.29 r.m.s.d . for non - hydrogen atoms 0.93 average numbers of noe violations > 0.5 ( per structure ) 1.5 average number of dihedral angle violations > 5 ( per structure ) 0 average structureensembleramachandran plot regions most favored ( % ) 88.4 92.4 additional allowed ( % ) 11.6 7.6 generously allowed ( % ) 0.0 0.0 disallowed ( % ) 0.0 0.0 athe r.m.s.d . from the mean structure calculated over residues 201222 , 228238 , and 245261bcalculated with procheck - nmr ( 36 ) structural statistics on the nmr - derived structures of sbi - iv the r.m.s.d . from the mean structure calculated over residues 201222 , 228238 , and 245261 calculated with procheck - nmr ( 36 ) sbi - iv forms a three - helix bundle sbi - iv adopts a three - helix bundle fold ( fig . 1 ) with approximate overall dimensions of 45 25 25 . helix 1 ( residues glu-201 to asn-222 ) is connected by a loop to helix 2 ( residues glu-228 to asn-238 ) , which is connected by a loop to helix 3 ( residues lys-245 to ala-261 ) . none of the residues 198206 and 257266 is involved in long range noes , and these residues make essentially no contribution to the tertiary structure of the domain . the core of the bundle comprises hydrophobic residues with hydrophilic side chains oriented toward solvent . a similar three - helix bundle fold has been observed in the other s. aureus complement modulatory proteins efb - c ( 12 ) , ehp ( 13 ) , scin ( 25 ) , and spa ( 26 ) ( fig . a , backbone ( n , c , and c ) trace of the 20 lowest energy structures colored as a continuum from blue at the n terminus to red at the c terminus . b , ribbon diagram of the average structure colored as in a. the -helices and n and c termini are labeled . a , backbone ( n , c , and c ) trace of the 20 lowest energy structures colored as a continuum from blue at the n terminus to red at the c terminus . b , ribbon diagram of the average structure colored as in a. the -helices and n and c termini are labeled . sbi - iv ( pdb code 2jvh ) , efb - c ( pdb 2gom ) , ehp ( pdb 2noj ) , scin ( pdb 2qff ) , and protein a domain b ( pdb 1ss1 ) are shown in ribbon and schematic topology representations . sbi - iv ( pdb code 2jvh ) , efb - c ( pdb 2gom ) , ehp ( pdb 2noj ) , scin ( pdb 2qff ) , and protein a domain b ( pdb 1ss1 ) are shown in ribbon and schematic topology representations . this compares with 56 peaks expected based on the amino acid sequence of the construct including the three n - terminal residues , gam , from the expression vector . this almost exact 1:1 correspondence with the expected number of peaks indicates that sbi - iii adopts a single conformation in solution , but the narrow h chemical shift dispersion , with every backbone nh peak except three located in the range 8.08.5 ppm , indicates that this conformation lacks significant tertiary structure , i.e. sbi - iii is natively unfolded . the peak patterns in the h - n hsqc spectra of sbi - iv ( supplemental fig . s3 ) indicate that sbi - iii remains unfolded in the presence of sbi - iv . we can not preclude , however , the possibility that sbi - iii adopts a different conformation in full - length sbi where its conformation may be influenced by one or more of the other domains . sbi - iv and sbi - iii - iv bind c3dg with 1:1 stoichiometry thermodynamics of the interactions between sbi - iv and c3dg and between sbi - iii - iv and c3dg were measured by reciprocal ( sbi : c3dg and c3dg : sbi ) isothermal titration calorimetry experiments ( fig . 3 and table 2 ) . the itc results indicate that the stoichiometry of both sbi - iv : c3dg and sbi - iii - iv : c3dg interactions is 1:1 with kd values of 0.35 m and 0.28 m ( averages of results from reciprocal itc titrations ) . these affinities are lower than the nanomolar to subnanomolar c3dg affinities previously measured by spr and itc for efb - c ( 12 ) and ehp ( 13 ) . table 2assessment of c3dg binding to sbi - iii - iv and sbi - iv by isothermal titration calorimetrytitrantcellnkdhs mkcal / molcal / mol / k sbi - iii - iv c3dg 1.04 0.40 4.6 13.9 sbi - iv c3dg 1.15 0.36 3.8 16.7 c3dg sbi - iii - iv 0.92 0.17 3.9 17.8 c3dg sbi - iv 0.93 0.34 4.6 14.0 an , molar ratio of titrant molecule bound to ligand molecule in the cell at satration assessment of c3dg binding to sbi - iii - iv and sbi - iv by isothermal titration calorimetry n , molar ratio of titrant molecule bound to ligand molecule in the cell at satration figure 3.isothermal titration calorimetry analyses of the sbi - iv : c3dg ( a ) and sbi - iii - iv : c3dg ( b ) interactions . in a , the concentration of c3dg in the cell was 10.4 m , and the concentration of the sbi - iv injectant in the syringe was 203 m . in b , the concentration of c3dg in the cell was 12.0 m , and the concentration of the sbi - iii - iv injectant in the syringe was 173 m . in each case , the data were fit to a single class of binding site model , and the best fit parameters for n ( stoichiometry ) , kd , and h , along with the error estimates of the fit , are provided as an inset to each panel . the reciprocal experiments in which c3dg was titrated into sbi - iv or sbi - iii - iv were also performed ( results summarized in table 2 ) . isothermal titration calorimetry analyses of the sbi - iv : c3dg ( a ) and sbi - iii - iv : c3dg ( b ) interactions . in a , the concentration of c3dg in the cell was 10.4 m , and the concentration of the sbi - iv injectant in the syringe was 203 m . in b , the concentration of c3dg in the cell was 12.0 m , and the concentration of the sbi - iii - iv injectant in the syringe was 173 m . in each case , the data were fit to a single class of binding site model , and the best fit parameters for n ( stoichiometry ) , kd , and h , along with the error estimates of the fit , are provided as an inset to each panel . the reciprocal experiments in which c3dg was titrated into sbi - iv or sbi - iii - iv were also performed ( results summarized in table 2 ) . efb residues important for c3d binding align with the corresponding residues in sbi - iv the sequence identity between sbi - iv ( 69 residues ) and efb - c ( 72 residues ) is only 19% over a 43-residue stretch , with five of eight identical residues located in helix 2 ( fig . , sbi - iv helix 2 residues arg-231 and asn-238 align with two efb residues , arg-131 and asn-138 , that are particularly important for formation of the efb - c - c3d complex ( 12 ) . two other efb residues that contact c3d , his-130 and lys-135 , are conservatively substituted by arginines in sbi - iv . in the efb - c - c3d complex , arg-131 lies deep in an acidic pocket of c3d , and asn-138 is involved in an intermolecular hydrogen bonding network . alignment with arg-131 and asn-138 of efb suggests that sbi residues arg-231 and asn-238 are key residues for sbi interaction with c3 . r231a and n238a mutations abolish sbi - iv binding to c3dg as a consequence of the sequence alignment results mentioned in the previous section , sbi - iv r231a and sbi - iv n238a single mutants and sbi - iv r231a - n238a double mutant were made . as shown by affinity pulldown experiments , both single and double mutations effectively abolished binding between sbi - iv and c3dg - containing c3 derivatives present in serum ( fig . 6 ) , where the binding interaction between sbi - iv and biosensor - bound c3dg was abolished when any of the sbi - iv single or double mutants was used as an analyte . a similar effect was previously observed upon double mutation of efb residues arg-131 and asn-138 ( 12 ) , but the results of similar experiments with ehp were complicated by the presence of a second lower affinity binding site on ehp ( 13 ) . at about 0.6 m when sbi - iv is the analyte ( this study , data not shown ) and 0.8 m when c3dg is the analyte ( 23 ) , the wild - type sbi - iv : c3dg affinity measured by spr is slightly lower than the 0.35 m affinity measured by itc ( see above ) . this may simply reflect the superimposed surface effects of spr due to the need to chemically couple one of the binding partners to the biosensor surface , relative to the pure solution phase interaction measured in the itc experiment . figure 4.sequence alignment of sbi - iv , efb - c , ehp , scin , and spa - b . sbi - iv residues arg-231 and asn-238 are highlighted by green rectangles ; the corresponding efb - c residues are arg-131 and asn-138 , and the corresponding ehp residues are arg-75 and asn-82 . sequence alignment of sbi - iv , efb - c , ehp , scin , and spa - b . sbi - iv residues arg-231 and asn-238 are highlighted by green rectangles ; the corresponding efb - c residues are arg-131 and asn-138 , and the corresponding ehp residues are arg-75 and asn-82 . sds - page analysis of an affinity pulldown assay with human serum as prey and various individual and combined sbi domains as bait proteins immobilized on an nhs - sepharose column . the first lane is a ladder of molecular weight marker proteins with molecular weights indicated . the next two lanes show capture of igg heavy- ( h , 50 kda ) and light - chain ( l , 25 kda ) fragments by sbi - ii and sbi - i - ii . the fifth and sixth lanes show that sbi - iii - iv and sbi - iv capture fragments of complement component c3 with approximate molecular masses of 119 , 75 , 65 , 43 , and 40 kda ( c3 chain origin of the fragments is indicated ) . the last three lanes show that sbi - iv mutants r231a , n238a , and r231a - n238a fail to capture c3 and instead capture albumin . sds - page analysis of an affinity pulldown assay with human serum as prey and various individual and combined sbi domains as bait proteins immobilized on an nhs - sepharose column . the first lane is a ladder of molecular weight marker proteins with molecular weights indicated . the next two lanes show capture of igg heavy- ( h , 50 kda ) and light - chain ( l , 25 kda ) fragments by sbi - ii and sbi - i - ii . the fifth and sixth lanes show that sbi - iii - iv and sbi - iv capture fragments of complement component c3 with approximate molecular masses of 119 , 75 , 65 , 43 , and 40 kda ( c3 chain origin of the fragments is indicated ) . the last three lanes show that sbi - iv mutants r231a , n238a , and r231a - n238a fail to capture c3 and instead capture albumin . spr sensorgrams are shown for sbi - iv wild - type and sbi - iv mutants r231a , n238a , and r231a - n238a . spr sensorgrams are shown for sbi - iv wild - type and sbi - iv mutants r231a , n238a , and r231a - n238a . r231a and n238a mutations abolish sbi - iv inhibition of the alternative complement pathway sbi - iv inhibited the alternative pathway in a dose - dependent manner ( fig . all of the r231a , n238a , and r231a - n238a mutations abolished sbi - iv inhibition of the alternative pathway ( fig . 7 ) in the same way that double mutation of efb residues arg-131 and asn-138 abolished alternative pathway blockade by efb ( 12 ) . the ic50 value for sbi - iv alternative pathway inhibition was 10 m compared with 0.56 m for efb , 0.41 m for efb - c ( 12 ) and 0.12 m for ehp ( 13 ) . s. aureus has evolved a complement system subversion strategy incorporating several secreted proteins that inhibit the complement cascade at different points using a variety of mechanisms ( 911 , 1315 , 17 , 20 , 38 ) . sbi is one such protein ( 23 ) , although it was originally discovered in the late 1990s as a binder of igg ( 21 ) and 2-glycoprotein i ( 22 ) , a membrane adhesion protein implicated in blood coagulation . the same authors subsequently showed that expression of sbi is induced by human igg ( 39 ) . we have recently characterized in some detail the igg binding properties of sbi but have found no evidence that sbi binds 2-glycoprotein i ( 24 ) . dose response residual complement activity for sbi - iv and sbi - iv mutants r231a , n238a , and double mutant r231a - n238a were assessed in a wielisa assay . dose response residual complement activity for sbi - iv and sbi - iv mutants r231a , n238a , and double mutant r231a - n238a were assessed in a wielisa assay . sbi can form a precipitate with human igg through interaction between the first two sbi domains and fc of igg . this contrasts with spa - mediated formation of insoluble immune complexes , which involves fab in addition to fc ( 24 ) . innate immune modulation involves the third and fourth sbi domains : sbi - iv and sbi - iii - iv are both able to bind the central protein of the complement system , c3 , through its thioester - containing c3dg and anaphylatoxin c3a subunits . sbi - iii - iv and sbi - e ( containing domains i through iv ) both activate the alternative pathway , leading to rapid consumption of c3 , whereas neither sbi - iii nor sbi - iv in isolation significantly activates complement ( 23 ) ; sbi - iv , indeed , inhibits the alternative pathway in a dose - dependent fashion ( fig . sbi - iv resembles ehp , which specifically inhibits the alternative pathway ( 13 ) , and efb , which operates predominantly on the alternative pathway ( 12 ) . sbi - iv is structurally similar to efb - c ( 12 ) , ehp ( 13 ) , scin ( 25 ) , and domains of spa ( figs . 1 and 2 ) . mutation of either or both of sbi - iv residues arg-231 and asn-238 to alanine , moreover , essentially abolishes both sbi - iv binding to c3dg ( figs . 5 and 6 ) and sbi - iv inhibition of the alternative complement pathway ( fig . 7 ) . the correlation between abolition of c3dg binding and abolition of alternative pathway inhibition as a result of r231a and n238a mutations further establishes the importance of direct sbi - c3 interaction for complement subversion by sbi ( 23 ) . the effect of the r231a and n238a mutations also indicates that the mode of interaction between sbi - iv and c3dg has similarities with that observed for efb - c in the crystal structure of its complex with c3d ( 12 ) , although the sequence identity between sbi - iv and efb - c of 19% makes it difficult to assess whether sbi and efb have evolved a similar interface with c3d by convergent or divergent evolution . notably , both efb and sbi have a binding preference for native c3 over c3b ( 12 , 23 ) . analysis of the ehp - c3d interaction by mutation and isothermal titration calorimetry indicated that ehp has two binding sites for c3d , a high affinity site resembling that of efb and a low affinity site that retains an asparagine corresponding to asn-238 in sbi and asn-138 in efb but lacks the arginine corresponding to sbi arg-231/efb arg-131 ( 13 ) . this two site binding by ehp was ascribed to the presence of tandem repeats of -aq(k / r)avnl- . although there is a repeat of sorts in sbi - iv ( -rvksand- at positions 210216 and -aqrevnk- at positions 233239 ) , the repeat similarity is much weaker and sequence spacing between the repeats is four residues longer than in ehp . as expected then , analysis of the sbi - iv : c3dg and sbi - iii - iv : c3dg interaction by itc ( fig . 3 ) indicates that the stoichiometry is 1:1 ( with kd values of 0.35 m and 0.28 m for sbi - iv : c3dg and sbi - iii - iv : c3dg ) , and as was the case in our earlier spr measurements of the binding affinity between sbi - iv and c3dg ( 23 ) , the itc data were well fit to a single class of binding site model . the ic50 value for sbi - iv inhibition of the alternative pathway is 10 m compared with 0.56 m for efb , 0.41 m for efb - c ( 12 ) and 0.12 m for ehp ( 13 ) , indicating that sbi - iv is less efficient as an inhibitor of the alternative pathway than efb and ehp . the previously measured ic50 values for sbi - iii - iv and sbi - e were 0.1 m and 2 m ( 23 ) , the lower values than sbi - iv reflecting the occurrence of rapid c3 consumption in addition to inhibition of the alternative pathway . taken together , the affinity and ic50 results suggest that , while the mutation data discussed above imply that the sbi - iv - c3 and efb - c3 interactions share important characteristics , there are also differences that may have functional significance . sbi - iii does not bind c3 and h - n hsqc nmr spectra indicate that isolated sbi - iii is natively unfolded , and it remains so when combined with sbi - iv in the sbi - iii - iv construct , yet the presence of sbi - iii is essential for sbi activation of the alternative pathway with resulting consumption of c3 . ( any effect of other domains of sbi on the conformation of sbi - iii remains to be investigated . ) when sbi - iii - iv but not sbi - iv is incubated with serum , a significant fraction of the c3 activated becomes covalently bound to sbi - iii - iv , presumably via the c3d thioester . thus it seems that sbi - iii provides the transacylation target hydroxyl group for sbi - c3b covalent adduct formation ( only the nascently activated c3b form of c3 can undergo covalent adduct formation ) . a hypothesis as to how sbi - iii : c3 covalent adduct formation leads to c3 consumption has been proposed ( 23 ) . specifically , the sbi - iii : c3 adduct may provide a nidus for the assembly of the alternative pathway c3 convertase , c3bbb , that is at least transiently resistant to inactivation by the serum regulatory molecules fh and fi , thereby facilitating the consumptive cleavage of c3 . the n - terminal domains sbi - i and sbi - ii may further assist in the complement consumption process by recruiting igg molecules that are also suitable targets for transacylation by c3b . previous studies have shown that alternative pathway c3 convertases covalently attached to igg are in a protected environment with respect to their rate of inactivation by fh and fi ( 40 , 41 ) . to date the c3 binding characteristics of sbi , efb ( 11 , 12 ) , and ehp ( 13 ) have been examined using recombinant proteins generated from s. aureus strains isolated from human patients . we have compared the nucleotide sequences of 23 sbi genes from a sample of 28 strain isolates from human and animal ( pig , cow , goat , ewe , rabbit , and poultry ) hosts plus those from five complete genome sequences ( 24 ) . no changes at all occurred in sbi - iii and very few changes occurred in sbi - iv . the positions of the sbi - iv polymorphic amino acids ( 222224 and 244246 ) correspond to the loop between helices 1 and 2 and the 2-3 loop and n terminus of helix 3 . this conservation of sbi - iii and sbi - iv amino acid sequences across more than 30 strains isolated from human and animal hosts suggests that the unique mechanism of complement system subversion by sbi is a feature of both human and animal infections . identification of the structures and active sites of the small complement inhibitory proteins secreted by s. aureus has lead to consideration of the potential therapeutic exploitation of these proteins ( 9 ) , including efb ( 12 ) , ehp ( 13 ) , and scin ( 25 ) . like efb and ehp , it appears that a discrete region of the surface of sbi - iv involving a handful of residues forms the interface with c3 , the central protein of the complement system . sbi - iv itself is therefore a potentially attractive basis either for structure - based design of small molecule or peptide - based therapeutic molecules or for development of protein - based therapeutics for complement - mediated acute inflammatory diseases .

among the recently discovered staphylococcus aureus immune evasion proteins , sbi is unique in its ability to interact with components of both the adaptive and innate immune systems of the host . sbi domains i and ii ( sbi - i and sbi - ii ) bind igg . sbi domain iv ( residues 198266 ) binds the central complement protein c3 . when linked to sbi - iii , sbi - iv induces a futile consumption of complement via alternative pathway activation , whereas isolated sbi - iv specifically inhibits the alternative pathway without complement consumption . here we have determined the three - dimensional structure of sbi - iv by nmr spectroscopy , showing that sbi - iv adopts a three - helix bundle fold similar to those of the s. aureus complement inhibitors efb - c , ehp , and scin . the 1h-15n hsqc spectrum of sbi - iii indicates that this domain , essential for futile complement consumption , is natively unfolded , at least when isolated from the rest of sbi . sbi - iv and sbi - iii - iv both bind c3dg with 1:1 stoichiometry and submicromolar affinity . despite low overall sequence identity , sbi possesses the same residues as efb at two positions essential for efb - c binding to c3d . mutation to alanine of either of these residues , arg-231 and asn-238 , abolishes both sbi - iv binding to c3dg and sbi - iv alternative pathway inhibition . the almost complete conservation of sbi - iii and sbi - iv amino acid sequences across more than 30 strains isolated from human and animal hosts indicates that the unique mechanism of sbi in complement system subversion is a feature of infections of both humans and economically important animals .

EXPERIMENTAL PROCEDURES RESULTS DISCUSSION Supplementary Material

cells were grown at 37 c to an a600 value of 0.6 when they were induced by addition of isopropyl-1-thio--d - galactopyranoside ( melford ) to 0.2 mm and cultured for a further 4 h. n - labeled sbi - iii and n - labeled and n / c - labeled sbi - iv for nmr spectroscopy were produced by expression in minimal medium with nh4cl and nh4cl / c6-d - glucose ( spectra stable isotopes ) as the sole nitrogen and nitrogen / carbon sources . nmr sample generation and nmr spectroscopy of sbi - iii and sbi - iv purified sbi - iii and sbi - iv were exchanged into 5 mm mes , 100 mm nacl , 1 mm edta , 1 mm benzamidine , ph 5.5 . sequence - specific backbone resonance assignments of sbi - iv were made using three - dimensional hncacb , cbca(co)nh , hnco , hn(ca)co , hnha , and hbha(cbcaco)nh data sets . side chain resonance assignments of sbi - iv were made using ccc - tocsy - nnh , hcc - tocsy - nnh , hcch tocsy , and n - edited tocsy ( 12.1 , 12.1 , 15.6 , and 50 ms mixing times ) . sbi - iv , sbi - iii - iv , and c3dg were exchanged into 20 mm tris , 150 mm nacl , ph 8.0 . for each interaction , reciprocal experiments were done , one with c3dg in the cell and sbi - iv or sbi - iii - iv in the syringe and the other with sbi - iv or sbi - iii - iv in the cell and c3dg in the syringe . purified sbi - i - ii , sbi - ii , sbi - iii , sbi - iv , sbi - iv r231a , sbi - iv n238a , and sbi - iv r231a - n238a were each covalently coupled to a 1-ml nhs - activated sepharose high performance column ( ge healthcare ) according to the manufacturer 's instructions . ( 37 ) , was used to measure inhibition of the alternative complement pathway by a range of concentrations of sbi - iv , sbi - iv r231a , sbi - iv n238a , and sbi - iv r231-n238a , using a procedure similar to that described previously ( 23 ) . sbi - iv structure determination a semi - automated procedure for iterative noe assignment was used to determine the structure of sbi - iv . from the mean structure calculated over residues 201222 , 228238 , and 245261bcalculated with procheck - nmr ( 36 ) structural statistics on the nmr - derived structures of sbi - iv the r.m.s.d . from the mean structure calculated over residues 201222 , 228238 , and 245261 calculated with procheck - nmr ( 36 ) sbi - iv forms a three - helix bundle sbi - iv adopts a three - helix bundle fold ( fig . none of the residues 198206 and 257266 is involved in long range noes , and these residues make essentially no contribution to the tertiary structure of the domain . a similar three - helix bundle fold has been observed in the other s. aureus complement modulatory proteins efb - c ( 12 ) , ehp ( 13 ) , scin ( 25 ) , and spa ( 26 ) ( fig . sbi - iv ( pdb code 2jvh ) , efb - c ( pdb 2gom ) , ehp ( pdb 2noj ) , scin ( pdb 2qff ) , and protein a domain b ( pdb 1ss1 ) are shown in ribbon and schematic topology representations . sbi - iv ( pdb code 2jvh ) , efb - c ( pdb 2gom ) , ehp ( pdb 2noj ) , scin ( pdb 2qff ) , and protein a domain b ( pdb 1ss1 ) are shown in ribbon and schematic topology representations . this compares with 56 peaks expected based on the amino acid sequence of the construct including the three n - terminal residues , gam , from the expression vector . this almost exact 1:1 correspondence with the expected number of peaks indicates that sbi - iii adopts a single conformation in solution , but the narrow h chemical shift dispersion , with every backbone nh peak except three located in the range 8.08.5 ppm , indicates that this conformation lacks significant tertiary structure , i.e. sbi - iii is natively unfolded . the peak patterns in the h - n hsqc spectra of sbi - iv ( supplemental fig . s3 ) indicate that sbi - iii remains unfolded in the presence of sbi - iv . we can not preclude , however , the possibility that sbi - iii adopts a different conformation in full - length sbi where its conformation may be influenced by one or more of the other domains . sbi - iv and sbi - iii - iv bind c3dg with 1:1 stoichiometry thermodynamics of the interactions between sbi - iv and c3dg and between sbi - iii - iv and c3dg were measured by reciprocal ( sbi : c3dg and c3dg : sbi ) isothermal titration calorimetry experiments ( fig . the itc results indicate that the stoichiometry of both sbi - iv : c3dg and sbi - iii - iv : c3dg interactions is 1:1 with kd values of 0.35 m and 0.28 m ( averages of results from reciprocal itc titrations ) . these affinities are lower than the nanomolar to subnanomolar c3dg affinities previously measured by spr and itc for efb - c ( 12 ) and ehp ( 13 ) . table 2assessment of c3dg binding to sbi - iii - iv and sbi - iv by isothermal titration calorimetrytitrantcellnkdhs mkcal / molcal / mol / k sbi - iii - iv c3dg 1.04 0.40 4.6 13.9 sbi - iv c3dg 1.15 0.36 3.8 16.7 c3dg sbi - iii - iv 0.92 0.17 3.9 17.8 c3dg sbi - iv 0.93 0.34 4.6 14.0 an , molar ratio of titrant molecule bound to ligand molecule in the cell at satration assessment of c3dg binding to sbi - iii - iv and sbi - iv by isothermal titration calorimetry n , molar ratio of titrant molecule bound to ligand molecule in the cell at satration figure 3.isothermal titration calorimetry analyses of the sbi - iv : c3dg ( a ) and sbi - iii - iv : c3dg ( b ) interactions . in a , the concentration of c3dg in the cell was 10.4 m , and the concentration of the sbi - iv injectant in the syringe was 203 m . in b , the concentration of c3dg in the cell was 12.0 m , and the concentration of the sbi - iii - iv injectant in the syringe was 173 m . the reciprocal experiments in which c3dg was titrated into sbi - iv or sbi - iii - iv were also performed ( results summarized in table 2 ) . isothermal titration calorimetry analyses of the sbi - iv : c3dg ( a ) and sbi - iii - iv : c3dg ( b ) interactions . in a , the concentration of c3dg in the cell was 10.4 m , and the concentration of the sbi - iv injectant in the syringe was 203 m . in b , the concentration of c3dg in the cell was 12.0 m , and the concentration of the sbi - iii - iv injectant in the syringe was 173 m . the reciprocal experiments in which c3dg was titrated into sbi - iv or sbi - iii - iv were also performed ( results summarized in table 2 ) . efb residues important for c3d binding align with the corresponding residues in sbi - iv the sequence identity between sbi - iv ( 69 residues ) and efb - c ( 72 residues ) is only 19% over a 43-residue stretch , with five of eight identical residues located in helix 2 ( fig . , sbi - iv helix 2 residues arg-231 and asn-238 align with two efb residues , arg-131 and asn-138 , that are particularly important for formation of the efb - c - c3d complex ( 12 ) . in the efb - c - c3d complex , arg-131 lies deep in an acidic pocket of c3d , and asn-138 is involved in an intermolecular hydrogen bonding network . alignment with arg-131 and asn-138 of efb suggests that sbi residues arg-231 and asn-238 are key residues for sbi interaction with c3 . r231a and n238a mutations abolish sbi - iv binding to c3dg as a consequence of the sequence alignment results mentioned in the previous section , sbi - iv r231a and sbi - iv n238a single mutants and sbi - iv r231a - n238a double mutant were made . 6 ) , where the binding interaction between sbi - iv and biosensor - bound c3dg was abolished when any of the sbi - iv single or double mutants was used as an analyte . figure 4.sequence alignment of sbi - iv , efb - c , ehp , scin , and spa - b . sbi - iv residues arg-231 and asn-238 are highlighted by green rectangles ; the corresponding efb - c residues are arg-131 and asn-138 , and the corresponding ehp residues are arg-75 and asn-82 . sequence alignment of sbi - iv , efb - c , ehp , scin , and spa - b . sbi - iv residues arg-231 and asn-238 are highlighted by green rectangles ; the corresponding efb - c residues are arg-131 and asn-138 , and the corresponding ehp residues are arg-75 and asn-82 . the next two lanes show capture of igg heavy- ( h , 50 kda ) and light - chain ( l , 25 kda ) fragments by sbi - ii and sbi - i - ii . the fifth and sixth lanes show that sbi - iii - iv and sbi - iv capture fragments of complement component c3 with approximate molecular masses of 119 , 75 , 65 , 43 , and 40 kda ( c3 chain origin of the fragments is indicated ) . the last three lanes show that sbi - iv mutants r231a , n238a , and r231a - n238a fail to capture c3 and instead capture albumin . the next two lanes show capture of igg heavy- ( h , 50 kda ) and light - chain ( l , 25 kda ) fragments by sbi - ii and sbi - i - ii . the fifth and sixth lanes show that sbi - iii - iv and sbi - iv capture fragments of complement component c3 with approximate molecular masses of 119 , 75 , 65 , 43 , and 40 kda ( c3 chain origin of the fragments is indicated ) . the last three lanes show that sbi - iv mutants r231a , n238a , and r231a - n238a fail to capture c3 and instead capture albumin . spr sensorgrams are shown for sbi - iv wild - type and sbi - iv mutants r231a , n238a , and r231a - n238a . spr sensorgrams are shown for sbi - iv wild - type and sbi - iv mutants r231a , n238a , and r231a - n238a . r231a and n238a mutations abolish sbi - iv inhibition of the alternative complement pathway sbi - iv inhibited the alternative pathway in a dose - dependent manner ( fig . all of the r231a , n238a , and r231a - n238a mutations abolished sbi - iv inhibition of the alternative pathway ( fig . the ic50 value for sbi - iv alternative pathway inhibition was 10 m compared with 0.56 m for efb , 0.41 m for efb - c ( 12 ) and 0.12 m for ehp ( 13 ) . dose response residual complement activity for sbi - iv and sbi - iv mutants r231a , n238a , and double mutant r231a - n238a were assessed in a wielisa assay . dose response residual complement activity for sbi - iv and sbi - iv mutants r231a , n238a , and double mutant r231a - n238a were assessed in a wielisa assay . innate immune modulation involves the third and fourth sbi domains : sbi - iv and sbi - iii - iv are both able to bind the central protein of the complement system , c3 , through its thioester - containing c3dg and anaphylatoxin c3a subunits . sbi - iii - iv and sbi - e ( containing domains i through iv ) both activate the alternative pathway , leading to rapid consumption of c3 , whereas neither sbi - iii nor sbi - iv in isolation significantly activates complement ( 23 ) ; sbi - iv , indeed , inhibits the alternative pathway in a dose - dependent fashion ( fig . sbi - iv resembles ehp , which specifically inhibits the alternative pathway ( 13 ) , and efb , which operates predominantly on the alternative pathway ( 12 ) . sbi - iv is structurally similar to efb - c ( 12 ) , ehp ( 13 ) , scin ( 25 ) , and domains of spa ( figs . mutation of either or both of sbi - iv residues arg-231 and asn-238 to alanine , moreover , essentially abolishes both sbi - iv binding to c3dg ( figs . 5 and 6 ) and sbi - iv inhibition of the alternative complement pathway ( fig . the effect of the r231a and n238a mutations also indicates that the mode of interaction between sbi - iv and c3dg has similarities with that observed for efb - c in the crystal structure of its complex with c3d ( 12 ) , although the sequence identity between sbi - iv and efb - c of 19% makes it difficult to assess whether sbi and efb have evolved a similar interface with c3d by convergent or divergent evolution . although there is a repeat of sorts in sbi - iv ( -rvksand- at positions 210216 and -aqrevnk- at positions 233239 ) , the repeat similarity is much weaker and sequence spacing between the repeats is four residues longer than in ehp . as expected then , analysis of the sbi - iv : c3dg and sbi - iii - iv : c3dg interaction by itc ( fig . 3 ) indicates that the stoichiometry is 1:1 ( with kd values of 0.35 m and 0.28 m for sbi - iv : c3dg and sbi - iii - iv : c3dg ) , and as was the case in our earlier spr measurements of the binding affinity between sbi - iv and c3dg ( 23 ) , the itc data were well fit to a single class of binding site model . the ic50 value for sbi - iv inhibition of the alternative pathway is 10 m compared with 0.56 m for efb , 0.41 m for efb - c ( 12 ) and 0.12 m for ehp ( 13 ) , indicating that sbi - iv is less efficient as an inhibitor of the alternative pathway than efb and ehp . the previously measured ic50 values for sbi - iii - iv and sbi - e were 0.1 m and 2 m ( 23 ) , the lower values than sbi - iv reflecting the occurrence of rapid c3 consumption in addition to inhibition of the alternative pathway . taken together , the affinity and ic50 results suggest that , while the mutation data discussed above imply that the sbi - iv - c3 and efb - c3 interactions share important characteristics , there are also differences that may have functional significance . sbi - iii does not bind c3 and h - n hsqc nmr spectra indicate that isolated sbi - iii is natively unfolded , and it remains so when combined with sbi - iv in the sbi - iii - iv construct , yet the presence of sbi - iii is essential for sbi activation of the alternative pathway with resulting consumption of c3 . when sbi - iii - iv but not sbi - iv is incubated with serum , a significant fraction of the c3 activated becomes covalently bound to sbi - iii - iv , presumably via the c3d thioester . specifically , the sbi - iii : c3 adduct may provide a nidus for the assembly of the alternative pathway c3 convertase , c3bbb , that is at least transiently resistant to inactivation by the serum regulatory molecules fh and fi , thereby facilitating the consumptive cleavage of c3 . the n - terminal domains sbi - i and sbi - ii may further assist in the complement consumption process by recruiting igg molecules that are also suitable targets for transacylation by c3b . to date the c3 binding characteristics of sbi , efb ( 11 , 12 ) , and ehp ( 13 ) have been examined using recombinant proteins generated from s. aureus strains isolated from human patients . we have compared the nucleotide sequences of 23 sbi genes from a sample of 28 strain isolates from human and animal ( pig , cow , goat , ewe , rabbit , and poultry ) hosts plus those from five complete genome sequences ( 24 ) . no changes at all occurred in sbi - iii and very few changes occurred in sbi - iv . this conservation of sbi - iii and sbi - iv amino acid sequences across more than 30 strains isolated from human and animal hosts suggests that the unique mechanism of complement system subversion by sbi is a feature of both human and animal infections . identification of the structures and active sites of the small complement inhibitory proteins secreted by s. aureus has lead to consideration of the potential therapeutic exploitation of these proteins ( 9 ) , including efb ( 12 ) , ehp ( 13 ) , and scin ( 25 ) . like efb and ehp , it appears that a discrete region of the surface of sbi - iv involving a handful of residues forms the interface with c3 , the central protein of the complement system .

the continued growth of microbial strain collections used for natural product discovery demands a high - throughput method for rapid strain prioritization . a sensitive and reliable survey of microbial genomes in a strain collection for genes characteristic for biosynthesis of the targeted class of natural products would identify the most promising ones for strain prioritization , thereby significantly increasing the likelihood of discovering the targeted class of natural products . our method exploits the capability of real - time pcr , in a high - throughput manner , to detect dna regions , encoding biosynthesis of the targeted class of natural products , from the genomic dna ( gdna ) samples of a microbial strain collection for strain prioritization . subsequent genome sequencing and genome mining of the high - priority hit strains promise to accelerate natural product discovery ( figure 1b ) . in real - time pcr applications , fluorescence levels , which correspond to the amount of accumulating products , are generated by fluorophores that intercalate double - stranded ( ds ) dna or by fluorophore - conjugated oligonucleotides that prime to dna targets . we chose to use sybr green i as the dna - intercalating fluorophore , as it enabled us to apply melting curve analysis of the pcr products . melting curve analysis reveals the apparent melting temperature ( tm ) of the pcr products , a value dependent on product length , base composition , and nucleotide mismatch . the tm values therefore can be exploited as an indicator of pcr product specificity without the need for gel electrophoresis . to demonstrate the utility and effectiveness of our strain prioritization method by real - time pcr , we chose to target the diterpene synthase genes to search for diterpenoid producers in general and ptm and ptn producers in particular from the actinomycete strain collection at the scripps research institute . diterpene synthases catalyze the critical steps in diterpenoid biosynthesis by morphing geranylgeranyl diphosphate into one of the many diterpenoid scaffolds , further transformations of which by pathway - specific tailoring enzymes afford the vast structural diversity known for diterpenoid natural products ( figure 2a ) . pathways for the biosynthesis of bacterial diterpenoids , highlighting the four diterpene - related synthases and the tailoring enzymes leading to platensimycin ( ptm ) , platencin ( ptn ) , viguiepinol , oxaloterpin , and other known diterpenoids in bacteria . we have previously cloned and characterized the biosynthetic gene clusters for ptm and ptn production in s. platensis ma7327 and ma7339 ( figure s1 ) . among the four diterpene - related synthases from the ptm and ptn biosynthetic machineries , geranylgeranyl diphosphate synthase ( ptmt4/ptnt4 ) is common for all bacterial diterpenoids , ent - copalyl diphosphate synthase ( ptmt2/ptnt2 ) is shared by both ptm and ptn , but ent - kaurene synthase ( ptmt3 ) or ent - atiserene synthase ( ptmt1/ptnt1 ) is specific for ptm or ptn , respectively ( figure 2a , s1 ) . we reasoned that targeting ptmt3 or ptmt1/ptnt1 should specifically narrow our search to producers of ent - kaurene - derived or ent - atiserene - derived diterpenoids , such as ptm or ptn , while targeting ptm / ptnt2 or ptm / ptnt4 could broaden our search to include producers that biosynthesize other ent - copalyl diphosphate - derived or all geranylgeranyl diphosphate - derived diterpenoids , respectively ( figure 2 ) . the primers were designed based on the conserved nucleotide sequences of ptmt1/ptnt1 , ptmt2/ptnt2 , ptmt3 , and ptmt / ptnt4 within the ptm and ptn gene clusters , as well as known homologues of actinomycete origin , and one of each primer for t1 and t3 was selected based on the atypical amino acid motifs ( dxxxd ) found in bacterial type i diterpene synthases ( figures s1 , s2 , and table s1 ) . the sizes for each of the pcr products were predicted to be 696 bp , 559 bp , 461 bp , or 411 bp , with the calculated tm of 92.1 , 91.6 , 91.6 , or 91.8 c for t1 , t2 , t3 , or t4 , respectively , according to the ptmt1/ptnt1 , ptmt2/ptnt2 , ptmt3 , and ptmt / ptnt4 sequences ( figure s1 ) . we arbitrarily set the specificity cutoff for each of the targeted pcr products at tm 0.8 c , where tm is the melting temperature of the positive control , to identify putative hits . smaller deviations will surely decrease the chance of false positives but might result in the miss of potential hits . we selected 1911 strains from our actinomycete collection and applied the method described above to prioritize the strains for diterpenoid producers in general and ptm and ptn producers in particular . the gdna of the 1911 strains were individually prepared , normalized , and arrayed in 384-well plates . each of the plates also contained one blank ( i.e. , no template dna ) as a negative control and one with the gdna of s. platensis ma7327 as a positive control . real - time pcrs were carried out with each of the four sets of primers ( figure s1 and table s1 ) , and the resultant pcr products were subjected to melting curve analysis to identify putative hits ( figure 3a ) . strain prioritization for natural product discovery by a high - throughput real - time pcr method , showcasing the identification of platensimycin , platencin , viguiepinol , oxaloterpin , and other diterpenoid producers by targeting selected diterpene - related synthases from the actinomycete collection at the scripps research institute . ( a ) high - throughput real - time pcr method targeting ( i ) t4 , ( ii ) t2 , ( iii ) t1 , and ( iv ) t3 and melting curve analysis of the resultant products to identify putative diterpenoid producers from a collection of 1911 strains . each panel depicts the melting curves with f/t ( y - axis ) representing the rate of change in fluorescence as a function of temperature . solid lines with open circles represent the positive controls with a normalized melting temperature ( tm ) . solid lines represent hits found during the melting step of the real - time pcr experiment with the tm range at tm 0.8 c . insets show pcr products of the hits that were analyzed by agarose gel electrophoresis and confirmed by dna sequencing . only the melting curves for one of the five 384-well plates were shown for t4 , which yielded 71 putative hits . for t1 , t2 , and t3 , the melting curves of the respective hits from all five 384-well plates were combined and depicted together . ( b ) euler diagram depicting the 488 putative diterpenoid producers identified from the 1911 strains by targeting t4 , among which nine , six , and six were co - identified by targeting t2 , t1 , and t3 , respectively , and confirmed by dna sequencing . ( c ) morphology of the three new platensimycin and platencin overproducers sb12026 ( cb00739/ptmr1 ) , sb12027 ( cb00765/ptmr1 ) , and sb12028 ( cb00775/ptmr1 ) in comparison with sb12001 ( ma7327/ptmr1 ) and sb12002 ( ma7327/ptmr1 ) , derived from s. platensis ma7327 , and sb12600 ( ma7339/ptmr1 ) derived from s. platensis ma7339 on an isp4 agar plate . while sb12001 failed to sporulate and sb12002 sporulated poorly under all conditions examined , sb12026 and sb12027 sporulated well on several media , as exemplified with isp4 . as summarized in figure 3b , 488 , 17 , 6 , and 6 out of the 1911 strains were identified as putative hits using the primers , targeting t4 , t2 , t3 , and t1 , respectively . although the 488 hits identified using the t4 primers represented a hit rate that was too high to justify detailed follow - up analysis of each hit , confirmatory pcr on selected hits , using the same t4 primers followed by agarose gel electrophoresis of the resultant products , yielded specific bands with the predicted size ( figure 3a , panel i ) . however , the resultant pcr products from each of the t4 hits were not all sequenced ( figure s3a ) , and the possibility of false positives due to nonspecific pcr amplification therefore can not be excluded . for the 17 hits identified using the t2 primers , follow - up analysis by pcr using the same t2 primers all afforded specific products with the predicted size ( figure 3a , panel ii ) . dna sequencing of the resultant products revealed that nine out of the 17 hits were true hits , which contain genes encoding putative ent - copalyl diphosphate synthase ( figure s3b ) , with the remaining eight hits resulting from nonspecific pcr amplification . finally , each set of the six hits identified using t1 or t3 primers and , remarkably , both set of six hits afforded specific pcr products with the predicted size ( figure 2b , panels iii and iv , respectively ) . dna sequencing of the resultant products confirmed that the six hits were true diterpenoid producers that contain genes encoding the putative ent - kaurene synthase ( figure s3c ) and ent - atiserene synthase ( figure s3d ) . cross examination of all hits revealed that six strains , cb00739 , cb00765 , cb00775 , cb00789 , cb02289 , and cb02304 , harbored all four targeted genes . the fact that they possessed both t1 and t3 , in addition to t2 and t4 , would predict all six hits as ptm ptn dual producers , a genetic disposition that resembled the ptm ptn dual producer s. platensis ma7327 but contrasted with the ptn - only producer s. platensis ma7339 ( s1 ) . of the remaining three hits identified using t2 , they ( cb00028 , cb00830 , and cb01059 ) all also harbored t4 , as would be expected for diterpenoid producers ( figure 2 ) . in fact , we previously isolated from cb00830 viguiepinol and oxaloterpins e and c , diterpenoids whose biosynthesis was confirmed to involve t2 and t4 . therefore , cb00028 and cb01059 are promising producers of new diterpenoid natural products featuring ent - copalyl diphosphate - derived scaffolds ( figure 2 ) . to verify each of the six hits possessing a functional ptm ptn dual biosynthetic cluster gene , selected intact genes , encoding the four diterpene synthases t1 , t2 , t3 , and t4 , as well as the pathway - specific negative transcriptional regulator ptmr1 , were first amplified by pcr using primers that were designed according to the ptm ptn dual biosynthetic gene cluster from s. platensis ma7327 ( table s1 ) . dna sequencing of the resultant products confirmed that each of the targeted genes was highly homologous ( > 95% ) to those of the known ptm phylogenetic analysis based on four selected housekeeping genes , 16s rrna , reca , rpob , and trpb(21,22 ) ( table s1 ) , assigned all six hits as streptomyces platensis ( figure s5a ) . close examination of the six hit strains , in comparison with s. platensis ma7327 and ma7339 , however , revealed that the new strains were distinct , as judged by the morphology and appearance of pigmented spores ( figure s5b ) . two of the hit strains , cb00739 and cb00765 , were selected and subjected to genome sequencing , confirming that these strains contained complete ptm ptn gene clusters . the ptm ptn dual biosynthetic gene clusters from cb00739 and cb00765 have the same number of open reading frames and same genetic organization as those from s. platensis ma7327 , and the overall nucleotide sequence identities among the three clusters are > 97% ( figure s4 ) . to validate that the identified ptm ptn dual biosynthetic gene clusters are functional , the six hit strains were first subjected to fermentation optimization for ptm and ptn production and confirmation . under the same conditions used for s. platensis ma7327 and ma7339 , ptm and ptn production was confirmed for all six hit strains ( figure s6 ) , with ptm and ptn titers varying in the ranges 0.6610 and < 0.111 mg l , respectively ( table 1 ) . these titers correlated well with those reported previously from the s. platensis ma7327 and ma7339 wild - type strains . unless otherwise noted , values are the averages of at least three independent trials and the ptn peak at 240 was too small to calculate a reliable titer ; ptn was detected by eic ( m / z at 426.20 for the [ ptn + h ] ion ) and is shown in figure s6 . we previously demonstrated that removal of the pathway - specific , negatively acting transcriptional regulator ptmr1 or ptnr1 from s. platensis ma7327 and ma7339 afforded recombinant strains that dramatically overproduced ptm , ptn , or both . however , the recombinant strains sporulated poorly under all conditions examined ( figure 3c ) . this has prevented us from further engineering ptm and ptn biosynthesis in these overproducers . we similarly inactivated ptmr1 in three of the six hits , cb00739 , cb00765 , and cb00775 ( table s2 ) , and the genotypes of the resultant recombinant strains sb12026 , sb12027 , and sb12028 , respectively , were confirmed by pcr ( figure s7 ) . under the same conditions for ptm and ptn production with the wild - type strains as controls , sb12026 , sb12027 , and sb12028 indeed overproduced ptm and ptn up to 310-fold , consistent with what has been observed for the s. platensis ma7327 and ma7339 strains ( table 1 and figure s6 ) . gratifyingly , sb12026 and sb12027 sporulate well ( figure 3c ) , opening up the opportunity to further manipulate ptm and ptn biosynthesis in these overproducers for pathway characterization and structural diversity . in spite of the indisputable track record of natural products as drugs and drug leads and small - molecule probes , the traditional approach to natural product discovery , with its elements of serendipity , demands too much time , effort , and resources . we now report a high - throughput method using real - time pcr for strain prioritization to identify the most promising strains from a microbial strain collection for natural product discovery . resources could then be devoted preferentially to the strains that hold the highest promise in producing novel natural products , thereby accelerating detection and isolation of the targeted natural products and cutting the time and cost associated with traditional natural product discovery programs . central to our method is the application of real - time pcr , targeting genes characteristic to the biosynthetic machinery of natural products with distinct scaffolds , in a high - throughput format and with superior sensitivity and specificity . the practicality and effectiveness of our method were showcased by targeting four diterpene - related synthase genes , t1 , t2 , t3 , and t4 , to prioritize 1911 strains , selected from the actinomycete strain collection at the scripps research institute , for diterpenoid producers in general and ptm and ptn producers in particular ( figure 2 ) . we chose to search for diterpenoid producers because they are underrepresented among microbial natural products and for new ptm and ptn producers because of the heroic effort in the discovery of the original s. platensis ma7327 and ma7339 strains , as well as the need for alternative producers with better genetic amenability . we rapidly identified a total of 488 potential diterpenoid producers , among which six were confirmed as ptm ptn dual producers and one as a viguiepinol and oxaloterpin producer . although the new ptm ptn dual producers are all s. platensis strains on the basis of polyphasic taxonomy , they exhibit district morphology , three of which showed superior genetic amenability to the original s. platensis ma7327 and ma7339 strains . genetic amenability of the producing organisms is of paramount importance in applying microbial genomics to natural product discovery , and new strains , alternatives to genetically recalcitrant original producers for the same natural products or families of natural products with similar scaffolds , represent an innovative solution to this critical challenge . the 25% ( i.e. , 488 out of 1911 ) hit rate of diterpenoid producers is higher than expected , given the small number of bacterial diterpenoids known to date , but is consistent with an early survey of our strain collection . taken together , these findings support our early proposal that the biosynthetic potential of diterpenoids in bacteria is significantly underestimated . further prioritization of the hits identified in this study promises the discovery of novel diterpenoid natural products . natural products occupy tremendous chemical structural space that is unmatched by any other small - molecule libraries . while the rich functionality of natural products is , without doubt , one of their great strengths , providing potency and selectivity , the biosynthetic machineries for each of the major molecular scaffolds are highly conserved across the entire family of natural products , as exemplified by the diterpene - related synthases t1 , t2 , t3 , and t4 , for diterpenoids . variations among the myriad of tailoring enzymes associated with each of the biosynthetic machineries further imbue the remarkable structural diversity within each of the natural product families ( figure 2a ) . although we have showcased our method by targeting diterpenoids , variation of the method should be readily applicable to the discovery of other classes of natural products by targeting conserved genes within each of the major biosynthetic machineries . applications of genome sequencing and genome mining to the high - priority strains could essentially eliminate the chance elements from traditional discovery programs and fundamentally change how natural products are discovered . the tm range used in this study was 0.8 c , and this range allowed us to identify hits with 9798% , 6398% , 9697% , and 3898% identity to ptmt1 , ptmt2 , ptmt3 , and ptmt4 , respectively ( figure s3 ) . the range of tm can be exploited to address the needs of each experiment , and a larger range of tm will surely increase the likelihood of finding diverse hits , albeit with a concomitant increase of nonspecific hits . finally , we used a dna intercalating fluorophore in the current method , allowing detection and subsequent melting temperature analysis of the amplified products . one could envision variations of the method with fluorophore - labeled oligonucleotides or molecular beacons in a multiplex approach of real - time pcr to target multiple genes simultaneously within a single reaction , further expanding its utility in strain prioritization , thereby accelerating natural product discovery . the actinomycete collection at the scripps research institute consists of strains isolated from various unexplored and underexplored ecological niches . the concentrations of gdna samples were estimated using a microplate fluorescence assay with minor modifications . briefly , gdna samples were diluted in 10 mm tris - hcl , ph 8.0 . sybr green i dye ( sigma - aldrich ) was added to each dilution of dna , mixed , and incubated at room temperature for 10 min in the dark . fluorescence levels were measured using a spectramax m5multi - mode microplate reader ( molecular devices ) with excitation and emission wavelengths of 450 and 520 nm , respectively . after the normalization of gdna concentration , samples were arrayed in 384-well masterblock deep - well plates as working stocks and transferred to microplates using a biomek fx workstation ( beckman coulter ) for each real - time pcr experiment . real - time pcr was performed with an applied biosystems 7900ht fast real - time pcr system . primer design was based on the ptmt3 sequence and the conserved sequences of ptmt1 , ptmt2 , and ptmt4 homologues ( figures s1 , s2 ) . genomic dna ( 1100 ng ) , 0.1 l of each primer ( 10 m stock ) , 0.5 l of dmso , 0.5 l of 10 sybr green i dye , 5 l of taq 2x master mix , and h2o were mixed to give a reaction volume of 10 l per well . a no template negative control and one positive control with the gdna of s. platensis ma7327 were included in each 384-well plate . the reaction conditions consisted of a background check at 50 c for 2 min ; initial denaturation at 95 c for 7 min ; and 3740 cycles of denaturation at 95 c for 30 s , primer annealing at 6468 c for 15 s , extension at 68 c for 3060 s , and melting at 95 c for 15 s with a ramp rate of 2% from 68 to 95 c . for t4 amplification , three additional cycles of denaturation at 95 c for 30 s , temperature ramping to 30 c , and extension at 68 c for 30 s were included after the initial denaturation step mentioned above . each tm was normalized to a theoretical tm calculated using the nearest neighbor model . for determining each theoretical tm , the sequence of the target gene was used assuming an initial dna amount of 100 ng and salt concentration of 50 mm . samples with tm 0.8 c compared to the positive control were considered hits . to confirm hits , regular pcr was performed , and the resulting products were analyzed by 1% agarose gel electrophoresis , purified by gel extraction , and sequenced . the t1 , t2 , t3 , t4 , and r1 genes of the six new ptm ptn dual producers ( cb00739 , cb00765 , cb00775 , cb00789 , cb02289 , and cb02304 ) were amplified using primers designed according to the ptm ptn dual biosynthetic gene cluster from s. platensis ma7327 ( table s1 ) . regions of the four housekeeping genes , 16s rrna , reca , rpob , and trpb , were amplified using primers listed in table s1 . the draft genome sequences of s. platensis cb00739 and cb00765 were obtained using the ion 316 chips and the ion pgm sequencing 300 kit ( life technologies ) following the manufacturer s instructions . fragment libraries ( 300 bp inserts ) and matepair libraries ( 5 kb inserts ) were sequenced . for s. platensis cb00739 , a final sequence assembly of 5 955 880 reads afforded a 9 441 068 bp draft genome consisting of 23 scaffolds . for s. platensis cb00765 , a final sequence assembly of 5 293 288 reads yielded a 9 404 443 bp draft genome consisting of 20 scaffolds . all dna sequences were deposited in the ncbi database ( see accession codes ) . alignment of the concatenated housekeeping genes ( partial sequences , 2975-bp total ) was generated using megalign 10.1 . the phylogenetic tree was constructed using the tamura - nei evolutionary distance method and 1000 bootstrap replications . strains and plasmids / cosmids used in this study are summarized in table s2 . gene disruption of ptmr1 was performed in e. coli bw25113/pij790 carrying appropriate cosmids by following the red - mediated pcr - targeting mutagenesis method as described previously . the mutant cosmids were introduced into the s. platensis cb00739 , cb00765 , and cb00775 by intergeneric conjugation after passaging the mutant cosmids through the nonmethylating e. coli et12567/puz8002 . apramycin selection and kanamycin sensitivity on isp4 medium were used to determine double - crossover mutants of the ptmr1 genes . the mutations were confirmed by pcr analysis using primers ptmridf and ptmridr ( table s1 ) . spores were inoculated into seed medium and incubated for 48 h , and 2 ml of seed culture was used to inoculate 50 ml of ptm medium supplemented with 10 ml l trace elements and 1.5 g of amberlite xad-16 resin ( sigma - aldrich ) . the resin was harvested by centrifugation , washed three times with h2o , and extracted with actone ( 3 10 ml ) . acetone was removed under reduced pressure , and the resulting oil was resuspended in 1.5 ml of meoh . hplc was carried out on a varian liquid chromatography system consisting of varian prostar 210 pumps and a prostar 330 photodiode array detector equipped with an apollo c18 column ( 250 mm 4.6 mm , 5 m , grace davison discovery sciences , deerfield , il , hplc analysis of ptm and ptn was performed using a 20 min solvent gradient ( 1 ml min ) from 15% ch3cn in h2o containing 0.1% formic acid to 90% ch3cn in h2o containing 0.1% formic acid , and ptm and ptn were eluted with retention times of 14.0 and 17.4 min , respectively . the peak area at 240 nm was used to quantify ptm and ptn production on the basis of calibration curves with authentic ptm and ptn standards . liquid chromatography mass spectrometry ( lcms ) was carried out on an agilent 1260 infinity lc coupled to a 6230 tof equipped with an agilent extend c18 column ( 50 mm 2.1 mm , 1.8 m ) . liquid chromatography was performed using a 17 min solvent gradient ( 0.4 ml min ) from 10% ch3cn in h2o containing 0.1% formic acid to 100% ch3cn containing 0.1% formic acid , and ptm and ptn were eluted with retention times of 7.4 and 9.7 min , respectively . extracted ion ( m / z at 442.1863 for the [ ptm + h ] ion and m / z at 426.1914 for the [ ptn + h ] ion ) chromatograms ( m / z at [ m + h ] 0.5 ) verified ptm and ptn production .

natural products offer unmatched chemical and structural diversity compared to other small - molecule libraries , but traditional natural product discovery programs are not sustainable , demanding too much time , effort , and resources . here we report a strain prioritization method for natural product discovery . central to the method is the application of real - time pcr , targeting genes characteristic to the biosynthetic machinery of natural products with distinct scaffolds in a high - throughput format . the practicality and effectiveness of the method were showcased by prioritizing 1911 actinomycete strains for diterpenoid discovery . a total of 488 potential diterpenoid producers were identified , among which six were confirmed as platensimycin and platencin dual producers and one as a viguiepinol and oxaloterpin producer . while the method as described is most appropriate to prioritize strains for discovering specific natural products , variations of this method should be applicable to the discovery of other classes of natural products . applications of genome sequencing and genome mining to the high - priority strains could essentially eliminate the chance elements from traditional discovery programs and fundamentally change how natural products are discovered .

Results and Discussion Experimental Section

the continued growth of microbial strain collections used for natural product discovery demands a high - throughput method for rapid strain prioritization . a sensitive and reliable survey of microbial genomes in a strain collection for genes characteristic for biosynthesis of the targeted class of natural products would identify the most promising ones for strain prioritization , thereby significantly increasing the likelihood of discovering the targeted class of natural products . our method exploits the capability of real - time pcr , in a high - throughput manner , to detect dna regions , encoding biosynthesis of the targeted class of natural products , from the genomic dna ( gdna ) samples of a microbial strain collection for strain prioritization . subsequent genome sequencing and genome mining of the high - priority hit strains promise to accelerate natural product discovery ( figure 1b ) . in real - time pcr applications , fluorescence levels , which correspond to the amount of accumulating products , are generated by fluorophores that intercalate double - stranded ( ds ) dna or by fluorophore - conjugated oligonucleotides that prime to dna targets . melting curve analysis reveals the apparent melting temperature ( tm ) of the pcr products , a value dependent on product length , base composition , and nucleotide mismatch . to demonstrate the utility and effectiveness of our strain prioritization method by real - time pcr , we chose to target the diterpene synthase genes to search for diterpenoid producers in general and ptm and ptn producers in particular from the actinomycete strain collection at the scripps research institute . diterpene synthases catalyze the critical steps in diterpenoid biosynthesis by morphing geranylgeranyl diphosphate into one of the many diterpenoid scaffolds , further transformations of which by pathway - specific tailoring enzymes afford the vast structural diversity known for diterpenoid natural products ( figure 2a ) . pathways for the biosynthesis of bacterial diterpenoids , highlighting the four diterpene - related synthases and the tailoring enzymes leading to platensimycin ( ptm ) , platencin ( ptn ) , viguiepinol , oxaloterpin , and other known diterpenoids in bacteria . we have previously cloned and characterized the biosynthetic gene clusters for ptm and ptn production in s. platensis ma7327 and ma7339 ( figure s1 ) . the primers were designed based on the conserved nucleotide sequences of ptmt1/ptnt1 , ptmt2/ptnt2 , ptmt3 , and ptmt / ptnt4 within the ptm and ptn gene clusters , as well as known homologues of actinomycete origin , and one of each primer for t1 and t3 was selected based on the atypical amino acid motifs ( dxxxd ) found in bacterial type i diterpene synthases ( figures s1 , s2 , and table s1 ) . the sizes for each of the pcr products were predicted to be 696 bp , 559 bp , 461 bp , or 411 bp , with the calculated tm of 92.1 , 91.6 , 91.6 , or 91.8 c for t1 , t2 , t3 , or t4 , respectively , according to the ptmt1/ptnt1 , ptmt2/ptnt2 , ptmt3 , and ptmt / ptnt4 sequences ( figure s1 ) . we arbitrarily set the specificity cutoff for each of the targeted pcr products at tm 0.8 c , where tm is the melting temperature of the positive control , to identify putative hits . smaller deviations will surely decrease the chance of false positives but might result in the miss of potential hits . we selected 1911 strains from our actinomycete collection and applied the method described above to prioritize the strains for diterpenoid producers in general and ptm and ptn producers in particular . the gdna of the 1911 strains were individually prepared , normalized , and arrayed in 384-well plates . , no template dna ) as a negative control and one with the gdna of s. platensis ma7327 as a positive control . real - time pcrs were carried out with each of the four sets of primers ( figure s1 and table s1 ) , and the resultant pcr products were subjected to melting curve analysis to identify putative hits ( figure 3a ) . strain prioritization for natural product discovery by a high - throughput real - time pcr method , showcasing the identification of platensimycin , platencin , viguiepinol , oxaloterpin , and other diterpenoid producers by targeting selected diterpene - related synthases from the actinomycete collection at the scripps research institute . ( a ) high - throughput real - time pcr method targeting ( i ) t4 , ( ii ) t2 , ( iii ) t1 , and ( iv ) t3 and melting curve analysis of the resultant products to identify putative diterpenoid producers from a collection of 1911 strains . each panel depicts the melting curves with f/t ( y - axis ) representing the rate of change in fluorescence as a function of temperature . solid lines represent hits found during the melting step of the real - time pcr experiment with the tm range at tm 0.8 c . for t1 , t2 , and t3 , the melting curves of the respective hits from all five 384-well plates were combined and depicted together . ( b ) euler diagram depicting the 488 putative diterpenoid producers identified from the 1911 strains by targeting t4 , among which nine , six , and six were co - identified by targeting t2 , t1 , and t3 , respectively , and confirmed by dna sequencing . ( c ) morphology of the three new platensimycin and platencin overproducers sb12026 ( cb00739/ptmr1 ) , sb12027 ( cb00765/ptmr1 ) , and sb12028 ( cb00775/ptmr1 ) in comparison with sb12001 ( ma7327/ptmr1 ) and sb12002 ( ma7327/ptmr1 ) , derived from s. platensis ma7327 , and sb12600 ( ma7339/ptmr1 ) derived from s. platensis ma7339 on an isp4 agar plate . as summarized in figure 3b , 488 , 17 , 6 , and 6 out of the 1911 strains were identified as putative hits using the primers , targeting t4 , t2 , t3 , and t1 , respectively . although the 488 hits identified using the t4 primers represented a hit rate that was too high to justify detailed follow - up analysis of each hit , confirmatory pcr on selected hits , using the same t4 primers followed by agarose gel electrophoresis of the resultant products , yielded specific bands with the predicted size ( figure 3a , panel i ) . however , the resultant pcr products from each of the t4 hits were not all sequenced ( figure s3a ) , and the possibility of false positives due to nonspecific pcr amplification therefore can not be excluded . for the 17 hits identified using the t2 primers , follow - up analysis by pcr using the same t2 primers all afforded specific products with the predicted size ( figure 3a , panel ii ) . finally , each set of the six hits identified using t1 or t3 primers and , remarkably , both set of six hits afforded specific pcr products with the predicted size ( figure 2b , panels iii and iv , respectively ) . dna sequencing of the resultant products confirmed that the six hits were true diterpenoid producers that contain genes encoding the putative ent - kaurene synthase ( figure s3c ) and ent - atiserene synthase ( figure s3d ) . cross examination of all hits revealed that six strains , cb00739 , cb00765 , cb00775 , cb00789 , cb02289 , and cb02304 , harbored all four targeted genes . the fact that they possessed both t1 and t3 , in addition to t2 and t4 , would predict all six hits as ptm ptn dual producers , a genetic disposition that resembled the ptm ptn dual producer s. platensis ma7327 but contrasted with the ptn - only producer s. platensis ma7339 ( s1 ) . of the remaining three hits identified using t2 , they ( cb00028 , cb00830 , and cb01059 ) all also harbored t4 , as would be expected for diterpenoid producers ( figure 2 ) . to verify each of the six hits possessing a functional ptm ptn dual biosynthetic cluster gene , selected intact genes , encoding the four diterpene synthases t1 , t2 , t3 , and t4 , as well as the pathway - specific negative transcriptional regulator ptmr1 , were first amplified by pcr using primers that were designed according to the ptm ptn dual biosynthetic gene cluster from s. platensis ma7327 ( table s1 ) . dna sequencing of the resultant products confirmed that each of the targeted genes was highly homologous ( > 95% ) to those of the known ptm phylogenetic analysis based on four selected housekeeping genes , 16s rrna , reca , rpob , and trpb(21,22 ) ( table s1 ) , assigned all six hits as streptomyces platensis ( figure s5a ) . two of the hit strains , cb00739 and cb00765 , were selected and subjected to genome sequencing , confirming that these strains contained complete ptm ptn gene clusters . we similarly inactivated ptmr1 in three of the six hits , cb00739 , cb00765 , and cb00775 ( table s2 ) , and the genotypes of the resultant recombinant strains sb12026 , sb12027 , and sb12028 , respectively , were confirmed by pcr ( figure s7 ) . under the same conditions for ptm and ptn production with the wild - type strains as controls , sb12026 , sb12027 , and sb12028 indeed overproduced ptm and ptn up to 310-fold , consistent with what has been observed for the s. platensis ma7327 and ma7339 strains ( table 1 and figure s6 ) . gratifyingly , sb12026 and sb12027 sporulate well ( figure 3c ) , opening up the opportunity to further manipulate ptm and ptn biosynthesis in these overproducers for pathway characterization and structural diversity . in spite of the indisputable track record of natural products as drugs and drug leads and small - molecule probes , the traditional approach to natural product discovery , with its elements of serendipity , demands too much time , effort , and resources . we now report a high - throughput method using real - time pcr for strain prioritization to identify the most promising strains from a microbial strain collection for natural product discovery . resources could then be devoted preferentially to the strains that hold the highest promise in producing novel natural products , thereby accelerating detection and isolation of the targeted natural products and cutting the time and cost associated with traditional natural product discovery programs . central to our method is the application of real - time pcr , targeting genes characteristic to the biosynthetic machinery of natural products with distinct scaffolds , in a high - throughput format and with superior sensitivity and specificity . the practicality and effectiveness of our method were showcased by targeting four diterpene - related synthase genes , t1 , t2 , t3 , and t4 , to prioritize 1911 strains , selected from the actinomycete strain collection at the scripps research institute , for diterpenoid producers in general and ptm and ptn producers in particular ( figure 2 ) . we chose to search for diterpenoid producers because they are underrepresented among microbial natural products and for new ptm and ptn producers because of the heroic effort in the discovery of the original s. platensis ma7327 and ma7339 strains , as well as the need for alternative producers with better genetic amenability . we rapidly identified a total of 488 potential diterpenoid producers , among which six were confirmed as ptm ptn dual producers and one as a viguiepinol and oxaloterpin producer . although the new ptm ptn dual producers are all s. platensis strains on the basis of polyphasic taxonomy , they exhibit district morphology , three of which showed superior genetic amenability to the original s. platensis ma7327 and ma7339 strains . genetic amenability of the producing organisms is of paramount importance in applying microbial genomics to natural product discovery , and new strains , alternatives to genetically recalcitrant original producers for the same natural products or families of natural products with similar scaffolds , represent an innovative solution to this critical challenge . , 488 out of 1911 ) hit rate of diterpenoid producers is higher than expected , given the small number of bacterial diterpenoids known to date , but is consistent with an early survey of our strain collection . taken together , these findings support our early proposal that the biosynthetic potential of diterpenoids in bacteria is significantly underestimated . further prioritization of the hits identified in this study promises the discovery of novel diterpenoid natural products . natural products occupy tremendous chemical structural space that is unmatched by any other small - molecule libraries . while the rich functionality of natural products is , without doubt , one of their great strengths , providing potency and selectivity , the biosynthetic machineries for each of the major molecular scaffolds are highly conserved across the entire family of natural products , as exemplified by the diterpene - related synthases t1 , t2 , t3 , and t4 , for diterpenoids . variations among the myriad of tailoring enzymes associated with each of the biosynthetic machineries further imbue the remarkable structural diversity within each of the natural product families ( figure 2a ) . although we have showcased our method by targeting diterpenoids , variation of the method should be readily applicable to the discovery of other classes of natural products by targeting conserved genes within each of the major biosynthetic machineries . applications of genome sequencing and genome mining to the high - priority strains could essentially eliminate the chance elements from traditional discovery programs and fundamentally change how natural products are discovered . finally , we used a dna intercalating fluorophore in the current method , allowing detection and subsequent melting temperature analysis of the amplified products . one could envision variations of the method with fluorophore - labeled oligonucleotides or molecular beacons in a multiplex approach of real - time pcr to target multiple genes simultaneously within a single reaction , further expanding its utility in strain prioritization , thereby accelerating natural product discovery . after the normalization of gdna concentration , samples were arrayed in 384-well masterblock deep - well plates as working stocks and transferred to microplates using a biomek fx workstation ( beckman coulter ) for each real - time pcr experiment . real - time pcr was performed with an applied biosystems 7900ht fast real - time pcr system . primer design was based on the ptmt3 sequence and the conserved sequences of ptmt1 , ptmt2 , and ptmt4 homologues ( figures s1 , s2 ) . the reaction conditions consisted of a background check at 50 c for 2 min ; initial denaturation at 95 c for 7 min ; and 3740 cycles of denaturation at 95 c for 30 s , primer annealing at 6468 c for 15 s , extension at 68 c for 3060 s , and melting at 95 c for 15 s with a ramp rate of 2% from 68 to 95 c . samples with tm 0.8 c compared to the positive control were considered hits . to confirm hits , regular pcr was performed , and the resulting products were analyzed by 1% agarose gel electrophoresis , purified by gel extraction , and sequenced . the t1 , t2 , t3 , t4 , and r1 genes of the six new ptm ptn dual producers ( cb00739 , cb00765 , cb00775 , cb00789 , cb02289 , and cb02304 ) were amplified using primers designed according to the ptm ptn dual biosynthetic gene cluster from s. platensis ma7327 ( table s1 ) . regions of the four housekeeping genes , 16s rrna , reca , rpob , and trpb , were amplified using primers listed in table s1 . gene disruption of ptmr1 was performed in e. coli bw25113/pij790 carrying appropriate cosmids by following the red - mediated pcr - targeting mutagenesis method as described previously . the mutant cosmids were introduced into the s. platensis cb00739 , cb00765 , and cb00775 by intergeneric conjugation after passaging the mutant cosmids through the nonmethylating e. coli et12567/puz8002 . apramycin selection and kanamycin sensitivity on isp4 medium were used to determine double - crossover mutants of the ptmr1 genes . acetone was removed under reduced pressure , and the resulting oil was resuspended in 1.5 ml of meoh .

chlorinecontaining organics ( table 1 ) are often believed to originate exclusively from industrial pollution . however , many living organisms ( e.g. marine sponges or terrestrial antagonistic microorganisms as a part of their defence mechanisms ) produce them naturally whereas chlorinated compounds are also released as a result of , for example , eruptions of volcanoes , forest fires and geothermal processes ( griebler et al . , 2004 ; bengtson et al . , nevertheless , it is their extensive industrial ( e.g. solvent , metal degreasing , rubber production ) and agricultural ( e.g. pesticide component ) application over the past 50 years that resulted in their deposition in various environments , especially in soils , groundwater aquifers and sediments ( bailey , 2001 ; meijer et al . , 2003 ; barber et al . , 2005 ; due to their physicochemical properties ( table 1 ) , exposure to these compounds can have carcinogenic and lethal effects on biota . therefore , the production and application of most of these compounds is no longer allowed in 90 countries since the stockholm convention in 2001 ( decision no . 2455/2001/ec , 2001 ; unep , 2005 ) . remediation of soils and groundwater can be achieved via physicochemical methods such as thermal cleaning , chemical oxidation or adsorption of pollutants on activated carbon ( lai et al . , 2007 ) , whereas there are no in situ remediation technologies for sediments other than complete removal of the contaminated sediment ( wenning et al . , 2006 ) . moreover , the high ecological disturbance that these physicochemical treatment methods can cause in the environment makes them unsustainable solutions in the long term ( wenning et al . , 2006 ) . other than harsh physicochemical treatments , a far more preferable option is bioremediation . during bioremediation chlorinated contaminants phytoremediation , where plants are employed to assimilate , degrade , metabolize or detoxify chlorinated compounds , is an effective bioremediation method ( susarla et al . , 2002 ) . for example , poplar trees were shown to assimilate and degrade tce to 2,2,2trichloroethanol , trichloroacetic acid and dichloroacetic acid ( newman et al . , 1997 ) . recently , it has also been shown that the presence of these trees may stimulate the transformation of pce in the subsurface ( james et al . , 2009 ) . in this study , in the test location populated with hybrid poplar trees pce pollution was reduced by over 99% , in comparison with 2% removal in an unplanted control . moreover , pepo ( squash ) , were shown to extract milligrams of pcbs from soil in approximately 8 weeks time ( zeeb et al . , 2006 ) . lately the generation of transgenic plants to improve the phytoremediation of these pollutants resulted in several promising demonstrations of tce , 1,2dichloroethane ( dca ) and chlorophenol removal in several laboratory scale tests ( wang et al . , 2004 ; dowling and doty , 2009 ; james and strand , 2009 ) . most fungi are robust organisms and are generally tolerant to high levels of pollution ( singh , 2006 ) . fungal lignocellulolytic enzymes have been related to the degradation of various pollutants when used in combination with mediators and reactive radicals . being the most commonly studied example , whiterot fungi are able to detoxify a wide range of pollutants including chlorinated organics , with lignin and manganese peroxidases ( tortella et al . sources , biological impacts and physicochemical properties of chlorinated organic compounds that have been reported to be degraded by hexachlorobenzene [ agency for toxic substances and disease registry ( atsdr ) , 2002 ] . there are theoretically 209 different pcb congeners , although only about 130 of these were found in commercial pcb mixtures ( unep chemicals , 1999 ) . chlorophenols [ agency for toxic substances and disease registry ( atsdr ) , 1999 ] . only pce is illustrated . the bacterial degradation of chlorinated pollutants can be a result of fortuitous cometabolic conversion , or it may contribute to the energy metabolism of the degrading organism . during the latter metabolic processes , chlorinated compounds are used either as carbon source or as electron acceptor ( coupled to the oxidation of an electron donor ) , depending on the oxidation state of the compound . although many chlorinated compounds may be transformed under aerobic conditions , the majority of polychlorinated compounds , such as those discussed in this review , are recalcitrant to aerobic degradation . due to the electronegative nature of the chlorine atom , oxidation of the carbon backbone in the chlorinated compound becomes thermodynamically unfavourable ( wohlfarth and diekert , 1997 ) , especially in polychlorinated compounds . as a result they serve as energetically favourable electron acceptors in microbial metabolism in anoxic environments such as sediments , subsurface soils and groundwater aquifers . consequently , anaerobic bacteria , which can use these compounds as electron acceptors in a process termed organohalide respiration , are good candidates for bioremediation ( van eekert and schraa , 2001 ) . within the organohaliderespiring bacteria , dehalococcoides spp . and related isolates within the chloroflexi represent a special case in the anaerobic detoxification of halogenated organic contaminants . it has been shown that several other bacteria belonging to the and proteobacteria ( anaeromyxobacter , desulfuromonas , desulfomonile , desulfovibrio , geobacter , sulfurospirillum ) or to the lowgc grampositive bacteria ( desulfitobacterium , dehalobacter ) are also able to degrade chlorinated organic contaminants through organohalide respiration ( fig . 1 ) , none of these species are as specialized as dehalococcoides , and they are reported to grow as well , for example , by metal reduction , denitrification or fermentation . alignment and phylogenetic analysis were performed with the arb software using the most recent release of the arbsilva project ( silva 96 ) ( ludwig et al . , 2004 ; pruesse et al . , 2007 ) , and the tree was constructed using the neighbour joining method . dehalococcoides is a genus of strictly anaerobic gramnegative bacteria that to the best of our knowledge are restricted to gaining energy from the reduction of chlorinated compounds by organohalide respiration . isolates have an irregular , spherical shape ( approximately 0.5 m ) often referred to as coccoid . electron acceptors such as oxygen , nitrate or sulfate has never been reported ( kube et al . , 2005 ; seshadri et al . , 2005 ) . reductive dechlorination by dehalococcoides spp . occurs via the replacement of a chlorine atom in the chlorinated compound by hydrogen ( reductive hydrogenolysis ) and results in a net input of one proton and two electrons ( fig . 2 ) ( holliger et al . , 1998 ) . gibbs free energy ( g ) generated with reductive dechlorination of chlorinated compounds could range from 130 to 180 kj mol per chlorine removed . the redox potential thus generated is comparable to the redox potential of denitrification and higher than that generated by sulfate reduction . as a result it was suggested that reductively dechlorinating bacteria could outcompete sulfate reducers and methanogens for reducing equivalents when the formation of reducing equivalents is rate limiting ( dolfing , 2003 ) . are also capable of degrading chlorinated aliphatic compounds , i.e. 1,2dca , via socalled dihaloelimination . in this process two neighbouring chlorine atoms are concurrently replaced via the formation of a double bond between the two carbon atoms . dihaloelimination requires less h2 for the removal of chlorine atoms than reductive hydrogenolysis , thus its energy balance is more favourable under h2limited conditions ( smidt and de vos , 2004 ) . reductive dechlorination of hexachlorobenzene ( hcb ) to pentachlorobenzene ( qcb ) . as the ecologists ' quest prevails to delve becking and beijerinck 's long running argument : everything is everywhere , but the environment selects ( beijerinck , 1913 ; becking , 1934 ) , the application of biomolecular tools , including the pcr amplification and sequencing of 16s ribosomal rna ( rrna ) genes from environmental samples , enables to study the full extent of microbial diversity and describe the biogeographical patterns exhibited by microorganisms at large spatial scales ( fierer and jackson , 2006 ; martiny et al . , 2006 ) . with the growing interest in dehalococcoides ' presence and functioning in the environment , several studies were conducted using dehalococcoidesspecific 16s rrna genebased approaches in uncontaminated and chlorinated ethene contaminated sediments , soils and groundwater aquifers ( lffler et al . , 2000 ; hendrickson et al . , 2002 ; kittelmann and friedrich , 2008 ) . currently , more than 100 16s rrna gene sequences of cultured and uncultured dehalococcoides spp . have been deposited to the database of the national center for biotechnology information ( ncbi ) . 1 ) ; however , various studies showed that this group is functionally very diverse ( maymogatell et al . , 1997 ; adrian et al . , 2000 ; hendrickson et al . , 2002 ; he et al . eight dehalococcoides strains have been isolated , mainly for their ability to degrade chlorinated ethenes ( table 2 ) . functional differences in these isolates can be observed in the chlorinated compound transformed and in the transformation endproducts . for example , the first isolate of the genusdehalococcoides ethenogenes strain 195 can completely dechlorinate pce to ethene , although degradation of vinyl chloride ( vc ) to ethene is cometabolic ( maymogatell et al . , 1997 ) . dehalococcoides ethenogenes strain 195 can also dechlorinate hcb to 1,3dcb ( dichlorobenzene ) , 1,4dcb , 1,2dcb and 1,3,5tcb ( trichlorobenzene ) . cbdb1 dechlorinates hcb to 1,3dcb , 1,4dcb and 1,3,5tcb , and recently also transformation of pce and tce to transdce was observed ( adrian et al . , 2007b ) . are difficult to maintain in pure culture ( maymogatell et al . , 1997 ; adrian et al . , 2000 ; he et al . , 2003 ) ; they are more easily maintained in a microbial community , on which they depend for h2 supply , as long as ideal growth conditions are provided ( duhamel et al . examples include chlorinated ethene transforming cornell ( maymogatell et al . , 1997 ) , victoria ( hendrickson et al . , 2002 ) , pinellas ( harkness et al . , 1999 ) , kb1 ( duhamel et al . , 2002 ) and anas cultures ( richardson et al . , 2002 ) . , two other distantly related isolates within the chloroflexi have recently been obtained ( fig . 2 ) . dehalobium chlorocoercia df1 is able to dechlorinate a variety of pcbs ( may et al . this microorganism dechlorinates polychlorinated alkanes ( yan et al . , 2009 ) . like dehalococcoides spp . , both isolates are strictly hydrogenotrophic several enrichment studies showed the presence of dehalococcoides spp . in different locations and environments in the northern hemisphere ( dehalococcoidescontaining enrichment cultures originating from river sediments have been shown to dechlorinate pcb and dioxin congeners , pce , tce and a number of chlorinated benzenes ( ballerstedt et al . , 2004 ; yoshida et al . , 2005 ; bedard et al . , 2007 ; bunge et al . , 2007 ; besides sediment enrichments , dechlorination by dehalococcoides was also reported in groundwater aquifers ( bowman et al . , 2006 ; brgmann et al . , 2008 ; imfeld et al . , 2008 ; lee et al . , 2008 ; himmelheber et al . , 2009 ) and a denitrifying membranebiofilm reactor ( chung et al . , 2008 ) . few studies have demonstrated that the bioaugmentation with reductively dehalogenating cultures can result in complete dechlorination of pce and tce to ethene ( ellis et al . , 2000 ; major et al . , 2002 ; lendvay et al . , 2003 ; scheutz et al . , in pure and enrichment cultures are in the range of 0.20.4 day under laboratory conditions ( maymogatell et al . , 1997 ; cupples et al . , 2003 ; additionally , quantitative analyses of the dehalococcoides spp . 16s rrna gene at chlorinated ethene bioremediation sites ( soil and groundwater ) revealed abundances of 1010 copies per gram material ( lendvay et al . recently , a groundwater bioremediation simulation study showed that growth rates obtained in laboratory conditions could also be replicated in largescale experiments , which resulted in up to 10 16s rrna gene copies l ( vainberg et al . , 2009 ) . hence , these pilot as well as fieldscale bioremediation tests with dehalococcoidescontaining cultures offer promising results for the further use of these microorganisms . in spite of all the information obtained in physiological studies very little is known about the diversity , distribution and functioning of dehalococcoides in different environments although they were detected at several contaminated locations . hendrickson and coauthors have demonstrated the presence of dehalococcoides spp . in soil and groundwater samples from 24 sites scattered throughout north america and europe ( hendrickson et al . , 2002 ) . up to 200 m pce could be dechlorinated , and complete dechlorination to ethene could be correlated to the presence of dehalococcoides spp . in the sampling locations . recently , we conducted a largescale survey focusing on presence , activity and dechlorination potential of dehalococcoides spp . in river sediments and floodplain soils from different polluted locations in europe ( fig . almost all of the tested sediment and soil samples showed the capacity to dechlorinate hcb and/or chlorinated ethenes irrespective of the in situ contaminant levels . nevertheless , the hcb transformation rates observed in the laboratoryscale microcosms and the number of 16s rrna gene copies of dehalococcoides spp . in the environmental samples relative abundance was furthermore shown to change significantly along temporal and spatial gradients , but was also found to be influenced by other environmental factors such as water temperature ( taet al . , 2009 ) . summary of results from the locations studied byta ( 2009 ) with cultivationdependent and independent molecular methods . detection with 16s rrna and/or 16s rrna genetargeted methods ; : hcb transformation ; : chlorinated ethene transformation ; ( ) no detection or no transformation ; ( + /++/+++ ) low to high rrna copies or long to short lag phases in hcb and chlorinated ethene transformation ; na : not available ; ( ) soil and ( ) river sediment sample from schnberg , germany . map was redrawn from openstreetmap ( http://www.openstreetmap.org ) . as nonfermentative microorganisms dehalococcoides spp . and their organohaliderespiring relatives dehalobium chlorocoercia df1 and dehalogenimonas lykanthroporepellens dc9 depend on the h2 supply from other microorganisms for their growth ( smidt and de vos , 2004 ; may et al . recently , it has also been suggested that the activity of dehalococcoides spp . in in situ conditions is linked to the performance of fermentative communities ( rling et al . , 2007 ) . therefore , it is crucial to have insight in factors affecting nutrient fluxes and microbial communities involved in carbon , nitrogen and sulfur ( c , n , s ) cycling in the river basins to be able to understand the survival and functioning of dehalococcoides spp . in different geographical locations . because there are considerable differences between dechlorination capabilities of the known dehalococcoides strains despite 16s rrna identities of > 99% , their sole presence based on the detection of the 16s rrna gene in an environment does not guarantee successful in situ dechlorination of a specific pollutant . consequently , molecular tools that target metabolic activities of the entire microbial communities in the environment are needed to have a canonical assessment of the conditions . our knowledge gap concerning the properties of dehalococcoides spp . is closing rapidly with the developments in highthroughput sequencing technologies . nc_002936 ) and strain cbdb1 ( nc_007356 ) genomes are approximately 1.47 and 1.39 million base pairs ( mbp ) respectively . both genomes comprise single circular chromosomes with 1591 predicted protein coding sequences ( cds ) in strain 195 ( seshadri et al . , 2005 ) and 1458 cds in strain cbdb1 ( table 3 ) . up to 1217 of the cds from strain cbdb1 have orthologous genes in d. ethenogenes strain 195 ( 83.5% ) ( kube et al . , 2005 ) . strain bav1 ( nc_009455 ) has a genome of 1.34 mbp with 1385 cds based on information provided in the integrated microbial genomes ( img ) database , release march 2009 ( markowitz et al . , 2008 ) . for example , one copy of each rrna gene is present in all dehalococcoides genomes ( kube et al . , 2005 ; seshadri et al . , comparative analysis of available dehalococcoides genomes showed that 70% of all genes in these genomes have a high sequence and contextual conservation ( mcmurdie et al . , 2008 ) . interestingly , d. ethenogenes strain 195 possesses a nitrogenaseencoding operon , which is missing in strain cbdb1 . even though this finding suggests that d. ethenogenes strain 195 can fix nitrogen , diazotropic growth of the dehalococcoides strains has not yet been reported . comparison of wholegenome sequence statistics for reductively dechlorinating bacteria as presented in integrated microbial genomes ( img / m ) database , march 2009 ( markowitz et al . , 2008 ) . genes : total gene count ; cds : coding sequences ; rna : number of rrna , trna and other rna genes ; 16s : number of 16s rrna gene copies ; orthologues : number of genes in orthologues ; paralogues : number of genes in paralogues ; rdh genes : confirmed and predicted reductive dehalogenaseencoding genes . different dehalococcoides strains contain different numbers of rdh genes that encode protein , which have been proven or predicted to catalyse the dechlorination reaction . when compared with the genomes of other dechlorinating bacteria , dehalococcoides have the highest number of rdh genes in their genomes ( table 3 ) . genomes of strains 195 , cbdb1 and bav1 have 17 , 32 and 10 rdh genes , respectively , whereas only seven rdh genes were identified in the genome of desulfitobacterium hafniense dcb2 , four rdh genes in d. hafniense y51 and two rdh genes in geobacter lovleyi sz and anaeromyxobacter dehalogenans ( thomas et al . , the draft genome of strain vs contains the highest number of rdh genes ( 36 fulllength genes ) ever found in a single bacterial genome ( mcmurdie et al . , 2008 ) . similarly , 14 and 19 rdh genes were detected via pcr amplification in dehalococcoides sp . strains fl2 and dcmb5 respectively ( holscher et al . , 2004 ; bunge et al . , twelve rdh genes from strain cbdb1 have orthologues in d. ethenogenes strain 195 genome with 86.495.4% sequence identity . in d. ethenogenes strain 195 and strain cbdb1 genomes almost all of the rdh genes ( except det0079 , tce reductive dehalogenase tcea in d. ethenogenes strain 195 and cbdba1583 in strain cbdb1 ) were found to be located in close proximity to genes for transcription regulators , and were predicted to be transcribed in the direction of dna synthesis , which suggests tight regulation of rdh activity ( kube et al . , 2005 ; seshadri et al . , only two rdh genes from strain 195 , det0079 and det0318 , have been characterized as tce ( tcea ) and pce ( pcea ) reductive dehalogenases respectively ( fung et al . , 2007 ) . the cbdba84 gene from strain cbdb1 was recently designated as a chlorobenzene reductive dehalogenase ( cbra ) , which is involved in dechlorination of 1,2,3,4tecb and 1,2,3tcb ( adrian et al . additionally , two vc reductase genes were identified from strain bav1 ( bvcra , dehabav1_0847 ) ( krajmalnikbrown et al . , 2004 ) and strain vs ( vcra , genbank accession no . can not be inferred from dehalococcoides phylogeny , detection methods based on processspecific biomarkers are necessary to describe the bioremediation capacity and activity of dehalococcoides in the environment . therefore , genes like rdhs and the corresponding gene products that are specific to functions of interest can serve as useful biomarkers in monitoring of different dehalococcoides activities . in the past years microarrays were shown to be useful tools for such monitoring activities and characterization of microbial communities ( zhou , 2003 ; wang et al . , 2009 ) . furthermore , functional gene arrays ( fgas ) , which target functional genes such as nitrogenases , cellulases etc . , allow fast and comprehensive analysis of metabolic potential and activity of microbial communities in the environment by targeting a large number of genes or their transcripts in one single experiment ( wu et al . , 2001 ; taroncheroldenburg et al . , 2003 ; steward et al . , 2004 ; zhou et al . , 2008 ) up to date the most extensive fga platform is the geochip ( he et al . , 2007 ) , which targets approximately 10 000 catabolic genes involved in major biogeochemical cycles , including those of carbon , nitrogen and sulfur , as well as organic pollutant degradation . analysis of hcbcontaminated sediments in the ebro river basin ( spain ) using the geochip amended with probes targeting 153 rdh genes showed that rdh gene diversity changed significantly between different sampling locations ( taet al . , 2009 ) . more specifically , sediment samples taken at a site with high hcb pollution ( lacorte et al . , 2006 ) were dominated by rdh genes of dehalococcoides spp . in contrast samples , which were characterized by more diffuse pollution with a broader range of contaminants , a wide spectrum of rdh genes was detected including those from various other organohaliderespiring microorganisms ( fig . however , it should be noted that microarrays can only detect known sequences , which can cause an underestimation of functional gene diversity and abundance in environments for which limited sequence information is available . application of fgas in combination with newly developed techniques such as highthroughput nongelbased proteomics ( maron et al . , 2007 ) and strain cbdb1 transcriptomes suggested continuous transcription of rdh genes such as tcea ( johnson et al . , 2008 ) and cbra ( wagner et al . , 2009 ) during different growth phases . as a result gene transcripts of such genes can be studied using transcriptomic techniques with fgas , in combination with proteomics methods ( morris et al . , 2006 ; morris et al . , 2007 ) to identify the proteins with significant functional impact . hierarchical cluster analysis of rdh gene profiles based on geochip functional gene array hybridization signals for samples from flix and rice fields ( rf , river delta ) in the ebro river . hcb is reported to be the dominant chlorinated contaminant in ebro 's basin where location flix bares the highest hcb pollution ( lacorte et al . , 2006 ) . the greyscale intensity indicates differences in hybridization signal intensity , with black representing the strongest signals . samples are represented according to sampling month , year and sampling depth ( i.e. f06d5 : february 2006 depth 05 cm ; j04d15 : june 2004 depth 1015 cm ) . for accession numbers of rdh gene targets , see ta and colleagues ( 2009 ) . the broad aim of systems microbiology is to define and understand the relationships between the individual components that build a cellular organism , a community and an ecological niche ( vieites et al . , 2009 ) . as a result , in the past , the focus of systems microbiology was on microbial isolates or enrichments ( mchardy and rigoutsos , 2007 ) . to date , the majority of the research conducted in the field of reductive dechlorination has been predominantly focused on the identification of genes and proteins directly responsible for the dechlorination process ( cupples et al . , 2003 ; muller et al . , 2004 ; holmes et al . , 2006 ; adrian et al . , 2007a ; mcmurdie et al . , 2007 ; west et al . , 2008 ; wagner et al . , these experimental studies , so far , allowed the analysis and characterization of several key genes . however , it is becoming evident that to understand microbial functions or functioning of microbial communities one must study the entire system ( vieites et al . , 2009 ) . the body of research summarized in this review also supports this idea and suggests that with biomolecular assays targeting ribosomal and processspecific functional genes such as those encoding reductive dehalogenases , it will remain difficult to understand the full extent of the process , since the dechlorination process comprises an integral part of a complex web of metabolic and regulatory interactions ( rahm et al . , 2006 ; west et al . the application of novel , more comprehensive methods like whole genome shotgun ( wgs ) sequencing of environmental dna and mrna ( functional metagenomics ) ( tringe et al . , 2005 ; kalyuzhnaya et al . , 2008 ) , the establishment of largescale databases which contain metagenomic data from different environments ( seshadri et al . , 2007 ; , 2009 ; vogel et al . , 2009 ) as well as the development of new computational resources for comparative ( meta)genomic analyses ( peterson et al . , 2001 ; alm et al . , 2005 ; markowitz et al . , 2006 ) enable us to develop and analyse data sets ( and microbial networks ) which so far are believed to be the closest to the actual environmental situations . thus , today , it can be proposed to leave reductionist approaches that are limited to only one or a few selected biomarkers , and to study reductive dechlorination and the function of dehalococcoides spp . in larger communities and in the environments in which they belong . as the functional properties of such communities are elucidated , we will be able to assess the true role and importance of dehalococcoides spp .

summarythe fate and persistence of chlorinated organics in the environment have been a concern for the past 50 years . industrialization and extensive agricultural activities have led to the accumulation of these pollutants in the environment , while their adverse impact on various ecosystems and human health also became evident . this review provides an update on the current knowledge of specialized anaerobic bacteria , namely dehalococcoides spp . , which are dedicated to the transformation of various chlorinated organic compounds via reductive dechlorination . advances in microbiology and molecular techniques shed light into the diversity and functioning of dehalococcoides spp . in several different locations . recent genome sequencing projects revealed a large number of genes that are potentially involved in reductive dechlorination . molecular approaches towards analysis of diversity and expression especially of reductive dehalogenaseencoding genes are providing a growing body of knowledge on biodegradative pathways active in defined pure and mixed cultures as well as directly in the environment . moreover , several successful field cases of bioremediation strengthen the notion of dedicated degraders such as dehalococcoides spp . as key players in the restoration of contaminated environments .

Pollution of chlorinated compounds and their bioremediation The little bacteria that can: the genus Discoveries from Future perspectives: reductive dechlorination, systems microbiology and microbial networks

chlorinecontaining organics ( table 1 ) are often believed to originate exclusively from industrial pollution . , 2004 ; bengtson et al . pesticide component ) application over the past 50 years that resulted in their deposition in various environments , especially in soils , groundwater aquifers and sediments ( bailey , 2001 ; meijer et al . , 2003 ; barber et al . , 2005 ; due to their physicochemical properties ( table 1 ) , exposure to these compounds can have carcinogenic and lethal effects on biota . therefore , the production and application of most of these compounds is no longer allowed in 90 countries since the stockholm convention in 2001 ( decision no . remediation of soils and groundwater can be achieved via physicochemical methods such as thermal cleaning , chemical oxidation or adsorption of pollutants on activated carbon ( lai et al . moreover , the high ecological disturbance that these physicochemical treatment methods can cause in the environment makes them unsustainable solutions in the long term ( wenning et al . during bioremediation chlorinated contaminants phytoremediation , where plants are employed to assimilate , degrade , metabolize or detoxify chlorinated compounds , is an effective bioremediation method ( susarla et al . recently , it has also been shown that the presence of these trees may stimulate the transformation of pce in the subsurface ( james et al . in this study , in the test location populated with hybrid poplar trees pce pollution was reduced by over 99% , in comparison with 2% removal in an unplanted control . moreover , pepo ( squash ) , were shown to extract milligrams of pcbs from soil in approximately 8 weeks time ( zeeb et al . lately the generation of transgenic plants to improve the phytoremediation of these pollutants resulted in several promising demonstrations of tce , 1,2dichloroethane ( dca ) and chlorophenol removal in several laboratory scale tests ( wang et al . most fungi are robust organisms and are generally tolerant to high levels of pollution ( singh , 2006 ) . fungal lignocellulolytic enzymes have been related to the degradation of various pollutants when used in combination with mediators and reactive radicals . being the most commonly studied example , whiterot fungi are able to detoxify a wide range of pollutants including chlorinated organics , with lignin and manganese peroxidases ( tortella et al . sources , biological impacts and physicochemical properties of chlorinated organic compounds that have been reported to be degraded by hexachlorobenzene [ agency for toxic substances and disease registry ( atsdr ) , 2002 ] . there are theoretically 209 different pcb congeners , although only about 130 of these were found in commercial pcb mixtures ( unep chemicals , 1999 ) . the bacterial degradation of chlorinated pollutants can be a result of fortuitous cometabolic conversion , or it may contribute to the energy metabolism of the degrading organism . during the latter metabolic processes , chlorinated compounds are used either as carbon source or as electron acceptor ( coupled to the oxidation of an electron donor ) , depending on the oxidation state of the compound . although many chlorinated compounds may be transformed under aerobic conditions , the majority of polychlorinated compounds , such as those discussed in this review , are recalcitrant to aerobic degradation . due to the electronegative nature of the chlorine atom , oxidation of the carbon backbone in the chlorinated compound becomes thermodynamically unfavourable ( wohlfarth and diekert , 1997 ) , especially in polychlorinated compounds . as a result they serve as energetically favourable electron acceptors in microbial metabolism in anoxic environments such as sediments , subsurface soils and groundwater aquifers . consequently , anaerobic bacteria , which can use these compounds as electron acceptors in a process termed organohalide respiration , are good candidates for bioremediation ( van eekert and schraa , 2001 ) . within the organohaliderespiring bacteria , dehalococcoides spp . and related isolates within the chloroflexi represent a special case in the anaerobic detoxification of halogenated organic contaminants . it has been shown that several other bacteria belonging to the and proteobacteria ( anaeromyxobacter , desulfuromonas , desulfomonile , desulfovibrio , geobacter , sulfurospirillum ) or to the lowgc grampositive bacteria ( desulfitobacterium , dehalobacter ) are also able to degrade chlorinated organic contaminants through organohalide respiration ( fig . 1 ) , none of these species are as specialized as dehalococcoides , and they are reported to grow as well , for example , by metal reduction , denitrification or fermentation . dehalococcoides is a genus of strictly anaerobic gramnegative bacteria that to the best of our knowledge are restricted to gaining energy from the reduction of chlorinated compounds by organohalide respiration . electron acceptors such as oxygen , nitrate or sulfate has never been reported ( kube et al . reductive dechlorination by dehalococcoides spp . occurs via the replacement of a chlorine atom in the chlorinated compound by hydrogen ( reductive hydrogenolysis ) and results in a net input of one proton and two electrons ( fig . gibbs free energy ( g ) generated with reductive dechlorination of chlorinated compounds could range from 130 to 180 kj mol per chlorine removed . the redox potential thus generated is comparable to the redox potential of denitrification and higher than that generated by sulfate reduction . in this process two neighbouring chlorine atoms are concurrently replaced via the formation of a double bond between the two carbon atoms . dihaloelimination requires less h2 for the removal of chlorine atoms than reductive hydrogenolysis , thus its energy balance is more favourable under h2limited conditions ( smidt and de vos , 2004 ) . reductive dechlorination of hexachlorobenzene ( hcb ) to pentachlorobenzene ( qcb ) . as the ecologists ' quest prevails to delve becking and beijerinck 's long running argument : everything is everywhere , but the environment selects ( beijerinck , 1913 ; becking , 1934 ) , the application of biomolecular tools , including the pcr amplification and sequencing of 16s ribosomal rna ( rrna ) genes from environmental samples , enables to study the full extent of microbial diversity and describe the biogeographical patterns exhibited by microorganisms at large spatial scales ( fierer and jackson , 2006 ; martiny et al . with the growing interest in dehalococcoides ' presence and functioning in the environment , several studies were conducted using dehalococcoidesspecific 16s rrna genebased approaches in uncontaminated and chlorinated ethene contaminated sediments , soils and groundwater aquifers ( lffler et al . currently , more than 100 16s rrna gene sequences of cultured and uncultured dehalococcoides spp . have been deposited to the database of the national center for biotechnology information ( ncbi ) . eight dehalococcoides strains have been isolated , mainly for their ability to degrade chlorinated ethenes ( table 2 ) . functional differences in these isolates can be observed in the chlorinated compound transformed and in the transformation endproducts . , 1997 ) . cbdb1 dechlorinates hcb to 1,3dcb , 1,4dcb and 1,3,5tcb , and recently also transformation of pce and tce to transdce was observed ( adrian et al . , 2002 ) . 2 ) . like dehalococcoides spp . , both isolates are strictly hydrogenotrophic several enrichment studies showed the presence of dehalococcoides spp . in different locations and environments in the northern hemisphere ( dehalococcoidescontaining enrichment cultures originating from river sediments have been shown to dechlorinate pcb and dioxin congeners , pce , tce and a number of chlorinated benzenes ( ballerstedt et al . , 2005 ; bedard et al . , 2002 ; lendvay et al . , in pure and enrichment cultures are in the range of 0.20.4 day under laboratory conditions ( maymogatell et al . , 2003 ; additionally , quantitative analyses of the dehalococcoides spp . recently , a groundwater bioremediation simulation study showed that growth rates obtained in laboratory conditions could also be replicated in largescale experiments , which resulted in up to 10 16s rrna gene copies l ( vainberg et al . hence , these pilot as well as fieldscale bioremediation tests with dehalococcoidescontaining cultures offer promising results for the further use of these microorganisms . in spite of all the information obtained in physiological studies very little is known about the diversity , distribution and functioning of dehalococcoides in different environments although they were detected at several contaminated locations . hendrickson and coauthors have demonstrated the presence of dehalococcoides spp . up to 200 m pce could be dechlorinated , and complete dechlorination to ethene could be correlated to the presence of dehalococcoides spp . in the sampling locations . recently , we conducted a largescale survey focusing on presence , activity and dechlorination potential of dehalococcoides spp . in river sediments and floodplain soils from different polluted locations in europe ( fig . almost all of the tested sediment and soil samples showed the capacity to dechlorinate hcb and/or chlorinated ethenes irrespective of the in situ contaminant levels . nevertheless , the hcb transformation rates observed in the laboratoryscale microcosms and the number of 16s rrna gene copies of dehalococcoides spp . in the environmental samples relative abundance was furthermore shown to change significantly along temporal and spatial gradients , but was also found to be influenced by other environmental factors such as water temperature ( taet al . , 2009 ) . detection with 16s rrna and/or 16s rrna genetargeted methods ; : hcb transformation ; : chlorinated ethene transformation ; ( ) no detection or no transformation ; ( + /++/+++ ) low to high rrna copies or long to short lag phases in hcb and chlorinated ethene transformation ; na : not available ; ( ) soil and ( ) river sediment sample from schnberg , germany . as nonfermentative microorganisms dehalococcoides spp . and their organohaliderespiring relatives dehalobium chlorocoercia df1 and dehalogenimonas lykanthroporepellens dc9 depend on the h2 supply from other microorganisms for their growth ( smidt and de vos , 2004 ; may et al . recently , it has also been suggested that the activity of dehalococcoides spp . in in situ conditions is linked to the performance of fermentative communities ( rling et al . , 2007 ) . therefore , it is crucial to have insight in factors affecting nutrient fluxes and microbial communities involved in carbon , nitrogen and sulfur ( c , n , s ) cycling in the river basins to be able to understand the survival and functioning of dehalococcoides spp . because there are considerable differences between dechlorination capabilities of the known dehalococcoides strains despite 16s rrna identities of > 99% , their sole presence based on the detection of the 16s rrna gene in an environment does not guarantee successful in situ dechlorination of a specific pollutant . consequently , molecular tools that target metabolic activities of the entire microbial communities in the environment are needed to have a canonical assessment of the conditions . our knowledge gap concerning the properties of dehalococcoides spp . up to 1217 of the cds from strain cbdb1 have orthologous genes in d. ethenogenes strain 195 ( 83.5% ) ( kube et al . strain bav1 ( nc_009455 ) has a genome of 1.34 mbp with 1385 cds based on information provided in the integrated microbial genomes ( img ) database , release march 2009 ( markowitz et al . , comparative analysis of available dehalococcoides genomes showed that 70% of all genes in these genomes have a high sequence and contextual conservation ( mcmurdie et al . interestingly , d. ethenogenes strain 195 possesses a nitrogenaseencoding operon , which is missing in strain cbdb1 . genes : total gene count ; cds : coding sequences ; rna : number of rrna , trna and other rna genes ; 16s : number of 16s rrna gene copies ; orthologues : number of genes in orthologues ; paralogues : number of genes in paralogues ; rdh genes : confirmed and predicted reductive dehalogenaseencoding genes . different dehalococcoides strains contain different numbers of rdh genes that encode protein , which have been proven or predicted to catalyse the dechlorination reaction . when compared with the genomes of other dechlorinating bacteria , dehalococcoides have the highest number of rdh genes in their genomes ( table 3 ) . genomes of strains 195 , cbdb1 and bav1 have 17 , 32 and 10 rdh genes , respectively , whereas only seven rdh genes were identified in the genome of desulfitobacterium hafniense dcb2 , four rdh genes in d. hafniense y51 and two rdh genes in geobacter lovleyi sz and anaeromyxobacter dehalogenans ( thomas et al . , the draft genome of strain vs contains the highest number of rdh genes ( 36 fulllength genes ) ever found in a single bacterial genome ( mcmurdie et al . in d. ethenogenes strain 195 and strain cbdb1 genomes almost all of the rdh genes ( except det0079 , tce reductive dehalogenase tcea in d. ethenogenes strain 195 and cbdba1583 in strain cbdb1 ) were found to be located in close proximity to genes for transcription regulators , and were predicted to be transcribed in the direction of dna synthesis , which suggests tight regulation of rdh activity ( kube et al . , only two rdh genes from strain 195 , det0079 and det0318 , have been characterized as tce ( tcea ) and pce ( pcea ) reductive dehalogenases respectively ( fung et al . the cbdba84 gene from strain cbdb1 was recently designated as a chlorobenzene reductive dehalogenase ( cbra ) , which is involved in dechlorination of 1,2,3,4tecb and 1,2,3tcb ( adrian et al . can not be inferred from dehalococcoides phylogeny , detection methods based on processspecific biomarkers are necessary to describe the bioremediation capacity and activity of dehalococcoides in the environment . therefore , genes like rdhs and the corresponding gene products that are specific to functions of interest can serve as useful biomarkers in monitoring of different dehalococcoides activities . in the past years microarrays were shown to be useful tools for such monitoring activities and characterization of microbial communities ( zhou , 2003 ; wang et al . furthermore , functional gene arrays ( fgas ) , which target functional genes such as nitrogenases , cellulases etc . , allow fast and comprehensive analysis of metabolic potential and activity of microbial communities in the environment by targeting a large number of genes or their transcripts in one single experiment ( wu et al . , 2001 ; taroncheroldenburg et al . , 2007 ) , which targets approximately 10 000 catabolic genes involved in major biogeochemical cycles , including those of carbon , nitrogen and sulfur , as well as organic pollutant degradation . analysis of hcbcontaminated sediments in the ebro river basin ( spain ) using the geochip amended with probes targeting 153 rdh genes showed that rdh gene diversity changed significantly between different sampling locations ( taet al . , 2009 ) . more specifically , sediment samples taken at a site with high hcb pollution ( lacorte et al . , 2006 ) were dominated by rdh genes of dehalococcoides spp . in contrast samples , which were characterized by more diffuse pollution with a broader range of contaminants , a wide spectrum of rdh genes was detected including those from various other organohaliderespiring microorganisms ( fig . however , it should be noted that microarrays can only detect known sequences , which can cause an underestimation of functional gene diversity and abundance in environments for which limited sequence information is available . application of fgas in combination with newly developed techniques such as highthroughput nongelbased proteomics ( maron et al . , 2007 ) and strain cbdb1 transcriptomes suggested continuous transcription of rdh genes such as tcea ( johnson et al . , 2006 ; morris et al . , 2007 ) to identify the proteins with significant functional impact . hierarchical cluster analysis of rdh gene profiles based on geochip functional gene array hybridization signals for samples from flix and rice fields ( rf , river delta ) in the ebro river . for accession numbers of rdh gene targets , see ta and colleagues ( 2009 ) . as a result , in the past , the focus of systems microbiology was on microbial isolates or enrichments ( mchardy and rigoutsos , 2007 ) . to date , the majority of the research conducted in the field of reductive dechlorination has been predominantly focused on the identification of genes and proteins directly responsible for the dechlorination process ( cupples et al . however , it is becoming evident that to understand microbial functions or functioning of microbial communities one must study the entire system ( vieites et al . the body of research summarized in this review also supports this idea and suggests that with biomolecular assays targeting ribosomal and processspecific functional genes such as those encoding reductive dehalogenases , it will remain difficult to understand the full extent of the process , since the dechlorination process comprises an integral part of a complex web of metabolic and regulatory interactions ( rahm et al . the application of novel , more comprehensive methods like whole genome shotgun ( wgs ) sequencing of environmental dna and mrna ( functional metagenomics ) ( tringe et al . , 2009 ) as well as the development of new computational resources for comparative ( meta)genomic analyses ( peterson et al . , 2006 ) enable us to develop and analyse data sets ( and microbial networks ) which so far are believed to be the closest to the actual environmental situations . thus , today , it can be proposed to leave reductionist approaches that are limited to only one or a few selected biomarkers , and to study reductive dechlorination and the function of dehalococcoides spp . in larger communities and in the environments in which they belong . as the functional properties of such communities are elucidated , we will be able to assess the true role and importance of dehalococcoides spp .

epidemiological studies have estimated that rates of obesity worldwide have increased steadily between 1980 and 2013 . in the us , over one third of adults and one sixth of children and adolescents are currently considered obese.14 overweight and obesity are frequently coupled to other debilitating diseases such as type 2 diabetes , coronary heart disease , ischemic stroke , and several cancers.57 particularly concerning is the increasing prevalence of pediatric obesity which has been linked not only to metabolic disorders and cardiovascular risk factors but also to psychiatric illness , early pubertal onset , and orthopedic disorders.811 because of the comorbidities associated with increased body weight , obesity is considered to be a major public health concern with a significant economic burden to our health care system . it has been projected that by 2030 , costs associated with overweight and obesity will comprise 16%18% of total health care expenses in the us.3,12 surgical interventions , such as roux - en - y gastric bypass and sleeve gastrectomy , have been highly successful in promoting weight loss and correcting accompanying comorbidities like diabetes.13,14 however , due to the risks associated with surgery , particularly in a vulnerable population , surgical interventions are often considered to be a last resort and reserved for those patients who are morbidly obese or have failed at other weight loss strategies . clearly , there is a need for less invasive means to achieve sustainable weight loss in patients who are not prime candidates for bariatric surgery or desire nonsurgical alternatives . a number of pharmacological interventions have been marketed for weight loss with mixed or limited success . appetite suppressants , such as phentermine , were initially approved by the us food and drug administration ( fda ) in the 1950s for short - term use for weight loss ; however , limited data are available to support their long - term efficacy . more recently , a combination drug consisting of phentermine and topiramate ( marketed as qsymia ) was approved by the fda for longer term use and shown to produce approximately 10% loss in body weight as well as improvements in glucose levels , blood pressure , cholesterol , and other cardiovascular risk factors after 2 years of treatment.15,16 orlistat ( trade name xenical ) was approved for use by the fda in 1999 as a long - term treatment for weight loss and acts by interfering with pancreatic lipase , thereby decreasing the absorption of dietary fat . results from clinical trials with orlistat have reported only modest weight loss ( 57 pounds ) after continued use , though significant improvements were noted for several cardiovascular risk factors including decreased blood pressure and improved glucose tolerance.1719 other drugs approved by the fda for chronic weight management are lorcasersin ( trade name belviq),20 a selective serotonin-2c receptor agonist , and the combination drug naltrexone / bupropion ( trade name contrave),2123 both of which are proposed to reduce food intake by acting through the hypothalamic melanocortin system.22 the latter is currently being considered for approval by the european medicines agency ( ema ) under the trade name mysimba . analogs of the incretin hormone , glucagon - like peptide 1 ( glp-1 ) , have recently been introduced as potential weight loss medications . these analogs were initially used as drugs for the treatment of type 2 diabetes ; however , results from clinical trials have repeatedly demonstrated their ability to induce weight loss.24 one of these medications that has been approved by the ema and fda for the treatment of diabetes is liraglutide , a long - acting glp-1 receptor agonist developed by novo nordisk , and a number of trials have reported positive results on its efficacy to improve indices of glycemic and cardiovascular risk factors . on december 23 , 2014 , the fda announced approval of a higher dose version of liraglutide ( trade name saxenda ) for the treatment of chronic weight management . this drug was approved for adults with a body mass index ( bmi ) of 30 or higher or those with a bmi of 27 or higher who have at least one weight - related comorbid condition such as hypertension , type 2 diabetes , or elevated cholesterol . this review provides an overview of glp-1 and glp-1 receptor agonists in animal and human studies , with particular emphasis on liraglutide , and discusses their therapeutic potential for obesity treatment . glp-1 is derived from posttranslational processing of the preproglucagon gene and subsequently cleaved into its biologically active forms , glp-1 ( 736 ) amide which comprises approximately 80% of circulating glp-1 and glp-1 ( 737).25,26 glp-1 is secreted from endocrine l cells in the distal intestinal mucosa primarily in response to the presence of nutrients in the intestinal lumen . once in circulation , the half - life of glp-1 is less than 2 minutes due to rapid degradation by the enzyme dipeptidyl peptidase - iv ( dpp - iv).27,28 glp-1 is classified as an incretin hormone because it stimulates a decrease in blood glucose levels by increasing the amount of insulin released from pancreatic beta cells after eating , prior to the elevation of blood glucose levels.29 glp-1 is also effective in controlling blood glucose because it suppresses glucagon secretion and slows gastric emptying.30 because of glp-1 s effects on glucose homeostasis , it was an attractive target for drug development in the treatment of type 2 diabetes . glp-1 has a very short half - life in circulation , and therefore , its utility as a therapeutic agent in the treatment of diabetes was limited . the peptide , exendin-4 , is a 39-amino acid naturally occurring peptide with 53% homology to native glp-1 that was originally isolated from the saliva of the gila monster . it was found to exhibit glucose regulatory properties similar to glp-1 but resisted degradation by dpp - iv . its synthetic version , exenatide , was approved by the fda in 2005 and in europe in 2006 as an adjunct therapy for patients with type 2 diabetes who failed to respond to metformin or sulfonylureas.31 it is administered as a subcutaneous injection either twice daily ( byetta ) or in a more recently approved sustained - release weekly formulation ( bydureon ) . while it was initially marketed as a diabetes drug , clinical trials consistently found significant reductions in body weight due to its appetite - suppressing effects.32 several other glp-1 analogs are currently under investigation or are approved for the treatment of type 2 diabetes . these include lixsenatide ( sanofi aventis , trade name lyxumia ) and albiglutide ( glaxosmithkline , trade names epezan and tanseum ) which have amino acid modifications to resist degradation by dpp - iv . the recently fda - approved drug , dulaglutide ( eli lilly , trade name trulicity ) , an analog of human glp-1 , is covalently linked to an fc fragment of human igg4 enabling it to resist degradation and reduce renal clearance.3335 liraglutide , another long - acting glp-1 receptor agonist , was developed by novo nordisk and marketed under the brand name victoza . it shares 97% structural homology with human glp-1 and has a half - life of 1014 hours . the longer half - life is due to a modification of the peptide with an amino acid substitution and the addition of a fatty acid chain that allows it to bind to circulating plasma proteins , thus slowing absorption and rendering it resistant to degradation.36 it was approved for use by the ema in 2009 and by the fda in 2010 as a diabetes medication that is administered by once - daily subcutaneous injection as a monotherapy or in combination with other diabetes medications . in a study comparing exenatide and liraglutide , it was found that liraglutide was superior to exenatide in reducing hba1c , systolic blood pressure , fasting blood glucose , and triglycerides and free fatty acids ( liraglutide effect and action in diabetes-6 trial ) with fewer undesirable side effects.37 as mentioned above , glp-1 is secreted from endocrine ( l cells ) in the intestinal mucosa primarily in the ileum and distal colon . besides its presence in the gastrointestinal tract , glp-1 is found in the central nervous system localized to neurons primarily in the nucleus of the solitary tract ( nts ) in the caudal brainstem.38 neurons in the nts that express glp-1 send projections to several brain regions that participate in feeding behavior and energy homeostasis.3840 glp-1 s actions are mediated through the g - protein - coupled glp-1 receptor . in the rodent , high - affinity binding sites for glp-1 are found in the pancreas , enteric nerves , vagus nerve , heart , kidney , and adipose tissue.4143 in the brain , glp-1 receptor mrna is distributed in many brain areas associated with food intake and body weight including the arcuate and dorsomedial nuclei of the hypothalamus , parabrachial nuclei , nucleus accumbens ( nac ) , nts , and area postrema.44 numerous studies have demonstrated that systemic administration of glp-1 or glp-1 receptor agonists decreases food intake , slows gastric emptying , and reduces body weight.4549 blockade of peripheral glp-1 receptors by the selective receptor antagonist , exendin ( 939 ) , attenuated the reduction of food intake produced by voluntary consumption of a sucrose meal and by prior administration of glp-1 . the same reversal in the food intake suppression was not seen after central administration of exendin ( 939 ) supporting the interpretation that peripheral , not central , glp-1 receptors were mediating the effects on food intake.48 further studies examining the neural pathways responsible for these effects have shown that severing the connection between the gastrointestinal tract and the brain by total subdiaphragmatic vagotomy , or by chemical vagal deafferentation , prevented the suppression of food intake produced by glp-1 receptor activation.50,51 together , these studies support a role for peripheral glp-1 as a physiological satiety signal . while these studies implicate peripheral glp-1 in ingestive control , there is also substantial evidence to support a role for central glp-1 signaling in food intake and body weight regulation . early studies reported that third intracerebroventricular ( icv ) administration of glp-1 significantly reduced food intake and body weight , whereas administration of the glp-1 receptor antagonist exendin ( 939 ) increased food intake and body weight.47,52 while there has been much debate about whether glp-1 acts primarily at a peripheral or central site , there are now sufficient data to support the view that glp-1 may have multiple actions that include both peripheral and central glp-1 receptor populations.49,53 while most studies have used exendin-4 as an experimental tool to activate glp-1 receptors , the availability of newly developed long - acting agonists such as liraglutide has prompted investigations to determine if they share similar biological activity . a study comparing the efficacy of peripheral administration of liraglutide and exendin-4 to decrease food intake and body weight in nonobese rats reported that once - daily administration of both agonists produced a dose - dependent reduction in chow intake ; however , only liraglutide reduced body weight.54 the effects of liraglutide on food intake and body weight were greater in magnitude and latency when given by intraperitoneal compared to subcutaneous injection . the same study reported that in diet - induced obese rats fed a palatable high - fat / high - sugar diet , chronic 7-day peripheral delivery of exendin-4 and liraglutide elicited equivalent reductions in food intake and weight loss over the test period . a similar comparative study was also conducted to examine the anorectic effects of centrally administered liraglutide and exendin-4 in rats.55 the data demonstrated that the icv dosage of liraglutide needed to suppress food intake and induce the same degree of hypothalamic c - fos activation ( a marker of neuronal activity ) was tenfold greater than equivalent feeding suppression and c - fos activation following exendin-4 . when animals were pretreated centrally with the glp-1 receptor antagonist , exendin ( 936 ) , the suppression of food intake by both agonists was blocked substantiating that the effects of both agonists were mediated through glp-1 receptor activation . additional studies using both pharmacological and surgical methods were conducted to determine whether the site of action for peripheral administration of exendin-4 and liraglutide was localized to peripheral or central glp-1 receptors.56 the results from this study demonstrated that intraperitoneal administration of exendin ( 939 ) failed to block the effects of peripherally administered exendin-4 and liraglutide . by contrast , central administration of exendin ( 939 ) attenuated , but did not completely abolish , the suppression of food intake by both exendin-4 and liraglutide and reversed the body weight change seen with liraglutide . although animals with selective vagal deafferentation still suppressed intake after glp-1 receptor agonist administration , higher doses were required . together , these studies suggest that peripheral administration of exendin-4 and liraglutide produce their feeding and weight loss effects at both central and peripheral glp-1 receptors . the findings are in contrast to those reported for glp-148 and suggest that the mechanisms through which long - acting glp-1 receptor agonists produce their effects on food intake and body weight are not identical to those of native glp-1 . this difference is likely due to the longer half - life of glp-1 analogs resulting in sustained peripheral physiological effects . furthermore , there is evidence that these compounds are capable of diffusing across the blood brain barrier and thereby stimulating central glp-1 receptors.57 the mechanism(s ) by which long - acting glp-1 receptor agonists reduce food intake and body weight is actively under investigation . one potential mechanism for reducing body weight is through glp-1 s effects on energy expenditure . central injection of liraglutide in rodents stimulates brown - fat thermogenesis and induces browning of white adipose tissue independent of its ability to suppress food intake.58 site - specific injections of liraglutide into various hypothalamic nuclei identified the ventromedial hypothalamus ( vmh ) as mediating this effect . since ampk within the vmh has been implicated in brown adipose tissue thermogenesis , the importance of ampk in the mediation of liraglutide - induced brown adipose tissue thermogenesis was also evaluated . this study found that icv administration of aicar , a pharmacological activator of ampk , had no effect on the ability of icv liraglutide to reduce food intake but prevented the reduction in body weight produced by liraglutide . furthermore , liraglutide injected into the vmh produced weight loss that was completely blocked by overexpression of constitutively active ampk . activation of ampk in the vmh decreased liraglutide - induced ucp1 expression in adipose tissue supporting the view that ampk in the vmh participates in the actions of central glp-1 on brown and white adipose tissue . in a series of studies designed to evaluate the site of action of liraglutide - induced weight loss , it was demonstrated that the arcuate nucleus in the hypothalamus plays a critical role.57 rats that underwent subdiaphragmatic vagal deafferentation reduced body weight to the same extent as sham - operated controls after a 14-day regimen of liraglutide ( twice - daily subcutaneous injections , 200 g / kg ) indicating that the weight - reducing effects of liraglutide are not dependent upon an intact afferent vagus nerve . similarly , liraglutide treatment produced weight loss to the same degree in animals with lesions of the hindbrain area postrema compared to those with sham lesions . however , long - term liraglutide treatment significantly increased expression of cocaine and amphetamine - related transcript but did not increase neuropeptide y ( npy)/agouti - related protein ( agrp ) gene expression in the arcuate nucleus suggesting that liraglutide prevents the normal activation of the npy / agrp system to increase food intake during periods of weight loss . preventing the normal stimulation of food intake by npy / agrp that occurs during energy deficit would benefit sustained decreases in body weight . in a follow - up study , fluorescently labeled liraglutide was used to determine if peripherally administered liraglutide could directly access brain regions that might be involved in its effects on food intake and body weight . labeled liraglutide was detected in all circumventricular organs and several hypothalamic nuclei including the arcuate nucleus . surprisingly , the nts was devoid of a fluorescent signal , a brain region strongly implicated in glp-1 s effects on food intake . this would suggest that if the nts is involved in the effects of liraglutide on body weight , it may be through an indirect , rather than a direct , activation of glp-1 receptors . despite this , numerous animal studies have implicated the nts as mediating the effects of glp-1 receptor stimulation , and studies have demonstrated that peripheral treatment with liraglutide can produce a conditioned taste aversion and transient pica , two rodent models of nausea which are proposed to be mediated by nts glp-1 receptors.5962 other mechanisms by which liraglutide suppresses food intake and body weight have also been evaluated . as mentioned , glp-1 receptor agonists have potent effects on gastric emptying which could contribute to their feeding inhibitory effects . doses of exenatide and liraglutide that produce equivalent reductions in food intake significantly delay gastric emptying . it was shown that the effects on gastric emptying were markedly decreased following long - term ( 14-day ) treatment with liraglutide but remained the same with exenatide treatment . because weight loss was similar between the two treatment groups , this suggests that suppression of gastric emptying did not contribute significantly to the weight loss produced by liraglutide.63 recent neuroanatomical and behavioral evidence also support a role for glp-1 in reward and motivation.64,65 glp-1 neurons in the nts project to reward - associated brain areas such as the ventral tegmental area ( vta ) and nac.39,40,6668 glp-1 analogs injected directly into the nac have been demonstrated to decrease intake of high - fat and high - sucrose diets , presumably by reducing their palatability.40,68 a link between peripheral nutrient - related signals and central glp-1 s effects on reward processing was supported by data showing that the suppression of meal intake following an intestinal infusion of 40% sucrose could be attenuated by blockade of nac glp-1 receptors.68 a role for the glp-1 nts neuronal projection to the vta in food intake has also been suggested in an animal study showing that intra - vta injection of exendin-4 reduced intake of both chow and high - fat diet , while blockade of vta glp-1 receptors increased high - fat food intake , proposed to be mediated , in part , through vta dopamine signaling.39 several lines of evidence support the view that liraglutide , like exendin-4 , may act via mechanisms that alter the rewarding properties of food . studies to evaluate dietary effects on the ability of liraglutide to inhibit food intake have shown that rats maintained on a high - fat diet exhibited a delayed response to the anorectic effects of peripherally administered liraglutide . however , once intake was suppressed , it was sustained for a longer period of time compared to animals on a low - fat diet.69 when diet - obese animals were treated with liraglutide and allowed a choice between a highly palatable diet and chow , their preference shifted toward decreasing intake of the palatable diet and increasing intake of chow.70,71 this feature was not shared with either the dpp - iv inhibitor , vildagliptin , which increases postprandial glp-1 , or sibutramine , a 5ht agonist / reuptake inhibitor , which causes similar reductions in body weight.70,71 because evidence suggests that glp-1 affects food intake through both energy homeostatic and reward mechanisms , glp-1 receptor agonist medications , such as liraglutide , offer a multi - pronged approach for promoting weight loss . a recent study in rats examined the effects of combining low doses of leptin and liraglutide and demonstrated greater weight loss with the combination treatment than with either drug given alone.72 as well , diet - induced obese mice that were chronically treated with liraglutide and a melanocortin-4 receptor agonist exhibited greater weight loss and improved glycemic control and cholesterol metabolism than that achieved with each treatment alone.73 these findings support further exploration into the feasibility of combinatorial therapies that may produce greater weight loss with less possibility of adverse events . animal studies with glp-1 receptor agonists will provide critical insights into mechanisms of action that could be utilized to enhance or tailor treatment regimens to more effectively promote weight loss in humans . liraglutide was first approved for the treatment of diabetes after studies were conducted in a large population of subjects and found that it improved glycemic control and produced significant body weight loss with limited side effects . subsequently , additional long - term ( 2-year ) studies were conducted to examine safety and tolerability as a primary outcome and address issues of sustainability of weight loss as a secondary outcome.74,75 these measures were compared with those from individuals who received the fat - blocking drug , orlistat . the results indicated that weight loss was significantly greater in the group that received liraglutide compared with the group that received orlistat . body composition data revealed that the primary reduction came from a loss of body fat of approximately 15% . systolic and diastolic blood pressure was significantly decreased in both groups but significantly lower in the liraglutide group after the 2-year treatment period . the primary outcome measures of safety and tolerability found several self - reported cases of symptomatic hypoglycemia ; however , the majority of drug - related side effects attributed to liraglutide were transient nausea and vomiting . the general conclusion was that the drug was well tolerated over extended treatment periods and was effective in sustaining weight loss and improving cardiovascular risk factors . similar results were reported in a 2-year study examining liraglutide s effects on body weight and glycemic control in japanese type 2 diabetic subjects76 and in a study showing that liraglutide ( 3.0 mg / day ) in combination with a low - calorie diet and physical activity resulted in significantly greater weight loss in overweight and obese adults with type 2 diabetes than in those receiving placebo.77 a large multisite randomized trial ( duration-6 ) was conducted to compare the effects of sustained - release exenatide once weekly to liraglutide ( 1.8 mg / day ) once daily in patients with type 2 diabetes.78 the major outcome measure was a change in hba1c , an indicator of blood glucose concentration ; however , other secondary outcomes including changes in body weight , fasting blood glucose , and blood pressure were also examined . it was demonstrated that while both glp-1 analogs improved glycemic control and produced weight loss , the improvements in hba1c and body weight reduction were greater in the liraglutide group . both groups had similar improvements in blood pressure and other cardiovascular biomarkers . while generally well tolerated , the most frequent of these is nausea which is more problematic in the early stages of treatment and with shorter acting agonists such as exenatide due to higher peak concentrations than those produced with slow - release glp-1 agonists.37,78,79 other gastrointestinal complaints such as vomiting and diarrhea have also been reported , though with less frequency than nausea.37,78,80 while these side effects do not represent serious health issues , they may contribute to a patient s decision to discontinue therapy . other side effects include a small ( 24 beats / min ) but sustained increase in heart rate . despite this , preclinical studies have reported that glp-1 agonists have a beneficial effect on cardiovascular outcomes.81 additionally , since glp-1 receptor agonists induce pancreatic beta - cell proliferation , there is the possibility that long - term use could result in pancreatitis . however , in clinical trials evaluating approximately 4,500 patients , seven cases of pancreatitis were reported which was not beyond expected rates for this population , and reviews of clinical data by the ema concluded that glp-1 agonist treatments did not pose an increased risk for the development of pancreatitis.82 some concerns were also raised about the potential risk of thyroid cancer based on animal studies demonstrating proliferation of thyroid c - cells and tumor formation in rodents following long - term treatment with glp-1 agonist compounds.83 this finding was not consistent with data obtained from nonhuman primates who , like humans , exhibit a much lower density of glp-1 receptors on thyroid c cells.84 nevertheless , the effects of prolonged use of higher doses of liraglutide such as those used for weight loss treatment in humans have not been determined . although many studies have reported significant effects of liraglutide on body weight loss , there are limited data available that specifically evaluate the possible mechanisms underlying liraglutide s effects on body weight in humans . to address this , a recent study of obese individuals , without diabetes , examined the effects of once - daily administration of liraglutide on several parameters that may contribute to weight loss , including gastric emptying , glycemic control , appetite , and energy metabolism.85 subjects received 1.8 mg ( the dose currently approved for diabetes treatment ) , 3.0 mg ( the dose currently approved for weight loss ) , or a placebo . after 5 weeks of treatment , the effects on gastric emptying of a test meal were found to be equivalent for the two doses of liraglutide and for liraglutide versus placebo for the duration of the 5-hour testing period ; however , early ( 1 h ) gastric emptying was significantly delayed with the 3.0 mg liraglutide dose . in addition to evaluating the effects of liraglutide on gastric emptying , subjects were asked to consume a lunch meal ad libitum to determine energy intake and to assess feelings of satiety , fullness , and hunger . the study found that both doses of liraglutide equivalently reduced energy intake which was accompanied by increased postprandial satiety and fullness ratings and a decrease in hunger ratings . all groups had reduced 24-hour energy expenditure which was greater for liraglutide versus placebo , a difference that was attributed to a decrease in body weight . a shift toward increased fat oxidation and decreased carbohydrate oxidation was noted in the group receiving liraglutide . measures of glycemic control were also found to be significantly improved in both liraglutide treatment groups . the conclusion from these data was that the mechanism responsible for weight loss induced by liraglutide was due to a long - term reduction in appetite and food intake , as opposed to effects on energy expenditure . as mentioned above , experiments in rodents have shown that repeated doses of liraglutide result in a diminished ability to delay gastric emptying;63 however , this does not appear to be the case in humans , since there were still significant effects on emptying at the earlier time points with the highest dose . thus , delays in gastric emptying may partially contribute to the reduction in meal intake produced by liraglutide in humans . while the van can et al study85 examined liraglutide s effects in obese subjects without diabetes , similar parameters were also evaluated in obese subjects with diabetes . the results in diabetic subjects were in general agreement with liraglutide s effects on weight loss , satiety , gastric emptying , and energy expenditure in nondiabetic subjects.86 the failure of this , and other studies , to observe significant changes in energy expenditure after glp-1 analog administration is in contrast to recent data demonstrating an increase in resting energy expenditure in obese type 2 diabetic patients.58 the discrepancy in these results may be due , in part , to the increased length of treatment in this study in which patients were treated for 1 year with glp-1 receptor agonists . as in rodent studies , it has been postulated that glp-1 receptor agonists may reduce food intake in humans by modulating the rewarding properties of food . in a study using functional magnetic resonance imaging , obese type 2 diabetic patients exhibited increased brain responses to pictures of food in reward and appetite - related brain regions compared to lean control subjects . the enhanced response to food - related images in obese subjects was blocked by administration of a glp-1 receptor antagonist , supporting the view that glp-1 agonists such as liraglutide may promote weight loss by reducing the hyperresponsiveness to food cues to a pattern that is consistent with that of lean individuals.65 an interaction between the adipose - derived peptide , leptin , and glp-1 has been demonstrated in experiments in rodents showing that subthreshold doses of leptin increased the ability of glp-1 inhibit food intake.87 furthermore , glp-1 receptor agonists can reinstate leptin sensitivity in obese animals.88,89 as in animals , obese humans have high levels of circulating leptin but are leptin resistant and therefore do not respond appropriately to this signal to reduce energy intake . during weight loss , a drop in plasma leptin levels that results from decreased fat mass produces a rebound response that favors increased food intake . based on animal studies , it was postulated that administration of a glp-1 receptor agonist during diet - induced weight loss would inhibit decreases in soluble leptin receptor plasma concentrations ( one marker for leptin activity ) caused by decreased adiposity and thus prevent a regain in body weight.90 this study demonstrated that liraglutide ( 1.2 mg / day ) inhibited the increase in soluble leptin receptor that is normally seen in individuals undergoing weight loss . consequently , free leptin levels were increased , thereby providing a signal that would prevent an increase in food intake and a decrease in energy expenditure . supporting the hypothesis , the results indicated that the group receiving the liraglutide , in conjunction with reduced caloric intake , had greater weight loss during the maintenance period than the group that did not receive liraglutide . this study has important implications for the potential use of liraglutide as an adjunct to more traditional weight loss regimens and for the long - term sustainability of weight loss treatments . additionally , it suggests another avenue through which glp-1 receptor agonists contribute to weight loss . in summary , the glp-1 receptor agonist , liraglutide , has been demonstrated to reduce food intake , promote weight loss , and improve indices of metabolic function in both animal and human studies . the primary mechanisms associated with these effects are proposed to be due to actions of glp-1 on peripheral ( vagal ) and central pathways that affect food intake and metabolism via hindbrain and hypothalamic activation , as well as those brain areas associated with motivation and reward processes . with the high prevalence of obesity and its negative impact on quality of life , there is a critical need for therapies that will produce a sustainable loss of body weight . currently , the most effective treatment option is bariatric surgery . a recent comparison between the clinical efficacy of bariatric surgery and liraglutide found surgery to be superior to liraglutide for both body weight reduction and improvements in metabolic parameters.91 despite this , many individuals are not ideal candidates for surgery and would benefit from noninvasive medical treatments . liraglutide has been demonstrated to produce significant weight loss in humans , with and without type 2 diabetes , while producing minimal side effects and thus is an attractive treatment option . additional research is warranted for its potential in combinatorial weight loss treatments that may further enhance and sustain weight loss .

the prevalence of obesity worldwide has nearly doubled since 1980 with current estimates of 2.1 billion in 2013 . overweight and obesity lead to numerous adverse conditions including type 2 diabetes , cardiovascular disease , stroke , and certain cancers . the worldwide spread of obesity and associated comorbidities not only threatens quality of life but also presents a significant economic burden . while bariatric surgery has proven to be a viable treatment option for the morbidly obese , there is clearly a need for less invasive alternatives . recent research has suggested that long - acting analogs of the gut hormone , glucagon - like peptide 1 ( glp-1 ) , may have potential as an antiobesity treatment . the glp-1 receptor agonist , liraglutide ( trade name saxenda ) , was recently approved by the us food and drug administration as an obesity treatment option and shown in clinical trials to be effective in reducing and sustaining body weight loss . this review presents the basis for glp-1-based therapies with a specific focus on animal and human studies examining liraglutide s effects on food intake and body weight .

Introduction GLP-1 and GLP-1 analogs Effects of liraglutide on food intake and body weight: animal studies Effects of liraglutide on food intake and body weight: human studies Conclusion

in the us , over one third of adults and one sixth of children and adolescents are currently considered obese.14 overweight and obesity are frequently coupled to other debilitating diseases such as type 2 diabetes , coronary heart disease , ischemic stroke , and several cancers.57 particularly concerning is the increasing prevalence of pediatric obesity which has been linked not only to metabolic disorders and cardiovascular risk factors but also to psychiatric illness , early pubertal onset , and orthopedic disorders.811 because of the comorbidities associated with increased body weight , obesity is considered to be a major public health concern with a significant economic burden to our health care system . it has been projected that by 2030 , costs associated with overweight and obesity will comprise 16%18% of total health care expenses in the us.3,12 surgical interventions , such as roux - en - y gastric bypass and sleeve gastrectomy , have been highly successful in promoting weight loss and correcting accompanying comorbidities like diabetes.13,14 however , due to the risks associated with surgery , particularly in a vulnerable population , surgical interventions are often considered to be a last resort and reserved for those patients who are morbidly obese or have failed at other weight loss strategies . clearly , there is a need for less invasive means to achieve sustainable weight loss in patients who are not prime candidates for bariatric surgery or desire nonsurgical alternatives . appetite suppressants , such as phentermine , were initially approved by the us food and drug administration ( fda ) in the 1950s for short - term use for weight loss ; however , limited data are available to support their long - term efficacy . more recently , a combination drug consisting of phentermine and topiramate ( marketed as qsymia ) was approved by the fda for longer term use and shown to produce approximately 10% loss in body weight as well as improvements in glucose levels , blood pressure , cholesterol , and other cardiovascular risk factors after 2 years of treatment.15,16 orlistat ( trade name xenical ) was approved for use by the fda in 1999 as a long - term treatment for weight loss and acts by interfering with pancreatic lipase , thereby decreasing the absorption of dietary fat . results from clinical trials with orlistat have reported only modest weight loss ( 57 pounds ) after continued use , though significant improvements were noted for several cardiovascular risk factors including decreased blood pressure and improved glucose tolerance.1719 other drugs approved by the fda for chronic weight management are lorcasersin ( trade name belviq),20 a selective serotonin-2c receptor agonist , and the combination drug naltrexone / bupropion ( trade name contrave),2123 both of which are proposed to reduce food intake by acting through the hypothalamic melanocortin system.22 the latter is currently being considered for approval by the european medicines agency ( ema ) under the trade name mysimba . analogs of the incretin hormone , glucagon - like peptide 1 ( glp-1 ) , have recently been introduced as potential weight loss medications . these analogs were initially used as drugs for the treatment of type 2 diabetes ; however , results from clinical trials have repeatedly demonstrated their ability to induce weight loss.24 one of these medications that has been approved by the ema and fda for the treatment of diabetes is liraglutide , a long - acting glp-1 receptor agonist developed by novo nordisk , and a number of trials have reported positive results on its efficacy to improve indices of glycemic and cardiovascular risk factors . on december 23 , 2014 , the fda announced approval of a higher dose version of liraglutide ( trade name saxenda ) for the treatment of chronic weight management . this drug was approved for adults with a body mass index ( bmi ) of 30 or higher or those with a bmi of 27 or higher who have at least one weight - related comorbid condition such as hypertension , type 2 diabetes , or elevated cholesterol . this review provides an overview of glp-1 and glp-1 receptor agonists in animal and human studies , with particular emphasis on liraglutide , and discusses their therapeutic potential for obesity treatment . once in circulation , the half - life of glp-1 is less than 2 minutes due to rapid degradation by the enzyme dipeptidyl peptidase - iv ( dpp - iv).27,28 glp-1 is classified as an incretin hormone because it stimulates a decrease in blood glucose levels by increasing the amount of insulin released from pancreatic beta cells after eating , prior to the elevation of blood glucose levels.29 glp-1 is also effective in controlling blood glucose because it suppresses glucagon secretion and slows gastric emptying.30 because of glp-1 s effects on glucose homeostasis , it was an attractive target for drug development in the treatment of type 2 diabetes . its synthetic version , exenatide , was approved by the fda in 2005 and in europe in 2006 as an adjunct therapy for patients with type 2 diabetes who failed to respond to metformin or sulfonylureas.31 it is administered as a subcutaneous injection either twice daily ( byetta ) or in a more recently approved sustained - release weekly formulation ( bydureon ) . while it was initially marketed as a diabetes drug , clinical trials consistently found significant reductions in body weight due to its appetite - suppressing effects.32 several other glp-1 analogs are currently under investigation or are approved for the treatment of type 2 diabetes . the recently fda - approved drug , dulaglutide ( eli lilly , trade name trulicity ) , an analog of human glp-1 , is covalently linked to an fc fragment of human igg4 enabling it to resist degradation and reduce renal clearance.3335 liraglutide , another long - acting glp-1 receptor agonist , was developed by novo nordisk and marketed under the brand name victoza . in the rodent , high - affinity binding sites for glp-1 are found in the pancreas , enteric nerves , vagus nerve , heart , kidney , and adipose tissue.4143 in the brain , glp-1 receptor mrna is distributed in many brain areas associated with food intake and body weight including the arcuate and dorsomedial nuclei of the hypothalamus , parabrachial nuclei , nucleus accumbens ( nac ) , nts , and area postrema.44 numerous studies have demonstrated that systemic administration of glp-1 or glp-1 receptor agonists decreases food intake , slows gastric emptying , and reduces body weight.4549 blockade of peripheral glp-1 receptors by the selective receptor antagonist , exendin ( 939 ) , attenuated the reduction of food intake produced by voluntary consumption of a sucrose meal and by prior administration of glp-1 . the same reversal in the food intake suppression was not seen after central administration of exendin ( 939 ) supporting the interpretation that peripheral , not central , glp-1 receptors were mediating the effects on food intake.48 further studies examining the neural pathways responsible for these effects have shown that severing the connection between the gastrointestinal tract and the brain by total subdiaphragmatic vagotomy , or by chemical vagal deafferentation , prevented the suppression of food intake produced by glp-1 receptor activation.50,51 together , these studies support a role for peripheral glp-1 as a physiological satiety signal . while these studies implicate peripheral glp-1 in ingestive control , there is also substantial evidence to support a role for central glp-1 signaling in food intake and body weight regulation . early studies reported that third intracerebroventricular ( icv ) administration of glp-1 significantly reduced food intake and body weight , whereas administration of the glp-1 receptor antagonist exendin ( 939 ) increased food intake and body weight.47,52 while there has been much debate about whether glp-1 acts primarily at a peripheral or central site , there are now sufficient data to support the view that glp-1 may have multiple actions that include both peripheral and central glp-1 receptor populations.49,53 while most studies have used exendin-4 as an experimental tool to activate glp-1 receptors , the availability of newly developed long - acting agonists such as liraglutide has prompted investigations to determine if they share similar biological activity . a study comparing the efficacy of peripheral administration of liraglutide and exendin-4 to decrease food intake and body weight in nonobese rats reported that once - daily administration of both agonists produced a dose - dependent reduction in chow intake ; however , only liraglutide reduced body weight.54 the effects of liraglutide on food intake and body weight were greater in magnitude and latency when given by intraperitoneal compared to subcutaneous injection . the same study reported that in diet - induced obese rats fed a palatable high - fat / high - sugar diet , chronic 7-day peripheral delivery of exendin-4 and liraglutide elicited equivalent reductions in food intake and weight loss over the test period . when animals were pretreated centrally with the glp-1 receptor antagonist , exendin ( 936 ) , the suppression of food intake by both agonists was blocked substantiating that the effects of both agonists were mediated through glp-1 receptor activation . the findings are in contrast to those reported for glp-148 and suggest that the mechanisms through which long - acting glp-1 receptor agonists produce their effects on food intake and body weight are not identical to those of native glp-1 . furthermore , there is evidence that these compounds are capable of diffusing across the blood brain barrier and thereby stimulating central glp-1 receptors.57 the mechanism(s ) by which long - acting glp-1 receptor agonists reduce food intake and body weight is actively under investigation . however , long - term liraglutide treatment significantly increased expression of cocaine and amphetamine - related transcript but did not increase neuropeptide y ( npy)/agouti - related protein ( agrp ) gene expression in the arcuate nucleus suggesting that liraglutide prevents the normal activation of the npy / agrp system to increase food intake during periods of weight loss . in a follow - up study , fluorescently labeled liraglutide was used to determine if peripherally administered liraglutide could directly access brain regions that might be involved in its effects on food intake and body weight . surprisingly , the nts was devoid of a fluorescent signal , a brain region strongly implicated in glp-1 s effects on food intake . despite this , numerous animal studies have implicated the nts as mediating the effects of glp-1 receptor stimulation , and studies have demonstrated that peripheral treatment with liraglutide can produce a conditioned taste aversion and transient pica , two rodent models of nausea which are proposed to be mediated by nts glp-1 receptors.5962 other mechanisms by which liraglutide suppresses food intake and body weight have also been evaluated . because weight loss was similar between the two treatment groups , this suggests that suppression of gastric emptying did not contribute significantly to the weight loss produced by liraglutide.63 recent neuroanatomical and behavioral evidence also support a role for glp-1 in reward and motivation.64,65 glp-1 neurons in the nts project to reward - associated brain areas such as the ventral tegmental area ( vta ) and nac.39,40,6668 glp-1 analogs injected directly into the nac have been demonstrated to decrease intake of high - fat and high - sucrose diets , presumably by reducing their palatability.40,68 a link between peripheral nutrient - related signals and central glp-1 s effects on reward processing was supported by data showing that the suppression of meal intake following an intestinal infusion of 40% sucrose could be attenuated by blockade of nac glp-1 receptors.68 a role for the glp-1 nts neuronal projection to the vta in food intake has also been suggested in an animal study showing that intra - vta injection of exendin-4 reduced intake of both chow and high - fat diet , while blockade of vta glp-1 receptors increased high - fat food intake , proposed to be mediated , in part , through vta dopamine signaling.39 several lines of evidence support the view that liraglutide , like exendin-4 , may act via mechanisms that alter the rewarding properties of food . however , once intake was suppressed , it was sustained for a longer period of time compared to animals on a low - fat diet.69 when diet - obese animals were treated with liraglutide and allowed a choice between a highly palatable diet and chow , their preference shifted toward decreasing intake of the palatable diet and increasing intake of chow.70,71 this feature was not shared with either the dpp - iv inhibitor , vildagliptin , which increases postprandial glp-1 , or sibutramine , a 5ht agonist / reuptake inhibitor , which causes similar reductions in body weight.70,71 because evidence suggests that glp-1 affects food intake through both energy homeostatic and reward mechanisms , glp-1 receptor agonist medications , such as liraglutide , offer a multi - pronged approach for promoting weight loss . similar results were reported in a 2-year study examining liraglutide s effects on body weight and glycemic control in japanese type 2 diabetic subjects76 and in a study showing that liraglutide ( 3.0 mg / day ) in combination with a low - calorie diet and physical activity resulted in significantly greater weight loss in overweight and obese adults with type 2 diabetes than in those receiving placebo.77 a large multisite randomized trial ( duration-6 ) was conducted to compare the effects of sustained - release exenatide once weekly to liraglutide ( 1.8 mg / day ) once daily in patients with type 2 diabetes.78 the major outcome measure was a change in hba1c , an indicator of blood glucose concentration ; however , other secondary outcomes including changes in body weight , fasting blood glucose , and blood pressure were also examined . it was demonstrated that while both glp-1 analogs improved glycemic control and produced weight loss , the improvements in hba1c and body weight reduction were greater in the liraglutide group . despite this , preclinical studies have reported that glp-1 agonists have a beneficial effect on cardiovascular outcomes.81 additionally , since glp-1 receptor agonists induce pancreatic beta - cell proliferation , there is the possibility that long - term use could result in pancreatitis . however , in clinical trials evaluating approximately 4,500 patients , seven cases of pancreatitis were reported which was not beyond expected rates for this population , and reviews of clinical data by the ema concluded that glp-1 agonist treatments did not pose an increased risk for the development of pancreatitis.82 some concerns were also raised about the potential risk of thyroid cancer based on animal studies demonstrating proliferation of thyroid c - cells and tumor formation in rodents following long - term treatment with glp-1 agonist compounds.83 this finding was not consistent with data obtained from nonhuman primates who , like humans , exhibit a much lower density of glp-1 receptors on thyroid c cells.84 nevertheless , the effects of prolonged use of higher doses of liraglutide such as those used for weight loss treatment in humans have not been determined . although many studies have reported significant effects of liraglutide on body weight loss , there are limited data available that specifically evaluate the possible mechanisms underlying liraglutide s effects on body weight in humans . to address this , a recent study of obese individuals , without diabetes , examined the effects of once - daily administration of liraglutide on several parameters that may contribute to weight loss , including gastric emptying , glycemic control , appetite , and energy metabolism.85 subjects received 1.8 mg ( the dose currently approved for diabetes treatment ) , 3.0 mg ( the dose currently approved for weight loss ) , or a placebo . after 5 weeks of treatment , the effects on gastric emptying of a test meal were found to be equivalent for the two doses of liraglutide and for liraglutide versus placebo for the duration of the 5-hour testing period ; however , early ( 1 h ) gastric emptying was significantly delayed with the 3.0 mg liraglutide dose . the conclusion from these data was that the mechanism responsible for weight loss induced by liraglutide was due to a long - term reduction in appetite and food intake , as opposed to effects on energy expenditure . the results in diabetic subjects were in general agreement with liraglutide s effects on weight loss , satiety , gastric emptying , and energy expenditure in nondiabetic subjects.86 the failure of this , and other studies , to observe significant changes in energy expenditure after glp-1 analog administration is in contrast to recent data demonstrating an increase in resting energy expenditure in obese type 2 diabetic patients.58 the discrepancy in these results may be due , in part , to the increased length of treatment in this study in which patients were treated for 1 year with glp-1 receptor agonists . the enhanced response to food - related images in obese subjects was blocked by administration of a glp-1 receptor antagonist , supporting the view that glp-1 agonists such as liraglutide may promote weight loss by reducing the hyperresponsiveness to food cues to a pattern that is consistent with that of lean individuals.65 an interaction between the adipose - derived peptide , leptin , and glp-1 has been demonstrated in experiments in rodents showing that subthreshold doses of leptin increased the ability of glp-1 inhibit food intake.87 furthermore , glp-1 receptor agonists can reinstate leptin sensitivity in obese animals.88,89 as in animals , obese humans have high levels of circulating leptin but are leptin resistant and therefore do not respond appropriately to this signal to reduce energy intake . based on animal studies , it was postulated that administration of a glp-1 receptor agonist during diet - induced weight loss would inhibit decreases in soluble leptin receptor plasma concentrations ( one marker for leptin activity ) caused by decreased adiposity and thus prevent a regain in body weight.90 this study demonstrated that liraglutide ( 1.2 mg / day ) inhibited the increase in soluble leptin receptor that is normally seen in individuals undergoing weight loss . this study has important implications for the potential use of liraglutide as an adjunct to more traditional weight loss regimens and for the long - term sustainability of weight loss treatments . in summary , the glp-1 receptor agonist , liraglutide , has been demonstrated to reduce food intake , promote weight loss , and improve indices of metabolic function in both animal and human studies . with the high prevalence of obesity and its negative impact on quality of life , there is a critical need for therapies that will produce a sustainable loss of body weight . liraglutide has been demonstrated to produce significant weight loss in humans , with and without type 2 diabetes , while producing minimal side effects and thus is an attractive treatment option .

breast cancer is the most common female cancer , except for skin cancer , in the united states for every major ethnic group and the second most common cause of cancer death in women . even with the development of full - field digital mammography , the main limitation of mammography is its decreased sensitivity in women with dense breast tissue . dense breast tissue is present in 40 - 60% of all women . in reaction to this limitation published literature has demonstrated that dbt in combination with a conventional digital mammogram has demonstrated improved mass detection , decreased recall rates , and increased cancer detection . however , dbt has not eliminated mammographic breast compression . dedicated breast computed tomography ( dbct ) is a new imaging modality that provides three - dimensional ( 3d ) data that can be reconstructed into multiple imaging planes . the radiation dose level of dbct is similar to the dose of a conventional two - view mammogram . there is limited published data on dbct breast lesion conspicuity and no published data on radiologist reader lesion confidence in clinical trials . therefore , the objective of our study is to assess radiologist confidence in the characterization of suspicious breast lesions in a population of women going to biopsy with a dbct system in comparison to diagnostic two - dimensional ( 2d ) full - field digital mammography ( dxdm ) . the procedures followed in this study were in accordance with the ethical standards of our institutional review board ( irb ) and with the helsinki declaration of 1975 , as revised in 2000 . in our irb approved and health insurance portability and accountability act ( hippa)-compliant study , all study subjects provided informed written consent after the nature of the study had been explained . in this prospective study , we recruited twenty consecutive women from our breast clinic who were scheduled to undergo a breast biopsy for a mammographically detected lesion that was evaluated with diagnostic 2d full - field digital mammography ( dxdm ) from january 1 , 2011 to october 1 , 2011 . the inclusion criteria consisted of 40-year - old or older women , who were able to provide informed consent , had at least one detected mammographic abnormality that was confirmed as a breast imaging - reporting and data system ( bi - rads ) assessment category 4 or 5 lesion after completion of diagnostic workup , and who were scheduled to undergo a core needle biopsy at our institution . women were not eligible for this study if they were under 40 years of age , weighed > 350 pounds , were pregnant or lactating , had no mammographically evident lesion , or had a preexisting back , neck , shoulder , or general mobility difficulties . after the dbct imaging was complete , each subject was given a questionnaire to complete in order to evaluate her comfort level during dbct imaging to that of other breast imaging scans on a continuous scale of 1 ( much more comfortable ) to 5 ( much less comfortable ) . the dbct scans were performed using a prototype cone - beam dbct system designed and developed by zumatek , inc . the system uses an ergonomically designed bed where the subject scanned lies prone , which is shown in figure 1 . the head of the scan bed consists of a custom formed patient support that allows for full breast and chest wall access while maximizing subject comfort and consequently , minimizing subject motion . the design of the scan bed allows one breast to be imaged at a time . the diameter of the patient table opening allows for a cylindrical imaging volume of 20 cm in diameter 20 cm in height or 6283 cm . the breast to be imaged is noncompressed and hangs pendant through the opening of the table in the imaging field below the scan bed , as denoted by the arrow in figure 1 . in addition , there are radiation shielded panels all around the bed that can be opened below the head of the scanning bed that can allow the imaging technologist direct visualization if further positioning of the subject as needed . besides direct visualization , there is also a laser site that can aid with positioning . ( b ) the noncompressed breast to be imaged hangs pendant through the opening of the table ( arrow ) below the scan bed . this cone - beam ct system features a single 30 cm 30 cm , amorphous silicon flat - panel , digital detector with a thallium - doped cesium iodide scintillator ( paxscan 3030 , varian medical systems , palo alto , california , usa ) . the detector has 197-micron pixels , which can be binned in 2 2 or read out in 1 1 mode . the system uses a rotating tungsten rotating anode ( rad-70b , varian medical systems , palo alto , california , usa ) with a tungsten filter . the dbct system is powered by a high - frequency x - ray generator ( sedecal , madrid , spain ) , which allows for fast pulsed x - ray generation up to 30 frames per second . the x - ray tube and detector are fixed at a specific source - to - image distance of 70 cm ; therefore , there is no contact with the breast being imaged . the scanner operates in step and shoot mode whereby it is preprogrammed to move and stop at 300 specific positions around the noncompressed , pendant breast as individual images are taken . the scan time for one breast is 1 min 40 s. the average radiation dose is < 4.5 mgy for a bilateral dbct scan . motors in the system provide motion to allow the imaging components to be moved about a full 360 vertical rotation axis , and also 60 horizontal pivoting about the rotation axis is achieved around the noncompressed breast being imaged . this unique 3d orbiting capability allows for full breast and chest wall access with more complete cone beam sampling throughout the image volume . a third motion is possible to allow the system to be moved vertically up and down to position the system optimally below the bed . at the end of the scan , the 300 images obtained for each breast are sent to a computer processing workstation and are automatically processed with an iterative reconstruction algorithm that reconstructs ( 768 768 14 bits / projection ) the images into a 3d dbct dataset . the measured isotropic reconstructed pixels are 197 microns , and the measured reconstructed resolution of the line pairs with a 20 cm diameter catphan phantom ( phantom laboratory , salem , ny , usa ) is 2.5 line pairs per millimeter . the resulting digital imaging and communication in medicine ( dicom ) compliant images were viewed using clearcanvas software ( clearcanvas , toronto , ontario , canada ) . the dbct data was displayed in three simultaneous imaging planes ( coronal , sagittal , and axial planes ) , figure 2 . in addition , the slice thickness and other viewing features , such as window and leveling , could be adjusted on the viewing workstation . 54-year - old women with a mass in the medial right breast diagnosed with a fibroadenoma . dedicated breast computed tomography images viewed as three - dimensional data sets in multiple imaging planes ( coronal , sagittal and axial planes ) on a softcopy workstation demonstrate a 1 cm , oval , predominately circumscribed , noncalcified mass ( arrow ) . the principal investigator ( pi ) and the research technologist , certified in mammography , were both trained on the 3d dbct system and workstation by application and technical supervising personnel from zumatek , inc . , including how to use , acquire and transmit the dbct images . for training purposes in this internal irb approved and hippa - compliant study , five healthy volunteers were recruited prior to study accrual . the healthy volunteers had to be at least 40 years old , able to provide informed consent , and had a normal current ( < 60 days ) mammogram . a healthy volunteer was excluded if she was < 40 years of age , weighed > 350 pounds , pregnant or lactating , had preexisting back , neck , shoulder , or general mobility difficulties , and did not have a mammogram within 60 days or the recent mammogram study was abnormal . all of the healthy volunteers provided informed consent . if the healthy volunteer was of childbearing age and not surgically sterile , a urine pregnancy test the healthy volunteers underwent bilateral imaging with the dbct system only to test the operation of the system , to make any necessary adjustments to the system , and to train the technologist who operated the system . images of all healthy volunteers were reviewed jointly by our study investigators and sponsor quality control personnel to review image quality . eligible subjects were identified by research staff review of the clinical reports of women who were scheduled for at least one image - guided needle core breast biopsy . all diagnostic 2d digital mammographic images ( dxdm ) with additional magnification views as clinically needed ( senographe essential , general electric medical , milwaukee , wi , usa ) of all the eligible subjects were reviewed by the pi of the study prior to contacting potential subjects to ensure that the lesion recommended for biopsy was visualized on the dxdm exam . all eligible women who agreed to participate in the study were asked to come to our imaging research laboratory prior to their scheduled biopsy appointment to complete the informed consent process and to undergo the study dbct scan . for subjects of childbearing age , a negative urine pregnancy test was required prior to enrollment . the enrolled subjects subsequently underwent imaging of the breast(s ) recommended for biopsy using the dbct system . one breast at a time was placed in the table opening near the head of the table , and the noncompressed breast was allowed to hang pendant for the scan in the imaging field of view . prior to starting the scan , the technologist confirmed that the subject 's breast to be imaged was centered in the device from below the table . the technologist then closed the observation panels , asked the subject to relax and remain still , and commenced with the dbct scan . the actual scan time was 1 min 40 s per breast . during imaging , the patient was allowed to breathe normally . if the patient had a qualifying lesion in the contralateral breast that breast was also positioned and scanned in the same manner . the procedure to scan the opposite breast was performed without the subject having to leave the scanning bed . the dbct images were reviewed by the pi of the study and research technologist to ensure image quality . afterward , the enrolled study subjects were asked to complete a questionnaire about the comfort of the dbct machine . the research dbct exam was not interpreted prior to the conductance of the breast biopsy and , therefore , did not influence clinical decision - making . the research technologist electronically submitted to our medical imaging laboratory all de - identified clinical report forms as well as the de - identified breast ct exams , mammograms , breast ultrasounds ( if available ) , and breast magnetic resonance imaging ( mri ) ( if available ) of all eligible enrolled subjects that were obtained and contributed to the ultimate diagnosis leading to biopsy . the transmitted de - identified data were reviewed and correlated with the imaging findings , entered , and prepared for inclusion in the reader study . a reader study was conducted after the completion of patient accrual to the study to compare the visibility of lesion characteristics with the dbct compared to 2d full - field digital screening mammography and diagnostic mammography images . for training purposes , each radiologist was initially presented with four cases in order to become familiar with the workstation design , image display , and lesion examples . the training cases consisted of a mixture of benign and malignant cases with different lesion types with lesion descriptors . the breast lesions were annotated on all images of each case for each modality for the readers by the pi of the study . the dbct data was displayed in coronal , sagittal , and axial planes , and the dxdm data were displayed on dedicated workstations . the radiologists were allowed to roam , zoom , window , and level all the images in either modality if desired . in addition , the viewing software for dbct allowed the radiologist to adjust slice thickness of the images and multi - intensity projection images could also be used as part of the software display for interpretation . the study consisted of the dbct and mammography exams presented side - by - side for a given case . each reader was asked to compare side - by - side the dbct exam to the dxdm exam including the lesion recommended for biopsy . each reader was asked to specify the lesion type and bi - rads assessment category for each lesion for each modality and to compare the lesion characteristics : shape for masses or morphology for calcifications and margins for masses or distribution for calcifications between the two modalities using confidence scores ( 0100 ) . the specific descriptors that were used for lesion characteristics were consistent with the bi - rads mammography lexicon and were used consistently amongst the readers . statistical analysis was performed with sas statistical software , version pc 9.2 ( sas institute , inc . , the average confidence scores of each modality over the seven readers for each condition were calculated , and a linear mixed effects model was used to analyze the data . a random intercept was used in the model to account for the correlation among readers when reading the images from the same subject . the wald 's test , based on the model fit , was used to test whether the grand mean parameter was larger than zero . the procedures followed in this study were in accordance with the ethical standards of our institutional review board ( irb ) and with the helsinki declaration of 1975 , as revised in 2000 . in our irb approved and health insurance portability and accountability act ( hippa)-compliant study , all study subjects provided informed written consent after the nature of the study had been explained . in this prospective study , we recruited twenty consecutive women from our breast clinic who were scheduled to undergo a breast biopsy for a mammographically detected lesion that was evaluated with diagnostic 2d full - field digital mammography ( dxdm ) from january 1 , 2011 to october 1 , 2011 . the inclusion criteria consisted of 40-year - old or older women , who were able to provide informed consent , had at least one detected mammographic abnormality that was confirmed as a breast imaging - reporting and data system ( bi - rads ) assessment category 4 or 5 lesion after completion of diagnostic workup , and who were scheduled to undergo a core needle biopsy at our institution . women were not eligible for this study if they were under 40 years of age , weighed > 350 pounds , were pregnant or lactating , had no mammographically evident lesion , or had a preexisting back , neck , shoulder , or general mobility difficulties . after the dbct imaging was complete , each subject was given a questionnaire to complete in order to evaluate her comfort level during dbct imaging to that of other breast imaging scans on a continuous scale of 1 ( much more comfortable ) to 5 ( much less comfortable ) . the dbct scans were performed using a prototype cone - beam dbct system designed and developed by zumatek , inc . the system uses an ergonomically designed bed where the subject scanned lies prone , which is shown in figure 1 . the head of the scan bed consists of a custom formed patient support that allows for full breast and chest wall access while maximizing subject comfort and consequently , minimizing subject motion . the design of the scan bed allows one breast to be imaged at a time . the diameter of the patient table opening allows for a cylindrical imaging volume of 20 cm in diameter 20 cm in height or 6283 cm . the breast to be imaged is noncompressed and hangs pendant through the opening of the table in the imaging field below the scan bed , as denoted by the arrow in figure 1 . in addition , there are radiation shielded panels all around the bed that can be opened below the head of the scanning bed that can allow the imaging technologist direct visualization if further positioning of the subject as needed . besides direct visualization ( b ) the noncompressed breast to be imaged hangs pendant through the opening of the table ( arrow ) below the scan bed . this cone - beam ct system features a single 30 cm 30 cm , amorphous silicon flat - panel , digital detector with a thallium - doped cesium iodide scintillator ( paxscan 3030 , varian medical systems , palo alto , california , usa ) . the detector has 197-micron pixels , which can be binned in 2 2 or read out in 1 1 mode . the system uses a rotating tungsten rotating anode ( rad-70b , varian medical systems , palo alto , california , usa ) with a tungsten filter . the dbct system is powered by a high - frequency x - ray generator ( sedecal , madrid , spain ) , which allows for fast pulsed x - ray generation up to 30 frames per second . the x - ray tube and detector are fixed at a specific source - to - image distance of 70 cm ; therefore , there is no contact with the breast being imaged . the scanner operates in step and shoot mode whereby it is preprogrammed to move and stop at 300 specific positions around the noncompressed , pendant breast as individual images are taken . the scan time for one breast is 1 min 40 s. the average radiation dose is < 4.5 mgy for a bilateral dbct scan . motors in the system provide motion to allow the imaging components to be moved about a full 360 vertical rotation axis , and also 60 horizontal pivoting about the rotation axis is achieved around the noncompressed breast being imaged . this unique 3d orbiting capability allows for full breast and chest wall access with more complete cone beam sampling throughout the image volume . a third motion is possible to allow the system to be moved vertically up and down to position the system optimally below the bed . at the end of the scan , the 300 images obtained for each breast are sent to a computer processing workstation and are automatically processed with an iterative reconstruction algorithm that reconstructs ( 768 768 14 bits / projection ) the images into a 3d dbct dataset . the measured isotropic reconstructed pixels are 197 microns , and the measured reconstructed resolution of the line pairs with a 20 cm diameter catphan phantom ( phantom laboratory , salem , ny , usa ) is 2.5 line pairs per millimeter . the resulting digital imaging and communication in medicine ( dicom ) compliant images were viewed using clearcanvas software ( clearcanvas , toronto , ontario , canada ) . the dbct data was displayed in three simultaneous imaging planes ( coronal , sagittal , and axial planes ) , figure 2 . in addition , the slice thickness and other viewing features , such as window and leveling , could be adjusted on the viewing workstation . 54-year - old women with a mass in the medial right breast diagnosed with a fibroadenoma . dedicated breast computed tomography images viewed as three - dimensional data sets in multiple imaging planes ( coronal , sagittal and axial planes ) on a softcopy workstation demonstrate a 1 cm , oval , predominately circumscribed , noncalcified mass ( arrow ) . the principal investigator ( pi ) and the research technologist , certified in mammography , were both trained on the 3d dbct system and workstation by application and technical supervising personnel from zumatek , inc . , including how to use , acquire and transmit the dbct images . for training purposes in this internal irb approved and hippa - compliant study , the healthy volunteers had to be at least 40 years old , able to provide informed consent , and had a normal current ( < 60 days ) mammogram . a healthy volunteer was excluded if she was < 40 years of age , weighed > 350 pounds , pregnant or lactating , had preexisting back , neck , shoulder , or general mobility difficulties , and did not have a mammogram within 60 days or the recent mammogram study was abnormal . all of the healthy volunteers provided informed consent . if the healthy volunteer was of childbearing age and not surgically sterile , a urine pregnancy test the healthy volunteers underwent bilateral imaging with the dbct system only to test the operation of the system , to make any necessary adjustments to the system , and to train the technologist who operated the system . images of all healthy volunteers were reviewed jointly by our study investigators and sponsor quality control personnel to review image quality . eligible subjects were identified by research staff review of the clinical reports of women who were scheduled for at least one image - guided needle core breast biopsy . all diagnostic 2d digital mammographic images ( dxdm ) with additional magnification views as clinically needed ( senographe essential , general electric medical , milwaukee , wi , usa ) of all the eligible subjects were reviewed by the pi of the study prior to contacting potential subjects to ensure that the lesion recommended for biopsy was visualized on the dxdm exam . all eligible women who agreed to participate in the study were asked to come to our imaging research laboratory prior to their scheduled biopsy appointment to complete the informed consent process and to undergo the study dbct scan . for subjects of childbearing age , a negative urine pregnancy test was required prior to enrollment . the enrolled subjects subsequently underwent imaging of the breast(s ) recommended for biopsy using the dbct system . one breast at a time was placed in the table opening near the head of the table , and the noncompressed breast was allowed to hang pendant for the scan in the imaging field of view . prior to starting the scan , the technologist confirmed that the subject 's breast to be imaged was centered in the device from below the table . the technologist then closed the observation panels , asked the subject to relax and remain still , and commenced with the dbct scan . the actual scan time was 1 min 40 s per breast . during imaging , the patient was allowed to breathe normally . if the patient had a qualifying lesion in the contralateral breast that breast was also positioned and scanned in the same manner . the procedure to scan the opposite breast was performed without the subject having to leave the scanning bed . the dbct images were reviewed by the pi of the study and research technologist to ensure image quality . afterward , the enrolled study subjects were asked to complete a questionnaire about the comfort of the dbct machine . the research dbct exam was not interpreted prior to the conductance of the breast biopsy and , therefore , did not influence clinical decision - making . the research technologist electronically submitted to our medical imaging laboratory all de - identified clinical report forms as well as the de - identified breast ct exams , mammograms , breast ultrasounds ( if available ) , and breast magnetic resonance imaging ( mri ) ( if available ) of all eligible enrolled subjects that were obtained and contributed to the ultimate diagnosis leading to biopsy . the transmitted de - identified data were reviewed and correlated with the imaging findings , entered , and prepared for inclusion in the reader study . a reader study was conducted after the completion of patient accrual to the study to compare the visibility of lesion characteristics with the dbct compared to 2d full - field digital screening mammography and diagnostic mammography images . seven breast imaging radiologists reviewed the cases in the reader study . for training purposes , each radiologist was initially presented with four cases in order to become familiar with the workstation design , image display , and lesion examples . the training cases consisted of a mixture of benign and malignant cases with different lesion types with lesion descriptors . the breast lesions were annotated on all images of each case for each modality for the readers by the pi of the study . the dbct data was displayed in coronal , sagittal , and axial planes , and the dxdm data were displayed on dedicated workstations . the radiologists were allowed to roam , zoom , window , and level all the images in either modality if desired . in addition , the viewing software for dbct allowed the radiologist to adjust slice thickness of the images and multi - intensity projection images could also be used as part of the software display for interpretation . the study consisted of the dbct and mammography exams presented side - by - side for a given case . each reader was asked to compare side - by - side the dbct exam to the dxdm exam including the lesion recommended for biopsy . each reader was asked to specify the lesion type and bi - rads assessment category for each lesion for each modality and to compare the lesion characteristics : shape for masses or morphology for calcifications and margins for masses or distribution for calcifications between the two modalities using confidence scores ( 0100 ) . the specific descriptors that were used for lesion characteristics were consistent with the bi - rads mammography lexicon and were used consistently amongst the readers . statistical analysis was performed with sas statistical software , version pc 9.2 ( sas institute , inc . , cary , nc , usa ) . the average confidence scores of each modality over the seven readers for each condition were calculated , and a linear mixed effects model was used to analyze the data . a random intercept was used in the model to account for the correlation among readers when reading the images from the same subject . the wald 's test , based on the model fit , was used to test whether the grand mean parameter was larger than zero . there were a total of 24 biopsied lesions in 20 subjects included in this study . each patient underwent percutaneous biopsy , and the pathology reports were reviewed and correlated with the imaging findings . the mammographic findings of the biopsied lesions consisted of 17 ( 70.8% ) masses and 7 ( 29.2% ) calcifications . overall , 8 ( 28.6% ) lesions were malignant , and 16 ( 71.4% ) were benign . six ( 75.0% ) of the malignant lesions ( 5 invasive cancers and 1 ductal carcinoma in situ [ dcis ] ) were presented as masses . only 2 ( 28.6% ) of the 7 cases of calcifications were malignant , both dcis . the mean size of a malignant mass case and a malignant calcification case was 2.8 cm ( range 1.04.5 cm ) and 4.3 cm ( range 1.57.0 cm ) , respectively . the mean size of a benign mass case was 1.5 cm ( range 0.42.5 cm ) , and the mean size of a benign calcification case was 1.8 cm ( range 0.53.0 cm ) . mammographic subject breast density and lesion type , size and pathology three ( 15% ) subjects had bilateral lesions and 1 ( 5.0% ) subject had 2 lesions in the same breast . of the 3 subjects with bilateral lesions , 1 ( 33.3% ) subject had bilateral invasive cancer , and the other 2 ( 66.7% ) subjects had unilateral cancer ( 1 case invasive and 1 case dcis ) with a contralateral benign lesion . the one ( 5.0% ) subject with 2 lesions in the same breast had both a malignant lesion ( dcis ) and a benign lesion ( fibroadenoma ) . with regard to breast side of the lesion , 10 ( 41.7% ) were located in the right breast , and 14 ( 58.3% ) were located in the left breast . three ( 37.5% ) malignant lesions were located in the right breast , and 5 ( 62.5% ) were located in the left breast . the 20 subjects enrolled and imaged in the study consisted of 12 ( 60.0% ) caucasian and 8 ( 40.0% ) african american women with a mean age of 58.0 years ( range 4175 years ) . the mean age of a subject with a malignant lesion was 56.5 years and the mean age of a subject with a benign lesion was 58.0 years . six ( 30.0% ) of the subjects had a history of a benign biopsy : 3 ( 50.0% ) core needle biopsy and 3 ( 50.0% ) open surgical excision . one ( 5.0% ) subject had a prior breast cancer history of dcis that had been treated with a mastectomy . the bi - rads mammographic breast composition categories for the enrolled subjects were 8 ( 40.0% ) with scattered areas of fibroglandular density , 9 ( 45.0% ) with heterogeneously dense , and 3 ( 15.0% ) with extremely dense breasts , table 1 . when the latter two categories are combined , 60.0% of the cases were in subjects with dense breast tissue . for the 7 ( 35.0% ) subjects diagnosed with at least 1 cancer ( 1 subject had a bilateral invasive cancer ) , the bi - rads mammographic breast composition categories were 3 ( 42.9% ) with scattered areas of fibroglandular density , 3 ( 42.9% ) heterogeneously dense , and 1 ( 14.2% ) with extremely dense breasts . the one ( 14.3% ) subject with the bilateral breast cancer had heterogeneously dense breasts . the two ( 28.6% ) subjects that had cancer ( dcis ) present as calcifications had the mammographic breast composition of scattered areas of fibroglandular density . for the 13 ( 65.0% ) subjects with benign only pathology , 5 ( 38.5% ) had scattered areas of fibroglandular density , 6 ( 46.2% ) had heterogeneously dense breasts , and 2 ( 15.3% ) had extremely dense breasts mammographically . all radiologists were certified by the american board of radiology and met accreditation standards mandated by the mammography quality standard act and program . none of the radiologists had prior dbct research experience or clinical experience in interpretation of dbct . mean confidence radiologist reader scores of dbct compared to dxdm for the two conditions for all lesions regardless of lesion type , lesion pathology , and subject breast density are shown in table 2 . across all lesions , there was no significant difference in the margin / distribution ( = 0.99 , p = 0.84 ) and shape / morphology ( = 0.10 , p = 0.98 ) visualization confidence scores of ct in relation to dxdm . however , analysis by lesion type showed a statistically significant increase in reader shape ( = 11.34 , p = 0.013 ) and margin ( = 9.93 , p = 0.023 ) visualization confidence with ct versus dxdm for masses and significant decrease in reader morphology ( = 29.95 , p = 0.001 ) and distribution ( = 28.62 , p = 0.002 ) visualization confidence for calcifications , table 3 . the reader visualization confidence scores of benign and malignant lesions between the two modalities for subjects with dense breast tissue were not significantly different ( p > 0.1 ) . there was no significant difference ( p > 0.1 ) between the two modalities in the readers bi - rads assessment scores across all lesions , regardless of size or final pathology or subject breast density . examples of a benign and a malignant mass with mammographic and dbct correlation images are shown in figures 3 and 4 , respectively . in addition , mammographic and dbt correlation images for benign and malignant calcifications cases are demonstrated in figures 5 and 6 . reader confidence scores all lesions data reader confidence scores by lesion type 49-year - old women with a mass in the medial right breast diagnosed with a fibroadenoma . mammographic images of the right breast : ( a ) craniocaudal , ( b ) mediolateral oblique , ( c ) spot compression magnification craniocaudal view , ( d ) spot compression magnification mediolateral oblique view . the mammographic images demonstrate a 1 cm , oval , obscured mass ( arrow ) in the upper inner quadrant . dedicated breast computed tomography slice images of the right breast : ( e ) axial , ( f ) sagittal , ( g ) coronal , ( h ) enlarged sagittal image . the dedicated breast computed tomography images of the mass ( arrow ) show a circumscribed margin since there is no overlap of breast tissue . 72-year - old women with a mass in the medial left breast diagnosed with invasive ductal carcinoma . mammographic images of the left breast : ( a ) mediolateral oblique , ( b ) craniocaudal , ( c ) spot compression magnification mediolateral oblique view , ( d ) spot compression magnification craniocaudal view . the mammographic images show a 1.5 cm , irregular , partially obscured mass ( arrow ) in the medial aspect . dedicated breast computed tomography slice images of the left breast : ( e ) sagittal , ( f ) axial , ( g ) coronal , ( h ) enlarged axial image . the dedicated breast computed tomography images of the mass ( arrow ) demonstrate that the margins of the mass to be spiculated . 48-year - old women with grouped calcifications in the medial left breast diagnosed with fibrocystic changes with associated calcifications . mammographic images of the left breast : ( a ) spot compression magnification craniocaudal view , ( b ) 90 spot compression magnification view . the mammographic images demonstrate 1.5 cm , grouped , coarse heterogeneous and round calcifications ( arrow ) . dedicated breast computed tomography slice images : ( c ) axial image , ( d ) enlarged axial image , ( e ) enlarged sagittal image . the dedicated breast computed tomography images of the calcifications ( arrow ) demonstrate a similar appearance as the mammographic images . 60-year - old women with calcifications in the medial left breast diagnosed with ductal carcinoma in situ . mammographic images of the left breast : ( a ) spot compression magnification craniocaudal view , ( b ) 90 spot compression magnification view . the mammographic images show fine linear calcifications in a segmental distribution spanning 7 cm ( arrow ) . dedicated breast computed tomography slice images : ( c ) axial , ( d ) sagittal , ( e ) enlarged axial view , ( f ) enlarged sagittal view . the dedicated breast computed tomography images also demonstrate the same abnormal calcifications and distribution ( arrow ) as the mammographic images . the total number of mammographic images was also recorded for this study . for the 20 subjects , the mean number of images obtained for each subject 's diagnostic mammogram exam was 6 ( range 210 ) . for the 11 ( 55.0% ) subjects that originally underwent a screening mammogram and were recalled for further diagnostic evaluation , the mean number of both mammographic exam images acquired were 10 ( range 616 ) . in contrast , only 1 dbct scan was obtained per index breast per subject in this research study . results of the study subject questionnaire after completion of the dbct portion of the study demonstrate that all 20 ( 100% ) subjects were satisfied or very satisfied with the dbct scan . thirteen ( 65.0% ) subjects reported no discomfort and 6 ( 30.0% ) subjects reported minimal to mild discomfort during dbct scanning . only 1 ( 5.0% ) all discomfort that was reported was related to neck , back , or abdomen from the scanning table or arm positioning . in regard to scanning time and noise level of the dbct device , 19 ( 95.0% ) and 19 ( 95.0% ) of the subjects were satisfied or very satisfied , respectively . questionnaire results of subject comfort of a dbct scan versus a mammogram , handheld breast ultrasound ( hhbus ) , and contrast enhanced - mri ( ce - mri ) of the breasts were also collected . all 20 ( 100% ) of the subjects reported the dbct scan to be more comfortable than a mammogram . sixty - five percent ( 13 subjects ) reported the dbct scan was more comfortable than hhbus . thirty - five percent ( 7 subjects ) reported the dbct scan and hhbus to be equal in comfort . one ( 5% ) subject reported the dbct scan was less comfortable than a ce - mri , and one ( 5% ) subject reported the dbct scan and the ce - mri to be equal in comfort . our initial clinical experience with a prototype 3d dbct system demonstrates that overall radiologist reader confidence in the characterization of suspicious lesions recommended for biopsy is similar to that of diagnostic 2d digital mammography . radiologists were significantly more confident in mass shape and margin characterization on dbct images compared to 2d digital diagnostic mammography . even though all calcification lesions were visible on the dbct , reader confidence in calcification lesion characteristics was significantly less than diagnostic mammography , and this may have been related to our prototype dbct system . the dbct system used 197-micron pixels in contrast to digital mammography that may have pixel sizes between 50 and 100 microns depending on the manufacturer . our study used a digital mammography system with 100-micron pixels in addition to a magnification factor of 1.8 for the spot compression magnification views . thus , improvements in dbct detector resolution and specific postprocessing algorithms could help radiologists improve calcification characterization . the increase in reader confidence with masses on dbct is most likely the result of dbct 's 3d imaging capability . with 3d imaging of the breast , dbct has the advantage of being able to remove tissue overlap ( masking ) through the use of tomographic slice sections . in addition , the acquired image data obtained by dbct is used to reconstruct a volume dataset for image display and can provide further diagnostic information for the radiologist . since dbct is true 3d imaging and does not require breast compression , it allows for registered orthogonal views of the breast in any projection for viewing , similar to mri of the breast . the viewing software allows the radiologist to review images and lesions in multiple planes and adjust slice thickness of the images for interpretation . in addition , a multi - intensity projection image can also be visualized as part of the software display . consequently , these advantages of dbct may improve lesion characterization , which may aid in decreasing unnecessary biopsies and could possibly help in surgical planning in patients diagnosed with breast cancer . , in which they evaluated bi - rads 4 and 5 lesions with their prototype dbct system . their study consisted of 67 lesions : 52 ( 77.6% ) masses , 12 ( 17.9% ) calcifications , and 3 ( 4.5% ) other types . however , 9 ( 13.4% ) of 67 lesions in their study were not seen on dbct . overall , their results showed that breast ct was equal to screen film mammography for visualization of breast lesions . in addition , breast ct was significantly better than film - screen mammography for visualization of masses ( p < 0.002 ) but film - screen mammography out - performed breast ct for visualization of calcifications ( p < 0.006 ) . in contrast to their study , our subjects mammographic images were acquired with 2d full - field digital mammography , and we evaluated not only the reader confidence of lesion type but also confidence of lesion characteristics . in the study by oconnell and kawakyu - oconnor , two radiologists evaluated 36 patients with 37 abnormal mammographic and/or ultrasound categorized as bi - rads 4 or 5 lesions that were evaluated with their prototype dbct system prior to biopsy . their results demonstrated a high degree of correlation between the dbct and digital mammography across a variety of lesion types with 33 of 34 ( 97.1% ) mammographic lesions being scored as equal or better visualized on breast ct relative to diagnostic mammography . only one ( 2.9% ) lesion in their study was not seen on dbct and was attributed to be a mammographic summation shadow ( 22 ) . unlike their study criteria , all of our study lesions were visible with both diagnostic 2d digital mammography and dbct . we did not include any lesions visible on sonography since we were comparing dbct to diagnostic 2d digital mammography . in addition , our reader study included five more radiologists and also evaluated lesion features . , reported that 10.4% of their calcification findings were not seen on their prototype dbct system . all ( 100% ) calcification lesions were visualized with dbct in our study ; however , radiologist confidence in calcification characterization was significantly better with digital diagnostic mammography . 3d dbct may have several potential advantages compared to digital mammography in a diagnostic setting . currently , there is no maximum limit of breast radiation exposure with diagnostic mammography . only screening 2d mammography and dbt have dose limits . as with the prototype dbct system we used , the dbct radiation dose is equivalent to about 2 months of natural background radiation exposure by a person in 1 year in the united states . comparable to mammographic doses , the radiation doses from dbct are largely restricted to breast tissue . the second advantage of the dbct system is that the automated images of the uncompressed breast are obtained in a standardized manner with a short acquisition time . the dbct scan time was 1 min 40 s in our study and was comparable to other published studies . another benefit of our system is that due to the design of the imaging device even subjects with a large breast size ( ee cup size ) can be imaged without excluding breast tissue . our results support other studies in which higher degrees of subject comfort in the dbct examination relative to mammography were reported . first , none of the readers had prior research or clinical experience in interpretation of dbct images . another limitation was that only a small sample size of different lesion types was included in this single site study in which enrolled subjects had undergone a standard of care 2d digital mammographic diagnostic workup prior to being enrolled in this research study . none of the subjects had dbt since this was not available at our institution at the time of this study . moreover , the resolution of the detector used on our prototype dbct system was less than a full - field digital mammogram . finally , our dbct images were obtained without contrast but rather with a beam to optimize contrast differences between adipose and glandular tissues . the results of the study may have differed if the radiologists had dbct experience , a larger sample size , dbt use , a prototype dbct system with a finer spatial resolution and dedicated image processing algorithms and possibly the administration of intravenous contrast . nevertheless , future studies are needed in evaluating noncontrast dbct versus contrast dbct and dbt . first , none of the readers had prior research or clinical experience in interpretation of dbct images . another limitation was that only a small sample size of different lesion types was included in this single site study in which enrolled subjects had undergone a standard of care 2d digital mammographic diagnostic workup prior to being enrolled in this research study . none of the subjects had dbt since this was not available at our institution at the time of this study . moreover , the resolution of the detector used on our prototype dbct system was less than a full - field digital mammogram . finally , our dbct images were obtained without contrast but rather with a beam to optimize contrast differences between adipose and glandular tissues . the results of the study may have differed if the radiologists had dbct experience , a larger sample size , dbt use , a prototype dbct system with a finer spatial resolution and dedicated image processing algorithms and possibly the administration of intravenous contrast . nevertheless , future studies are needed in evaluating noncontrast dbct versus contrast dbct and dbt . dedicated 3d breast computed tomography is a novel well - tolerated imaging modality that allows for true isotropic , 3d imaging of the breast without mammographic compression . dbct is comparable to conventional mammography in terms of radiation dose and overall lesion visibility . dbct is superior in radiologists visualization and characterization of masses , but inferior to diagnostic mammography for calcifications . technical challenges remain , but 3d dbct may have potential clinical applications in breast cancer screening and diagnosis .

objectives : to assess radiologist confidence in the characterization of suspicious breast lesions with a dedicated three - dimensional breast computed tomography ( dbct ) system in comparison to diagnostic two - dimensional digital mammography ( dxdm).materials and methods : twenty women were recruited who were to undergo a breast biopsy for a breast imaging - reporting and data system ( bi - rads ) 4 or 5 lesion evaluated with dxdm in this institutional review board - approved study . the enrolled subjects underwent imaging of the breast(s ) of concern using dbct . seven radiologists reviewed the cases . each reader compared dbct to the dxdm and was asked to specify the lesion type and bi - rads score for each lesion and modality . they also compared lesion characteristics : shape for masses or morphology for calcifications ; and margins for masses or distribution for calcifications between the modalities using confidence scores ( 0100).results : twenty - four biopsied lesions were included in this study : 17 ( 70.8% ) masses and 7 ( 29.2% ) calcifications . eight ( 33.3% ) lesions were malignant , and 16 ( 66.7% ) were benign . across all lesions , there was no significant difference in the margin / distribution ( = 0.99 , p = 0.84 ) and shape / morphology ( = 0.10 , p = 0.98 ) visualization confidence scores of dbct in relation to dxdm . however , analysis by lesion type showed a statistically significant increase in reader shape ( = 11.34 , p = 0.013 ) and margin ( = 9.93 , p = 0.023 ) visualization confidence with dbct versus dxdm for masses and significant decrease in reader morphology ( = 29.95 , p = 0.001 ) and distribution ( = 28.62 , p = 0.002 ) visualization confidence for calcifications.conclusion:reader confidence in the characterization of suspicious masses is significantly improved with dbct , but reduced for calcifications . further study is needed to determine whether this technology can be used for breast cancer screening .

INTRODUCTION MATERIALS AND METHODS Subjects Dedicated breast computed tomography scanner Training and start up Image acquisition Postimaging Data submission Reader study Statistical analysis RESULTS DISCUSSION Limitations CONCLUSION Financial support and sponsorship Conflicts of interest

breast cancer is the most common female cancer , except for skin cancer , in the united states for every major ethnic group and the second most common cause of cancer death in women . in reaction to this limitation published literature has demonstrated that dbt in combination with a conventional digital mammogram has demonstrated improved mass detection , decreased recall rates , and increased cancer detection . dedicated breast computed tomography ( dbct ) is a new imaging modality that provides three - dimensional ( 3d ) data that can be reconstructed into multiple imaging planes . the radiation dose level of dbct is similar to the dose of a conventional two - view mammogram . therefore , the objective of our study is to assess radiologist confidence in the characterization of suspicious breast lesions in a population of women going to biopsy with a dbct system in comparison to diagnostic two - dimensional ( 2d ) full - field digital mammography ( dxdm ) . the procedures followed in this study were in accordance with the ethical standards of our institutional review board ( irb ) and with the helsinki declaration of 1975 , as revised in 2000 . in this prospective study , we recruited twenty consecutive women from our breast clinic who were scheduled to undergo a breast biopsy for a mammographically detected lesion that was evaluated with diagnostic 2d full - field digital mammography ( dxdm ) from january 1 , 2011 to october 1 , 2011 . the inclusion criteria consisted of 40-year - old or older women , who were able to provide informed consent , had at least one detected mammographic abnormality that was confirmed as a breast imaging - reporting and data system ( bi - rads ) assessment category 4 or 5 lesion after completion of diagnostic workup , and who were scheduled to undergo a core needle biopsy at our institution . the breast to be imaged is noncompressed and hangs pendant through the opening of the table in the imaging field below the scan bed , as denoted by the arrow in figure 1 . in addition , there are radiation shielded panels all around the bed that can be opened below the head of the scanning bed that can allow the imaging technologist direct visualization if further positioning of the subject as needed . motors in the system provide motion to allow the imaging components to be moved about a full 360 vertical rotation axis , and also 60 horizontal pivoting about the rotation axis is achieved around the noncompressed breast being imaged . at the end of the scan , the 300 images obtained for each breast are sent to a computer processing workstation and are automatically processed with an iterative reconstruction algorithm that reconstructs ( 768 768 14 bits / projection ) the images into a 3d dbct dataset . the measured isotropic reconstructed pixels are 197 microns , and the measured reconstructed resolution of the line pairs with a 20 cm diameter catphan phantom ( phantom laboratory , salem , ny , usa ) is 2.5 line pairs per millimeter . dedicated breast computed tomography images viewed as three - dimensional data sets in multiple imaging planes ( coronal , sagittal and axial planes ) on a softcopy workstation demonstrate a 1 cm , oval , predominately circumscribed , noncalcified mass ( arrow ) . if the healthy volunteer was of childbearing age and not surgically sterile , a urine pregnancy test the healthy volunteers underwent bilateral imaging with the dbct system only to test the operation of the system , to make any necessary adjustments to the system , and to train the technologist who operated the system . eligible subjects were identified by research staff review of the clinical reports of women who were scheduled for at least one image - guided needle core breast biopsy . all diagnostic 2d digital mammographic images ( dxdm ) with additional magnification views as clinically needed ( senographe essential , general electric medical , milwaukee , wi , usa ) of all the eligible subjects were reviewed by the pi of the study prior to contacting potential subjects to ensure that the lesion recommended for biopsy was visualized on the dxdm exam . all eligible women who agreed to participate in the study were asked to come to our imaging research laboratory prior to their scheduled biopsy appointment to complete the informed consent process and to undergo the study dbct scan . the enrolled subjects subsequently underwent imaging of the breast(s ) recommended for biopsy using the dbct system . one breast at a time was placed in the table opening near the head of the table , and the noncompressed breast was allowed to hang pendant for the scan in the imaging field of view . afterward , the enrolled study subjects were asked to complete a questionnaire about the comfort of the dbct machine . the research dbct exam was not interpreted prior to the conductance of the breast biopsy and , therefore , did not influence clinical decision - making . the research technologist electronically submitted to our medical imaging laboratory all de - identified clinical report forms as well as the de - identified breast ct exams , mammograms , breast ultrasounds ( if available ) , and breast magnetic resonance imaging ( mri ) ( if available ) of all eligible enrolled subjects that were obtained and contributed to the ultimate diagnosis leading to biopsy . the breast lesions were annotated on all images of each case for each modality for the readers by the pi of the study . in addition , the viewing software for dbct allowed the radiologist to adjust slice thickness of the images and multi - intensity projection images could also be used as part of the software display for interpretation . the study consisted of the dbct and mammography exams presented side - by - side for a given case . each reader was asked to compare side - by - side the dbct exam to the dxdm exam including the lesion recommended for biopsy . each reader was asked to specify the lesion type and bi - rads assessment category for each lesion for each modality and to compare the lesion characteristics : shape for masses or morphology for calcifications and margins for masses or distribution for calcifications between the two modalities using confidence scores ( 0100 ) . the specific descriptors that were used for lesion characteristics were consistent with the bi - rads mammography lexicon and were used consistently amongst the readers . , the average confidence scores of each modality over the seven readers for each condition were calculated , and a linear mixed effects model was used to analyze the data . the procedures followed in this study were in accordance with the ethical standards of our institutional review board ( irb ) and with the helsinki declaration of 1975 , as revised in 2000 . in this prospective study , we recruited twenty consecutive women from our breast clinic who were scheduled to undergo a breast biopsy for a mammographically detected lesion that was evaluated with diagnostic 2d full - field digital mammography ( dxdm ) from january 1 , 2011 to october 1 , 2011 . the inclusion criteria consisted of 40-year - old or older women , who were able to provide informed consent , had at least one detected mammographic abnormality that was confirmed as a breast imaging - reporting and data system ( bi - rads ) assessment category 4 or 5 lesion after completion of diagnostic workup , and who were scheduled to undergo a core needle biopsy at our institution . the breast to be imaged is noncompressed and hangs pendant through the opening of the table in the imaging field below the scan bed , as denoted by the arrow in figure 1 . in addition , there are radiation shielded panels all around the bed that can be opened below the head of the scanning bed that can allow the imaging technologist direct visualization if further positioning of the subject as needed . the measured isotropic reconstructed pixels are 197 microns , and the measured reconstructed resolution of the line pairs with a 20 cm diameter catphan phantom ( phantom laboratory , salem , ny , usa ) is 2.5 line pairs per millimeter . 54-year - old women with a mass in the medial right breast diagnosed with a fibroadenoma . dedicated breast computed tomography images viewed as three - dimensional data sets in multiple imaging planes ( coronal , sagittal and axial planes ) on a softcopy workstation demonstrate a 1 cm , oval , predominately circumscribed , noncalcified mass ( arrow ) . if the healthy volunteer was of childbearing age and not surgically sterile , a urine pregnancy test the healthy volunteers underwent bilateral imaging with the dbct system only to test the operation of the system , to make any necessary adjustments to the system , and to train the technologist who operated the system . eligible subjects were identified by research staff review of the clinical reports of women who were scheduled for at least one image - guided needle core breast biopsy . all diagnostic 2d digital mammographic images ( dxdm ) with additional magnification views as clinically needed ( senographe essential , general electric medical , milwaukee , wi , usa ) of all the eligible subjects were reviewed by the pi of the study prior to contacting potential subjects to ensure that the lesion recommended for biopsy was visualized on the dxdm exam . all eligible women who agreed to participate in the study were asked to come to our imaging research laboratory prior to their scheduled biopsy appointment to complete the informed consent process and to undergo the study dbct scan . the enrolled subjects subsequently underwent imaging of the breast(s ) recommended for biopsy using the dbct system . one breast at a time was placed in the table opening near the head of the table , and the noncompressed breast was allowed to hang pendant for the scan in the imaging field of view . afterward , the enrolled study subjects were asked to complete a questionnaire about the comfort of the dbct machine . the research dbct exam was not interpreted prior to the conductance of the breast biopsy and , therefore , did not influence clinical decision - making . the research technologist electronically submitted to our medical imaging laboratory all de - identified clinical report forms as well as the de - identified breast ct exams , mammograms , breast ultrasounds ( if available ) , and breast magnetic resonance imaging ( mri ) ( if available ) of all eligible enrolled subjects that were obtained and contributed to the ultimate diagnosis leading to biopsy . seven breast imaging radiologists reviewed the cases in the reader study . the breast lesions were annotated on all images of each case for each modality for the readers by the pi of the study . the dbct data was displayed in coronal , sagittal , and axial planes , and the dxdm data were displayed on dedicated workstations . in addition , the viewing software for dbct allowed the radiologist to adjust slice thickness of the images and multi - intensity projection images could also be used as part of the software display for interpretation . each reader was asked to compare side - by - side the dbct exam to the dxdm exam including the lesion recommended for biopsy . each reader was asked to specify the lesion type and bi - rads assessment category for each lesion for each modality and to compare the lesion characteristics : shape for masses or morphology for calcifications and margins for masses or distribution for calcifications between the two modalities using confidence scores ( 0100 ) . the specific descriptors that were used for lesion characteristics were consistent with the bi - rads mammography lexicon and were used consistently amongst the readers . the average confidence scores of each modality over the seven readers for each condition were calculated , and a linear mixed effects model was used to analyze the data . there were a total of 24 biopsied lesions in 20 subjects included in this study . the mammographic findings of the biopsied lesions consisted of 17 ( 70.8% ) masses and 7 ( 29.2% ) calcifications . overall , 8 ( 28.6% ) lesions were malignant , and 16 ( 71.4% ) were benign . six ( 75.0% ) of the malignant lesions ( 5 invasive cancers and 1 ductal carcinoma in situ [ dcis ] ) were presented as masses . only 2 ( 28.6% ) of the 7 cases of calcifications were malignant , both dcis . of the 3 subjects with bilateral lesions , 1 ( 33.3% ) subject had bilateral invasive cancer , and the other 2 ( 66.7% ) subjects had unilateral cancer ( 1 case invasive and 1 case dcis ) with a contralateral benign lesion . the one ( 5.0% ) subject with 2 lesions in the same breast had both a malignant lesion ( dcis ) and a benign lesion ( fibroadenoma ) . with regard to breast side of the lesion , 10 ( 41.7% ) were located in the right breast , and 14 ( 58.3% ) were located in the left breast . three ( 37.5% ) malignant lesions were located in the right breast , and 5 ( 62.5% ) were located in the left breast . the 20 subjects enrolled and imaged in the study consisted of 12 ( 60.0% ) caucasian and 8 ( 40.0% ) african american women with a mean age of 58.0 years ( range 4175 years ) . the bi - rads mammographic breast composition categories for the enrolled subjects were 8 ( 40.0% ) with scattered areas of fibroglandular density , 9 ( 45.0% ) with heterogeneously dense , and 3 ( 15.0% ) with extremely dense breasts , table 1 . for the 7 ( 35.0% ) subjects diagnosed with at least 1 cancer ( 1 subject had a bilateral invasive cancer ) , the bi - rads mammographic breast composition categories were 3 ( 42.9% ) with scattered areas of fibroglandular density , 3 ( 42.9% ) heterogeneously dense , and 1 ( 14.2% ) with extremely dense breasts . the one ( 14.3% ) subject with the bilateral breast cancer had heterogeneously dense breasts . for the 13 ( 65.0% ) subjects with benign only pathology , 5 ( 38.5% ) had scattered areas of fibroglandular density , 6 ( 46.2% ) had heterogeneously dense breasts , and 2 ( 15.3% ) had extremely dense breasts mammographically . mean confidence radiologist reader scores of dbct compared to dxdm for the two conditions for all lesions regardless of lesion type , lesion pathology , and subject breast density are shown in table 2 . across all lesions , there was no significant difference in the margin / distribution ( = 0.99 , p = 0.84 ) and shape / morphology ( = 0.10 , p = 0.98 ) visualization confidence scores of ct in relation to dxdm . however , analysis by lesion type showed a statistically significant increase in reader shape ( = 11.34 , p = 0.013 ) and margin ( = 9.93 , p = 0.023 ) visualization confidence with ct versus dxdm for masses and significant decrease in reader morphology ( = 29.95 , p = 0.001 ) and distribution ( = 28.62 , p = 0.002 ) visualization confidence for calcifications , table 3 . the reader visualization confidence scores of benign and malignant lesions between the two modalities for subjects with dense breast tissue were not significantly different ( p > 0.1 ) . there was no significant difference ( p > 0.1 ) between the two modalities in the readers bi - rads assessment scores across all lesions , regardless of size or final pathology or subject breast density . reader confidence scores all lesions data reader confidence scores by lesion type 49-year - old women with a mass in the medial right breast diagnosed with a fibroadenoma . dedicated breast computed tomography slice images of the right breast : ( e ) axial , ( f ) sagittal , ( g ) coronal , ( h ) enlarged sagittal image . the dedicated breast computed tomography images of the mass ( arrow ) show a circumscribed margin since there is no overlap of breast tissue . dedicated breast computed tomography slice images of the left breast : ( e ) sagittal , ( f ) axial , ( g ) coronal , ( h ) enlarged axial image . the dedicated breast computed tomography images of the mass ( arrow ) demonstrate that the margins of the mass to be spiculated . dedicated breast computed tomography slice images : ( c ) axial image , ( d ) enlarged axial image , ( e ) enlarged sagittal image . the dedicated breast computed tomography images of the calcifications ( arrow ) demonstrate a similar appearance as the mammographic images . the dedicated breast computed tomography images also demonstrate the same abnormal calcifications and distribution ( arrow ) as the mammographic images . in regard to scanning time and noise level of the dbct device , 19 ( 95.0% ) and 19 ( 95.0% ) of the subjects were satisfied or very satisfied , respectively . questionnaire results of subject comfort of a dbct scan versus a mammogram , handheld breast ultrasound ( hhbus ) , and contrast enhanced - mri ( ce - mri ) of the breasts were also collected . our initial clinical experience with a prototype 3d dbct system demonstrates that overall radiologist reader confidence in the characterization of suspicious lesions recommended for biopsy is similar to that of diagnostic 2d digital mammography . even though all calcification lesions were visible on the dbct , reader confidence in calcification lesion characteristics was significantly less than diagnostic mammography , and this may have been related to our prototype dbct system . the increase in reader confidence with masses on dbct is most likely the result of dbct 's 3d imaging capability . with 3d imaging of the breast , dbct has the advantage of being able to remove tissue overlap ( masking ) through the use of tomographic slice sections . , in which they evaluated bi - rads 4 and 5 lesions with their prototype dbct system . their study consisted of 67 lesions : 52 ( 77.6% ) masses , 12 ( 17.9% ) calcifications , and 3 ( 4.5% ) other types . in contrast to their study , our subjects mammographic images were acquired with 2d full - field digital mammography , and we evaluated not only the reader confidence of lesion type but also confidence of lesion characteristics . in the study by oconnell and kawakyu - oconnor , two radiologists evaluated 36 patients with 37 abnormal mammographic and/or ultrasound categorized as bi - rads 4 or 5 lesions that were evaluated with their prototype dbct system prior to biopsy . their results demonstrated a high degree of correlation between the dbct and digital mammography across a variety of lesion types with 33 of 34 ( 97.1% ) mammographic lesions being scored as equal or better visualized on breast ct relative to diagnostic mammography . we did not include any lesions visible on sonography since we were comparing dbct to diagnostic 2d digital mammography . all ( 100% ) calcification lesions were visualized with dbct in our study ; however , radiologist confidence in calcification characterization was significantly better with digital diagnostic mammography . another benefit of our system is that due to the design of the imaging device even subjects with a large breast size ( ee cup size ) can be imaged without excluding breast tissue . another limitation was that only a small sample size of different lesion types was included in this single site study in which enrolled subjects had undergone a standard of care 2d digital mammographic diagnostic workup prior to being enrolled in this research study . another limitation was that only a small sample size of different lesion types was included in this single site study in which enrolled subjects had undergone a standard of care 2d digital mammographic diagnostic workup prior to being enrolled in this research study . dedicated 3d breast computed tomography is a novel well - tolerated imaging modality that allows for true isotropic , 3d imaging of the breast without mammographic compression . dbct is superior in radiologists visualization and characterization of masses , but inferior to diagnostic mammography for calcifications . technical challenges remain , but 3d dbct may have potential clinical applications in breast cancer screening and diagnosis .

republic of korea does not yet have a universal criterion for disability evaluation . in order to develop a single reasonable and consistent criterion that could be widely accepted across the nation , eleven committees for each medical specialty were recruited under the supervision of korean academy of medical sciences ( kams ) : cardiopulmonary , digestive , endocrine , extremities , genitourinary , nervous system , pediatric development , psychiatric , skin & appearance , special sense , and spine . each committee consisted of about ten specialists . since both disability and developing disability evaluation are social processes in the sense that they are formed and changed by social norms and values , the steering committee developed a series of strategies from the very beginning that would help the evaluation criterion to be accepted as legitimate by majority of society . unfortunately due to the lack of time and budget , however , the strategies could not be fully exercised . we plan to apply our strategies more fully in the next year with more money and time available . this paper introduces this series of strategies in order to share korean experience . as widely acknowledged , in order for a certain impairment to be recognized as a disability , it must constrain a person in a way that substantially limits the activity , especially in relation to employment or education . in other words , a person with same impairment could be disabled or non - disabled , depending on time and space where he or she lives in . for example , substantial leg impairment could be fatal in a pre - modern society , but it is now much less critical in modern society where a variety of transportation tools are available . also under the strong emphasis of the social value on facial appearance disability is the combined product of impairment and diverse social environments , including technology , social norms , and cultural values . because of this social process of disability , we need to consider and even incorporate social norms and values of the current society when we evaluate disability . social values are constantly changing and also vary across societies or even groups within one society . thus , social surroundings of impairment must be recognized as an entity with fractured and diverse aspects rather than one simple universe . considering this social characteristic of disability , the best strategy would be to incorporate diverse and even conflicting values and pressures from the beginning of the evaluation rather than treat them as a separate entity from the evaluation . before we introduce those strategies , however , let me continue the debate on the social process of the evaluation procedure itself . but in many cases , this first step alone is not enough for successful evaluation procedure for two reasons . for any specialty field thus , the best approach might be to bring those unresolved issues to a bigger number of specialists : expanded experts . also , expanded experts are necessary to obtain stronger legitimacy . even if core experts could produce objective and scientific criteria to the full extent , as we discussed above , criteria themselves are social product , and thus , they need to be shared as many social members as possible to gain strong legitimacy and widely adopted by many experts later . second step would be , as we discussed above , to form a team of expanded experts . depending on the magnitude of unresolved grey areas among core experts , the size of expanded experts should be determined . however , more critical issue here is how to recruit the members of expanded experts . the goal of forming core experts is not to obtain a representative sample of experts , but to recruit top experts to build up evaluation criterion . we need a representative sample of experts as much as possible , because unresolved issues among core experts are not of technique but of social value or customs in many cases . especially , considering legitimacy issue that could be critical once the evaluation criteria announced , the representativeness of the expanded experts is essential . usually , there is no simple universal way of obtaining representative sample of medical experts and we will discuss this more later . the last step could be to probe and incorporate public opinions in various active ways . since this last step usually is not considered as a part of evaluation process , we will discuss this later in detail . as widely acknowledged , in order for a certain impairment to be recognized as a disability , it must constrain a person in a way that substantially limits the activity , especially in relation to employment or education . in other words , a person with same impairment could be disabled or non - disabled , depending on time and space where he or she lives in . for example , substantial leg impairment could be fatal in a pre - modern society , but it is now much less critical in modern society where a variety of transportation tools are available . also under the strong emphasis of the social value on facial appearance , some big permanent scars on face could lead to disability . disability is the combined product of impairment and diverse social environments , including technology , social norms , and cultural values . because of this social process of disability , we need to consider and even incorporate social norms and values of the current society when we evaluate disability . social values are constantly changing and also vary across societies or even groups within one society . thus , social surroundings of impairment must be recognized as an entity with fractured and diverse aspects rather than one simple universe . considering this social characteristic of disability , the best strategy would be to incorporate diverse and even conflicting values and pressures from the beginning of the evaluation rather than treat them as a separate entity from the evaluation . the kams guide tried to adopt a series of strategies based on this viewpoint . before we introduce those strategies , however , let me continue the debate on the social process of the evaluation procedure itself . but in many cases , this first step alone is not enough for successful evaluation procedure for two reasons . for any specialty field thus , the best approach might be to bring those unresolved issues to a bigger number of specialists : expanded experts . even if core experts could produce objective and scientific criteria to the full extent , as we discussed above , criteria themselves are social product , and thus , they need to be shared as many social members as possible to gain strong legitimacy and widely adopted by many experts later . second step would be , as we discussed above , to form a team of expanded experts . depending on the magnitude of unresolved grey areas among core experts , the size of expanded experts should be determined . however , more critical issue here is how to recruit the members of expanded experts . the goal of forming core experts is not to obtain a representative sample of experts , but to recruit top experts to build up evaluation criterion . we need a representative sample of experts as much as possible , because unresolved issues among core experts are not of technique but of social value or customs in many cases . especially , considering legitimacy issue that could be critical once the evaluation criteria announced , the representativeness of the expanded experts is essential . usually , there is no simple universal way of obtaining representative sample of medical experts and we will discuss this more later . the last step could be to probe and incorporate public opinions in various active ways . since this last step usually is not considered as a part of evaluation process , we will discuss this later in detail . usually , it could be done by recommendation of top experts or appointment by an official national medical association . once recruited , however , the consensus is the key to the successful evaluation procedure . because of the nature of the evaluation , if possible , consensus instead of majority rule would be much more desirable . reaching a consensus however , it is hard to find a perfect moderator who can balance between every different or even conflicting views . especially , expert committees like this also need an expert of the same field as a moderator , which makes him or her uneasy to act in a neutral way . although consensus usually gives more satisfaction among participants , we sometimes have to rely on voting due to the lack of time . if time permits , small number of experts like ten or so could reach a consensus . but , we do not usually have enough time for consensus regarding every issue . here we suggest a way as an example to reach a consensus with the minimum activity of moderator in a relatively short time . it is called multiperson decision making problem ( mdmp ) model ( 1 - 5 ) . 1 ) opinion expression : each expert expresses his or her own preference on the issue . based on each opinion , exist several alternatives for this measure including variance that was used in economics ( 5 - 8 ) . whatever measure we use , if it exceeds some pre - determined criterion , we can claim that we reach a consensus and all the following procedures are unnecessary . 2 ) proximity measure : if the amount of consensus is not satisfactory , we measure proximity of each opinion from the whole opinion . 3 ) feedback : based on proximity measure , people with proximity score that are furthest from the collective score are recommended to change their opinion so that their opinions become closer to the collective one . 4 ) changing the opinions : based on the feedback , some people change their opinion . 5 ) now , new consensus score is calculated , and it is compared to the criterion . 6 ) if it exceeds the criterion , satisfactory level of consensus is reached and no more action is needed . if not , we will repeat from step 2 ) again until we will have satisfactory amount of consensus . as we discussed above , the critical issue here is to obtain a representative sample of experts in specific medical field to resolve legitimacy issue . the most widely used method was a snow - ball sampling based on core experts as seeds . once we contact those additional experts , we ask the same favor : to enumerate other experts . although this recruitment strategy is convenient and natural way to collect a small number of target people , it usually ends up with strongly biased sample . for example , if we start with male experts , the resultant sample would be male - biased . usually , medical experts must be registered and korea is not an exception . if we can contact experts in a random way from the directory and do a survey with them , it would be a perfect representative sample . however , medical experts , especially in korea , are busiest men due to tight schedule of caring patients . thus , the response rate for survey is usually extremely low , and as result , the resultant final sample is also strongly biased . one possible alternative which we plan to implement is to adopt a respondent driven sampling ( rds ) method that adjusts biasedness introduced during snowball sampling ( 9 - 11 ) . this method is handy in the sense that it is really similar to traditional snowball sampling except we collect some network information of respondents so that we can adjust biasedness of each respondent later . this method is widely adopted since its debut in the 1990s , especially among the studies that targeted for hidden small population such as jazz singers , drug users , men who have sex with men ( msm ) , etc . we conducted a new survey of 400 experts ( doctors ) on their opinion on physical impairment rate for 16 different impairments via faxes and obtained 195 completed cases ( 48.8% response rate ) form september 2008 to january 2009 . although in general experts showed very consistent opinions , some impairments such as ' gastrointestinal fistulas ' and ' complex regional pain syndrome ' exposed substantial variance ( or standard errors ) among expert opinions . for example , response rate for ' complex regional pain syndrome ' ranges from 0% to 70% and also inter - quartile range is 30% , which is relatively large . this finding strongly suggests the necessity of developing strategies for successful consensus making among experts since simple mean could be ineffective and even illegitimate when there exist wide variations of opinions . we need to examine opinion of the public beyond expert group for two reasons . we need to examine public opinion when our decision requires more than one medical special field . even if the evaluation criteria and procedure are scientific and objective , the legitimacy of the criteria could be weak unless they are accepted by the public . especially , considering sharp conflicts between interest groups with regard to disability evaluation , it could be critical to secure public legitimacy . the first one is to conduct a survey of a representative sample of the public . the first approach is most widely used . also if we obtain a majority preference from this survey , it is the best strategy available . but what if we confirm the existence of sharp division , for example 52% vs. 48% ? it would be hard to draw conclusions from this type of neck to neck competing and sometimes even conflicting opinions . this survey is conducted in february 2009 on a 840 representative sample of korean adults through mobile phone text messaging . a survey research company named mbizone keeps the list of more than one million people who registered and agreed to be respondents for the surveys conducted by the company in the exchange of small amount of money . for example , the first question reads , " who is more disabled in korea , ' person who lost both arms ' or ' person who lost both legs ' ? " we coded 1 if a respondent chose the first one and coded 3 if he or she chose the latter one . the closest one is 1.94 for the comparison between ' amputation of one arm ' and ' multi - segmental fusion of the lumbar spine ' . for example , table 1 clearly showed that korean experts believed amputation of arms was more critical than amputation of legs ( 95% confidence intervals are ' 73.89 - 76.73 ' vs. ' 63.71 - 65.95 ' ) . additional table ( not shown ) revealed that only 4.5% of experts believed that amputation of legs was more critical while 70% believed that amputation of arms was more critical . the mean value of ' amputation of arms ' vs. ' amputation of legs ' was 2.15 , which means that public believed that amputation of legs was more critical . according to additional analysis ( not shown ) , the proportion of people who believed that amputation of legs was more critical was 44% while 30% of the respondent believed that amputation of arms was more serious . both findings robustly implicated the need of developing efficient and legitimate procedure of consensus formation . the second alternative to investigate public opinion is to integrate mdmp model with public survey . because mdmp model includes feedback and changing opinions , it is not possible to apply this to huge number of people in person . instead , it is possible to apply this to about one thousand people via mobile phone . once we select a representative sample from mbizone , we can give feedbacks and obtain new changing opinions through its system . considering the fact that mobile phone survey only takes about one day for about 1,000 people , this is a plausible strategy in the future . usually , it could be done by recommendation of top experts or appointment by an official national medical association . once recruited , however , the consensus is the key to the successful evaluation procedure . because of the nature of the evaluation , if possible , consensus instead of majority rule would be much more desirable . reaching a consensus however , it is hard to find a perfect moderator who can balance between every different or even conflicting views . especially , expert committees like this also need an expert of the same field as a moderator , which makes him or her uneasy to act in a neutral way . although consensus usually gives more satisfaction among participants , we sometimes have to rely on voting due to the lack of time . if time permits , small number of experts like ten or so could reach a consensus . but , we do not usually have enough time for consensus regarding every issue . here we suggest a way as an example to reach a consensus with the minimum activity of moderator in a relatively short time . it is called multiperson decision making problem ( mdmp ) model ( 1 - 5 ) . 1 ) opinion expression : each expert expresses his or her own preference on the issue . based on each opinion , exist several alternatives for this measure including variance that was used in economics ( 5 - 8 ) . whatever measure we use , if it exceeds some pre - determined criterion , we can claim that we reach a consensus and all the following procedures are unnecessary . 2 ) proximity measure : if the amount of consensus is not satisfactory , we measure proximity of each opinion from the whole opinion . 3 ) feedback : based on proximity measure , people with proximity score that are furthest from the collective score are recommended to change their opinion so that their opinions become closer to the collective one . 4 ) changing the opinions : based on the feedback , some people change their opinion . 5 ) now , new consensus score is calculated , and it is compared to the criterion . 6 ) if it exceeds the criterion , satisfactory level of consensus is reached and no more action is needed . if not , we will repeat from step 2 ) again until we will have satisfactory amount of consensus . as we discussed above , the critical issue here is to obtain a representative sample of experts in specific medical field to resolve legitimacy issue . the most widely used method was a snow - ball sampling based on core experts as seeds . once we contact those additional experts , we ask the same favor : to enumerate other experts . although this recruitment strategy is convenient and natural way to collect a small number of target people , it usually ends up with strongly biased sample . for example , if we start with male experts , the resultant sample would be male - biased . usually , medical experts must be registered and korea is not an exception . if we can contact experts in a random way from the directory and do a survey with them , it would be a perfect representative sample . however , medical experts , especially in korea , are busiest men due to tight schedule of caring patients . thus , the response rate for survey is usually extremely low , and as result , the resultant final sample is also strongly biased . one possible alternative which we plan to implement is to adopt a respondent driven sampling ( rds ) method that adjusts biasedness introduced during snowball sampling ( 9 - 11 ) . this method is handy in the sense that it is really similar to traditional snowball sampling except we collect some network information of respondents so that we can adjust biasedness of each respondent later . this method is widely adopted since its debut in the 1990s , especially among the studies that targeted for hidden small population such as jazz singers , drug users , men who have sex with men ( msm ) , etc . we conducted a new survey of 400 experts ( doctors ) on their opinion on physical impairment rate for 16 different impairments via faxes and obtained 195 completed cases ( 48.8% response rate ) form september 2008 to january 2009 . although in general experts showed very consistent opinions , some impairments such as ' gastrointestinal fistulas ' and ' complex regional pain syndrome ' exposed substantial variance ( or standard errors ) among expert opinions . for example , response rate for ' complex regional pain syndrome ' ranges from 0% to 70% and also inter - quartile range is 30% , which is relatively large . this finding strongly suggests the necessity of developing strategies for successful consensus making among experts since simple mean could be ineffective and even illegitimate when there exist wide variations of opinions . first , we need to weigh impairments across field . for example , we need to decide relative importance of eyes vs. legs . in this case we need to examine public opinion when our decision requires more than one medical special field . even if the evaluation criteria and procedure are scientific and objective , the legitimacy of the criteria could be weak unless they are accepted by the public . especially , considering sharp conflicts between interest groups with regard to disability evaluation , it could be critical to secure public legitimacy the first one is to conduct a survey of a representative sample of the public . the first approach is most widely used . also if we obtain a majority preference from this survey , it is the best strategy available . but what if we confirm the existence of sharp division , for example 52% vs. 48% ? it would be hard to draw conclusions from this type of neck to neck competing and sometimes even conflicting opinions . this survey is conducted in february 2009 on a 840 representative sample of korean adults through mobile phone text messaging . a survey research company named mbizone keeps the list of more than one million people who registered and agreed to be respondents for the surveys conducted by the company in the exchange of small amount of money . for example , the first question reads , " who is more disabled in korea , ' person who lost both arms ' or ' person who lost both legs ' ? " we coded 1 if a respondent chose the first one and coded 3 if he or she chose the latter one . the closest one is 1.94 for the comparison between ' amputation of one arm ' and ' multi - segmental fusion of the lumbar spine ' . for example , table 1 clearly showed that korean experts believed amputation of arms was more critical than amputation of legs ( 95% confidence intervals are ' 73.89 - 76.73 ' vs. ' 63.71 - 65.95 ' ) . additional table ( not shown ) revealed that only 4.5% of experts believed that amputation of legs was more critical while 70% believed that amputation of arms was more critical . the mean value of ' amputation of arms ' vs. ' amputation of legs ' was 2.15 , which means that public believed that amputation of legs was more critical . according to additional analysis ( not shown ) , the proportion of people who believed that amputation of legs was more critical was 44% while 30% of the respondent believed that amputation of arms was more serious . both findings robustly implicated the need of developing efficient and legitimate procedure of consensus formation . the second alternative to investigate public opinion is to integrate mdmp model with public survey . because mdmp model includes feedback and changing opinions , it is not possible to apply this to huge number of people in person . instead , it is possible to apply this to about one thousand people via mobile phone . once we select a representative sample from mbizone , we can give feedbacks and obtain new changing opinions through its system . considering the fact that mobile phone survey only takes about one day for about 1,000 people , this is a plausible strategy in the future . the kams realized that disability evaluation itself is a social product , therefore , tried to incorporate social property of evaluation into evaluation process from the beginning . due to the lack of budget and time we plan to apply strategy 1 , 2 , or 3 as time permits , since we now enter into the second year period of our project . evaluation guide must change across societies and times but we believe that our set of strategies could be an example of a general framework where opinions of experts and public are systematically examined and thus legitimacy could strongly be held .

the steering committee of the korean academy of medical sciences guideline for physical impairment was fully aware of the social processes of disability evaluation from the beginning and thus , developed a series of strategies to examine and incorporate social property of the evaluation into the evaluation guide . although those strategies could not be implemented to full extent because of lack of budget and time , we believe it worthwhile to share those in this paper as an example of general framework for developing disability evaluation . a series of strategies will be introduced and discussed that views the evaluation process as social per se , and propose a scheme that is designed to obtain growing legitimacy starting from core experts to expanded experts to general public . also preliminary analyses on surveys of public attitude and experts ' opinion with regard to the relative importance of each possible disability revealed the following three facts : 1 ) public had difficulty weighing relative importance of many impairments . 2 ) regarding some impairments including complex regional pain syndrome many doctors had varied opinions . 3 ) public attitude did not always consistent with doctor 's opinion . all these findings strongly suggest the need for developing strategies to draw consensus for legitimate and effective evaluation .

INTRODUCTION Disability and disability evaluation as social processes MATERIALS AND METHODS Consensus among core experts Consensus among expanded experts Public opinion DISCUSSION

republic of korea does not yet have a universal criterion for disability evaluation . in order to develop a single reasonable and consistent criterion that could be widely accepted across the nation , eleven committees for each medical specialty were recruited under the supervision of korean academy of medical sciences ( kams ) : cardiopulmonary , digestive , endocrine , extremities , genitourinary , nervous system , pediatric development , psychiatric , skin & appearance , special sense , and spine . since both disability and developing disability evaluation are social processes in the sense that they are formed and changed by social norms and values , the steering committee developed a series of strategies from the very beginning that would help the evaluation criterion to be accepted as legitimate by majority of society . unfortunately due to the lack of time and budget , however , the strategies could not be fully exercised . we plan to apply our strategies more fully in the next year with more money and time available . this paper introduces this series of strategies in order to share korean experience . as widely acknowledged , in order for a certain impairment to be recognized as a disability , it must constrain a person in a way that substantially limits the activity , especially in relation to employment or education . in other words , a person with same impairment could be disabled or non - disabled , depending on time and space where he or she lives in . for example , substantial leg impairment could be fatal in a pre - modern society , but it is now much less critical in modern society where a variety of transportation tools are available . also under the strong emphasis of the social value on facial appearance disability is the combined product of impairment and diverse social environments , including technology , social norms , and cultural values . because of this social process of disability , we need to consider and even incorporate social norms and values of the current society when we evaluate disability . social values are constantly changing and also vary across societies or even groups within one society . thus , social surroundings of impairment must be recognized as an entity with fractured and diverse aspects rather than one simple universe . considering this social characteristic of disability , the best strategy would be to incorporate diverse and even conflicting values and pressures from the beginning of the evaluation rather than treat them as a separate entity from the evaluation . before we introduce those strategies , however , let me continue the debate on the social process of the evaluation procedure itself . but in many cases , this first step alone is not enough for successful evaluation procedure for two reasons . for any specialty field thus , the best approach might be to bring those unresolved issues to a bigger number of specialists : expanded experts . also , expanded experts are necessary to obtain stronger legitimacy . even if core experts could produce objective and scientific criteria to the full extent , as we discussed above , criteria themselves are social product , and thus , they need to be shared as many social members as possible to gain strong legitimacy and widely adopted by many experts later . second step would be , as we discussed above , to form a team of expanded experts . depending on the magnitude of unresolved grey areas among core experts , the size of expanded experts should be determined . however , more critical issue here is how to recruit the members of expanded experts . the goal of forming core experts is not to obtain a representative sample of experts , but to recruit top experts to build up evaluation criterion . we need a representative sample of experts as much as possible , because unresolved issues among core experts are not of technique but of social value or customs in many cases . especially , considering legitimacy issue that could be critical once the evaluation criteria announced , the representativeness of the expanded experts is essential . usually , there is no simple universal way of obtaining representative sample of medical experts and we will discuss this more later . the last step could be to probe and incorporate public opinions in various active ways . since this last step usually is not considered as a part of evaluation process , we will discuss this later in detail . as widely acknowledged , in order for a certain impairment to be recognized as a disability , it must constrain a person in a way that substantially limits the activity , especially in relation to employment or education . in other words , a person with same impairment could be disabled or non - disabled , depending on time and space where he or she lives in . for example , substantial leg impairment could be fatal in a pre - modern society , but it is now much less critical in modern society where a variety of transportation tools are available . also under the strong emphasis of the social value on facial appearance , some big permanent scars on face could lead to disability . disability is the combined product of impairment and diverse social environments , including technology , social norms , and cultural values . because of this social process of disability , we need to consider and even incorporate social norms and values of the current society when we evaluate disability . social values are constantly changing and also vary across societies or even groups within one society . thus , social surroundings of impairment must be recognized as an entity with fractured and diverse aspects rather than one simple universe . considering this social characteristic of disability , the best strategy would be to incorporate diverse and even conflicting values and pressures from the beginning of the evaluation rather than treat them as a separate entity from the evaluation . the kams guide tried to adopt a series of strategies based on this viewpoint . before we introduce those strategies , however , let me continue the debate on the social process of the evaluation procedure itself . but in many cases , this first step alone is not enough for successful evaluation procedure for two reasons . for any specialty field thus , the best approach might be to bring those unresolved issues to a bigger number of specialists : expanded experts . even if core experts could produce objective and scientific criteria to the full extent , as we discussed above , criteria themselves are social product , and thus , they need to be shared as many social members as possible to gain strong legitimacy and widely adopted by many experts later . second step would be , as we discussed above , to form a team of expanded experts . depending on the magnitude of unresolved grey areas among core experts , the size of expanded experts should be determined . however , more critical issue here is how to recruit the members of expanded experts . the goal of forming core experts is not to obtain a representative sample of experts , but to recruit top experts to build up evaluation criterion . we need a representative sample of experts as much as possible , because unresolved issues among core experts are not of technique but of social value or customs in many cases . especially , considering legitimacy issue that could be critical once the evaluation criteria announced , the representativeness of the expanded experts is essential . usually , there is no simple universal way of obtaining representative sample of medical experts and we will discuss this more later . the last step could be to probe and incorporate public opinions in various active ways . since this last step usually is not considered as a part of evaluation process , we will discuss this later in detail . once recruited , however , the consensus is the key to the successful evaluation procedure . because of the nature of the evaluation , if possible , consensus instead of majority rule would be much more desirable . reaching a consensus however , it is hard to find a perfect moderator who can balance between every different or even conflicting views . especially , expert committees like this also need an expert of the same field as a moderator , which makes him or her uneasy to act in a neutral way . although consensus usually gives more satisfaction among participants , we sometimes have to rely on voting due to the lack of time . if time permits , small number of experts like ten or so could reach a consensus . but , we do not usually have enough time for consensus regarding every issue . here we suggest a way as an example to reach a consensus with the minimum activity of moderator in a relatively short time . 1 ) opinion expression : each expert expresses his or her own preference on the issue . based on each opinion , exist several alternatives for this measure including variance that was used in economics ( 5 - 8 ) . whatever measure we use , if it exceeds some pre - determined criterion , we can claim that we reach a consensus and all the following procedures are unnecessary . 2 ) proximity measure : if the amount of consensus is not satisfactory , we measure proximity of each opinion from the whole opinion . 3 ) feedback : based on proximity measure , people with proximity score that are furthest from the collective score are recommended to change their opinion so that their opinions become closer to the collective one . 5 ) now , new consensus score is calculated , and it is compared to the criterion . 6 ) if it exceeds the criterion , satisfactory level of consensus is reached and no more action is needed . if not , we will repeat from step 2 ) again until we will have satisfactory amount of consensus . as we discussed above , the critical issue here is to obtain a representative sample of experts in specific medical field to resolve legitimacy issue . the most widely used method was a snow - ball sampling based on core experts as seeds . once we contact those additional experts , we ask the same favor : to enumerate other experts . although this recruitment strategy is convenient and natural way to collect a small number of target people , it usually ends up with strongly biased sample . usually , medical experts must be registered and korea is not an exception . if we can contact experts in a random way from the directory and do a survey with them , it would be a perfect representative sample . however , medical experts , especially in korea , are busiest men due to tight schedule of caring patients . thus , the response rate for survey is usually extremely low , and as result , the resultant final sample is also strongly biased . one possible alternative which we plan to implement is to adopt a respondent driven sampling ( rds ) method that adjusts biasedness introduced during snowball sampling ( 9 - 11 ) . this method is handy in the sense that it is really similar to traditional snowball sampling except we collect some network information of respondents so that we can adjust biasedness of each respondent later . this method is widely adopted since its debut in the 1990s , especially among the studies that targeted for hidden small population such as jazz singers , drug users , men who have sex with men ( msm ) , etc . we conducted a new survey of 400 experts ( doctors ) on their opinion on physical impairment rate for 16 different impairments via faxes and obtained 195 completed cases ( 48.8% response rate ) form september 2008 to january 2009 . although in general experts showed very consistent opinions , some impairments such as ' gastrointestinal fistulas ' and ' complex regional pain syndrome ' exposed substantial variance ( or standard errors ) among expert opinions . for example , response rate for ' complex regional pain syndrome ' ranges from 0% to 70% and also inter - quartile range is 30% , which is relatively large . this finding strongly suggests the necessity of developing strategies for successful consensus making among experts since simple mean could be ineffective and even illegitimate when there exist wide variations of opinions . we need to examine opinion of the public beyond expert group for two reasons . we need to examine public opinion when our decision requires more than one medical special field . even if the evaluation criteria and procedure are scientific and objective , the legitimacy of the criteria could be weak unless they are accepted by the public . especially , considering sharp conflicts between interest groups with regard to disability evaluation , it could be critical to secure public legitimacy . the first one is to conduct a survey of a representative sample of the public . the first approach is most widely used . it would be hard to draw conclusions from this type of neck to neck competing and sometimes even conflicting opinions . this survey is conducted in february 2009 on a 840 representative sample of korean adults through mobile phone text messaging . a survey research company named mbizone keeps the list of more than one million people who registered and agreed to be respondents for the surveys conducted by the company in the exchange of small amount of money . for example , the first question reads , " who is more disabled in korea , ' person who lost both arms ' or ' person who lost both legs ' ? " the closest one is 1.94 for the comparison between ' amputation of one arm ' and ' multi - segmental fusion of the lumbar spine ' . for example , table 1 clearly showed that korean experts believed amputation of arms was more critical than amputation of legs ( 95% confidence intervals are ' 73.89 - 76.73 ' vs. ' 63.71 - 65.95 ' ) . additional table ( not shown ) revealed that only 4.5% of experts believed that amputation of legs was more critical while 70% believed that amputation of arms was more critical . the mean value of ' amputation of arms ' vs. ' amputation of legs ' was 2.15 , which means that public believed that amputation of legs was more critical . according to additional analysis ( not shown ) , the proportion of people who believed that amputation of legs was more critical was 44% while 30% of the respondent believed that amputation of arms was more serious . both findings robustly implicated the need of developing efficient and legitimate procedure of consensus formation . because mdmp model includes feedback and changing opinions , it is not possible to apply this to huge number of people in person . instead , it is possible to apply this to about one thousand people via mobile phone . once we select a representative sample from mbizone , we can give feedbacks and obtain new changing opinions through its system . considering the fact that mobile phone survey only takes about one day for about 1,000 people , this is a plausible strategy in the future . usually , it could be done by recommendation of top experts or appointment by an official national medical association . once recruited , however , the consensus is the key to the successful evaluation procedure . because of the nature of the evaluation , if possible , consensus instead of majority rule would be much more desirable . especially , expert committees like this also need an expert of the same field as a moderator , which makes him or her uneasy to act in a neutral way . although consensus usually gives more satisfaction among participants , we sometimes have to rely on voting due to the lack of time . if time permits , small number of experts like ten or so could reach a consensus . but , we do not usually have enough time for consensus regarding every issue . here we suggest a way as an example to reach a consensus with the minimum activity of moderator in a relatively short time . it is called multiperson decision making problem ( mdmp ) model ( 1 - 5 ) . 1 ) opinion expression : each expert expresses his or her own preference on the issue . based on each opinion , exist several alternatives for this measure including variance that was used in economics ( 5 - 8 ) . whatever measure we use , if it exceeds some pre - determined criterion , we can claim that we reach a consensus and all the following procedures are unnecessary . 2 ) proximity measure : if the amount of consensus is not satisfactory , we measure proximity of each opinion from the whole opinion . 3 ) feedback : based on proximity measure , people with proximity score that are furthest from the collective score are recommended to change their opinion so that their opinions become closer to the collective one . 4 ) changing the opinions : based on the feedback , some people change their opinion . 5 ) now , new consensus score is calculated , and it is compared to the criterion . if not , we will repeat from step 2 ) again until we will have satisfactory amount of consensus . as we discussed above , the critical issue here is to obtain a representative sample of experts in specific medical field to resolve legitimacy issue . the most widely used method was a snow - ball sampling based on core experts as seeds . once we contact those additional experts , we ask the same favor : to enumerate other experts . although this recruitment strategy is convenient and natural way to collect a small number of target people , it usually ends up with strongly biased sample . if we can contact experts in a random way from the directory and do a survey with them , it would be a perfect representative sample . however , medical experts , especially in korea , are busiest men due to tight schedule of caring patients . thus , the response rate for survey is usually extremely low , and as result , the resultant final sample is also strongly biased . one possible alternative which we plan to implement is to adopt a respondent driven sampling ( rds ) method that adjusts biasedness introduced during snowball sampling ( 9 - 11 ) . this method is handy in the sense that it is really similar to traditional snowball sampling except we collect some network information of respondents so that we can adjust biasedness of each respondent later . we conducted a new survey of 400 experts ( doctors ) on their opinion on physical impairment rate for 16 different impairments via faxes and obtained 195 completed cases ( 48.8% response rate ) form september 2008 to january 2009 . although in general experts showed very consistent opinions , some impairments such as ' gastrointestinal fistulas ' and ' complex regional pain syndrome ' exposed substantial variance ( or standard errors ) among expert opinions . for example , response rate for ' complex regional pain syndrome ' ranges from 0% to 70% and also inter - quartile range is 30% , which is relatively large . this finding strongly suggests the necessity of developing strategies for successful consensus making among experts since simple mean could be ineffective and even illegitimate when there exist wide variations of opinions . first , we need to weigh impairments across field . for example , we need to decide relative importance of eyes vs. legs . in this case we need to examine public opinion when our decision requires more than one medical special field . even if the evaluation criteria and procedure are scientific and objective , the legitimacy of the criteria could be weak unless they are accepted by the public . especially , considering sharp conflicts between interest groups with regard to disability evaluation , it could be critical to secure public legitimacy the first one is to conduct a survey of a representative sample of the public . but what if we confirm the existence of sharp division , for example 52% vs. 48% ? it would be hard to draw conclusions from this type of neck to neck competing and sometimes even conflicting opinions . a survey research company named mbizone keeps the list of more than one million people who registered and agreed to be respondents for the surveys conducted by the company in the exchange of small amount of money . for example , the first question reads , " who is more disabled in korea , ' person who lost both arms ' or ' person who lost both legs ' ? " the closest one is 1.94 for the comparison between ' amputation of one arm ' and ' multi - segmental fusion of the lumbar spine ' . for example , table 1 clearly showed that korean experts believed amputation of arms was more critical than amputation of legs ( 95% confidence intervals are ' 73.89 - 76.73 ' vs. ' 63.71 - 65.95 ' ) . the mean value of ' amputation of arms ' vs. ' amputation of legs ' was 2.15 , which means that public believed that amputation of legs was more critical . according to additional analysis ( not shown ) , the proportion of people who believed that amputation of legs was more critical was 44% while 30% of the respondent believed that amputation of arms was more serious . both findings robustly implicated the need of developing efficient and legitimate procedure of consensus formation . the second alternative to investigate public opinion is to integrate mdmp model with public survey . instead , it is possible to apply this to about one thousand people via mobile phone . once we select a representative sample from mbizone , we can give feedbacks and obtain new changing opinions through its system . the kams realized that disability evaluation itself is a social product , therefore , tried to incorporate social property of evaluation into evaluation process from the beginning . due to the lack of budget and time we plan to apply strategy 1 , 2 , or 3 as time permits , since we now enter into the second year period of our project . evaluation guide must change across societies and times but we believe that our set of strategies could be an example of a general framework where opinions of experts and public are systematically examined and thus legitimacy could strongly be held .

infections of the skin , particularly those caused by resistant pathogens , represent a clinical challenge to address . acute bacterial skin and skin structure infections ( absssi ) are a new classification of skin and skin structure infections ( sssis ) in accordance with health canada , the us food and drug administration , and the european medicines commission . absssi include cellulitis / erysipelas , wound infection , and major cutaneous abscess with a minimum lesion surface area of ~75 cm.1 absssi is one of several classifications of skin infections ( skin and soft tissue infection [ ssti ] , complicated sssi [ csssi ] , and complicated ssti [ cssti ] ) . a key distinction of absssi from other classifications is that it excludes less serious skin infections as well as infections needing more complex treatment regimens . as absssi is a recent classification , limited literature exists to inform the burden of illness for this patient population . however , several authors have reported data for ssti , sssi , and cssti , and , although these reports are for skin infections with a broader population base than absssi alone , the trends are informative for absssi patients . sstis are most frequently caused by gram - positive pathogens , with methicillin - resistant staphylococcus aureus ( mrsa ) being increasingly the predominant pathogen in the usa and europe.25 linked to the growing prevalence of mrsa , rates of hospitalization for ssti have increased in the last decade in the usa.6,7 reports from canada also highlight the increasing prevalence of ssti due to mrsa in outpatients , emergency departments ( ed ) , and hospitals.813 in a study of visits to the ed for ssti , the prevalence of mrsa varied significantly across the canadian provinces ( 11%100% ) , with a higher prevalence in western canada.12 data from the canadian nosocomial infection surveillance program from 1995 to 2007 showed that hospitalization for ssti caused by mrsa increased from 24% to 37% between 1994 and 2007.13 surveillance reports have also documented increased isolation of mrsa from hospitalized patients with ssti in north america , although rates were lower in canada than in the usa.2,14 the involvement of mrsa in skin infections adversely affects patient outcomes . skin infections caused by mrsa are associated with increased instances of treatment failure,1517 disease recurrence,16,18 increased risk of invasive disease,19 and higher mortality.15,2022 mrsa - positive skin infections also have a significant economic impact because of increased durations of hospitalization15,22,23 and higher hospital costs.15 real - world data on cost drivers in patients with skin infections due to mrsa are important , so that health care can be delivered effectively and efficiently in an increasingly cost - constrained environment . hospitalization is the major cost driver associated with mrsa infection.24,25 hospital length of stay ( los ) is an important outcomes measure for patients with cssti due to mrsa , as the majority of medical costs for this condition are related to hospital ward and intensive care unit ( icu ) stays.26,27 although ssti are among the most common infections necessitating hospitalization,6 in general , there is limited countrywide information on hospitalizations for patients with skin infections caused by mrsa . a retrospective medical chart review of patients hospitalized with cssti between 2010 and 2011 in 12 european countries showed a mean hospital los of 20.6 days.28 in the usa , a retrospective multicenter observational registry of patients with mrsa skin infections treated with daptomycin showed median antibiotic - related los of 48 days , depending on age and comorbidities.29 another large us database study that used 4-year hospital discharge data from the solucient database documented mean and median los for cssti due to mrsa of 12.6 ( standard deviation [ sd ] : 18.9 ) days and 7.0 days , respectively.21 in patients hospitalized with cssti due to infection with s. aureus , healthcare cost and utilization project ( hcup ) data for 2009 showed an los of 7.3 days,7 and another us study using the premier perspective database reported an average hospital los of 6.1 days for the 20052006 period.30 canadian data on absssi due to mrsa are limited . in 2003 , a small study of 89 patients with ssti due to mrsa reported a mean hospital los of 28.9 ( sd : 29.8 ) days in patients treated with intravenous ( iv ) vancomycin.31 a large retrospective database study conducted over a 5-year period ( 2004 through 2008 inclusive ) in ~65,000 patients hospitalized for cellulitis ( not mrsa - specific ) in canada found a mean hospital los of 7.1 ( sd : 8.2 ) days ; 29.5% of patients admitted for cellulitis experienced a prolonged los in hospital ( > 7 days).32 to narrow the information gap for canada , we conducted a retrospective database analysis to characterize a cohort of patients hospitalized with absssi due to mrsa across multiple provinces ( alberta , british columbia , manitoba , newfoundland , new brunswick , nova scotia , ontario , prince edward island , and saskatchewan ) grouped into four geographic regions . demographic and clinical features , los , treatment patterns , and time trends were analyzed . the dad is a national database containing information primarily related to hospital inpatient events ; the records represent 75% of all patient discharges in canada ( not including quebec ) . the dad collects , processes , and analyzes all discharges from acute care inpatient facilities , day surgery procedures , long - term care , and rehabilitation and psychiatry facilities , and contains data from 1979 to 2014 . the database includes demographic information , calculated los , admission and discharge dates , most responsible diagnosis ( this refers to the condition responsible for the longest los [ icd-10-ca coding system ] ) , up to ten secondary diagnoses ( secondary diagnoses included codes for preexisting , post - admitting , and admitting diagnoses ) , and diagnostic and therapeutic procedures ( canadian classification of interventions [ cci ] coding system ) . information within the dad is de - identified and maintains full compliance with the health insurance portability and accountability act regulations ; therefore , because this study was a retrospective database study using de - identified patient information , it is exempt from irb approval per regulatory guidance . patients were included if they were 18 years of age and older and were hospitalized between january 1 , 2008 and december 31 , 2014 , with icd-10-ca diagnosis codes consistent with absssi due to mrsa diagnosis ( see table s1 for absssi codes ) . the icd-10-ca codes selected were consistent with the codes used for the three absssi infection types described in the absssi fda1 definition and were cross - validated with those used in a retrospective population - based study in canada.32 they were also checked for consistency by medical personnel and canadian institute for health information ( cihi ) analysts . indications that are not part of the absssi domains with codes indicative of a more chronic condition were excluded from the analysis . of note , although there was no way of obtaining the size of the lesion , the approach described earlier was an efficient way to identify the absssi patient population in the absence of clinical data . the patient was assumed to have absssi caused by mrsa if both codes b95.6 and u82.1 were present in the hospital record , in addition to codes for absssi . data from patients initial ( or index ) absssi due to mrsa hospitalization were analyzed . to create an operational definition that represented acute and newly diagnosed disease , patients were excluded if their icd-10-ca codes indicated hospitalization for mrsa infection during the 30-day period preceding the index hospitalization . to reduce the potential for confounding due to coinfection , patients were excluded if they had any of the following coexisting conditions : pneumonia , septic ( pyogenic ) arthritis , neutropenia , bacteremia , and intra - abdominal infections , including appendicitis , diverticular disease with perforation and abscess , peritonitis , and cholecystitis , as identified by the icd-10-ca diagnosis codes ( any position ) on the index hospitalization record . patients were categorized into two subgroups depending on whether absssi due to mrsa was the primary or the secondary diagnosis . patients were assigned to the primary diagnosis group if they had at least one icd-10-ca code indicating either absssi or mrsa as the most responsible diagnosis on their hospital claim . patients were assigned to the secondary diagnosis group if the icd-10-ca codes were present for both absssi and mrsa as a secondary diagnosis and if there was at least one code for another condition or surgery coded as the most responsible diagnosis . demographic parameters analyzed were age and age - group ( both as of the index hospitalization ) , age distribution , sex , year of index absssi due to mrsa hospitalization , type of admission ( emergent or elective ) , and the responsible payer for hospital charges ( resident province / territory , other province / territory , canadian resident self - pay , other countries resident self - pay , and other ) . clinical characteristics included the absssi caused by mrsa subtype ( cellulitis / erysipelas , major or deep abscess , wound infection , and other [ pyoderma , pyoderma gangrenosum , and pyogenic granuloma ] ) , anatomical site of infection ( limb , trunk , head / neck / face , or multiple ) , major comorbid conditions ( these included important prevalent conditions that could impact medical resource use ) , and number of comorbidities . the main outcome measure was los for the index absssi due to mrsa admission , which was calculated from dates of admission and discharge on the hospital record . secondary outcomes were los in a specialty care facility ( nursing homes , residences for senior citizens , and chronic and long - term care and related facilities ) , main patient service ( general medicine , dermatology , general surgery , other surgery , infectious diseases ) that contributed to the longest portion of the patient s los , discharge status ( died in hospital , discharged to home , continuing care [ defined as ongoing care delivered to patients not ready for hospital discharge , either in a separate facility or co located with acute services in the hospital ] ) , administration of antibiotics , and surgical procedures ( incision , drainage , surgical debridement , wound excision , and amputation ) . an exploratory analysis of time trends for los descriptive statistics were used to summarize baseline patient characteristics , treatment patterns , and study outcomes , including los for each patient subgroup . mean , sd , median , interquartile range , and minimum and maximum values were reported for continuous variables . analyses were separately conducted for four specific regions of canada ( british columbia / alberta , ontario , canadian prairie [ manitoba and saskatchewan ] , and atlantic canada [ newfoundland , new brunswick , nova scotia , and prince edward island ] ) . analyses of los , use of surgical procedures , and main patient service utilization were performed according to the hospitalization year to examine time trends . the dad is a national database containing information primarily related to hospital inpatient events ; the records represent 75% of all patient discharges in canada ( not including quebec ) . the dad collects , processes , and analyzes all discharges from acute care inpatient facilities , day surgery procedures , long - term care , and rehabilitation and psychiatry facilities , and contains data from 1979 to 2014 . the database includes demographic information , calculated los , admission and discharge dates , most responsible diagnosis ( this refers to the condition responsible for the longest los [ icd-10-ca coding system ] ) , up to ten secondary diagnoses ( secondary diagnoses included codes for preexisting , post - admitting , and admitting diagnoses ) , and diagnostic and therapeutic procedures ( canadian classification of interventions [ cci ] coding system ) . information within the dad is de - identified and maintains full compliance with the health insurance portability and accountability act regulations ; therefore , because this study was a retrospective database study using de - identified patient information , it is exempt from irb approval per regulatory guidance . patients were included if they were 18 years of age and older and were hospitalized between january 1 , 2008 and december 31 , 2014 , with icd-10-ca diagnosis codes consistent with absssi due to mrsa diagnosis ( see table s1 for absssi codes ) . the icd-10-ca codes selected were consistent with the codes used for the three absssi infection types described in the absssi fda1 definition and were cross - validated with those used in a retrospective population - based study in canada.32 they were also checked for consistency by medical personnel and canadian institute for health information ( cihi ) analysts . indications that are not part of the absssi domains with codes indicative of a more chronic condition were excluded from the analysis . of note , although there was no way of obtaining the size of the lesion , the approach described earlier was an efficient way to identify the absssi patient population in the absence of clinical data . the patient was assumed to have absssi caused by mrsa if both codes b95.6 and u82.1 were present in the hospital record , in addition to codes for absssi . data from patients initial ( or index ) absssi due to mrsa hospitalization were analyzed . to create an operational definition that represented acute and newly diagnosed disease , patients were excluded if their icd-10-ca codes indicated hospitalization for mrsa infection during the 30-day period preceding the index hospitalization . to reduce the potential for confounding due to coinfection , patients were excluded if they had any of the following coexisting conditions : pneumonia , septic ( pyogenic ) arthritis , neutropenia , bacteremia , and intra - abdominal infections , including appendicitis , diverticular disease with perforation and abscess , peritonitis , and cholecystitis , as identified by the icd-10-ca diagnosis codes ( any position ) on the index hospitalization record . patients were categorized into two subgroups depending on whether absssi due to mrsa was the primary or the secondary diagnosis . patients were assigned to the primary diagnosis group if they had at least one icd-10-ca code indicating either absssi or mrsa as the most responsible diagnosis on their hospital claim . patients were assigned to the secondary diagnosis group if the icd-10-ca codes were present for both absssi and mrsa as a secondary diagnosis and if there was at least one code for another condition or surgery coded as the most responsible diagnosis . demographic parameters analyzed were age and age - group ( both as of the index hospitalization ) , age distribution , sex , year of index absssi due to mrsa hospitalization , type of admission ( emergent or elective ) , and the responsible payer for hospital charges ( resident province / territory , other province / territory , canadian resident self - pay , other countries resident self - pay , and other ) . clinical characteristics included the absssi caused by mrsa subtype ( cellulitis / erysipelas , major or deep abscess , wound infection , and other [ pyoderma , pyoderma gangrenosum , and pyogenic granuloma ] ) , anatomical site of infection ( limb , trunk , head / neck / face , or multiple ) , major comorbid conditions ( these included important prevalent conditions that could impact medical resource use ) , and number of comorbidities . the main outcome measure was los for the index absssi due to mrsa admission , which was calculated from dates of admission and discharge on the hospital record . secondary outcomes were los in a specialty care facility ( nursing homes , residences for senior citizens , and chronic and long - term care and related facilities ) , main patient service ( general medicine , dermatology , general surgery , other surgery , infectious diseases ) that contributed to the longest portion of the patient s los , discharge status ( died in hospital , discharged to home , continuing care [ defined as ongoing care delivered to patients not ready for hospital discharge , either in a separate facility or co located with acute services in the hospital ] ) , administration of antibiotics , and surgical procedures ( incision , drainage , surgical debridement , wound excision , and amputation ) . descriptive statistics were used to summarize baseline patient characteristics , treatment patterns , and study outcomes , including los for each patient subgroup . mean , sd , median , interquartile range , and minimum and maximum values analyses were separately conducted for four specific regions of canada ( british columbia / alberta , ontario , canadian prairie [ manitoba and saskatchewan ] , and atlantic canada [ newfoundland , new brunswick , nova scotia , and prince edward island ] ) . analyses of los , use of surgical procedures , and main patient service utilization were performed according to the hospitalization year to examine time trends . figure 1 shows the selection of patients from the database . among the 6,719 patients included in the study , 3,273 ( 48.7% ) had absssi due to mrsa as the primary reason for the hospital stay and 3,446 ( 51.3% ) had absssi due to mrsa as the secondary reason for the hospital stay . tables 13 summarize the demographic , clinical , and hospitalization characteristics of patients across all the geographic regions . the mean age of patients hospitalized with a primary diagnosis ranged from 49 to 55 years , while the mean age for patients with a secondary diagnosis ranged from 55 to 67 years , indicating that patients with a secondary diagnosis were older than those with a primary diagnosis ( table 1 ) . for all provinces except atlantic canada , patients aged 55 years and older were more likely to experience absssi as a result of mrsa as a secondary diagnosis . in atlantic canada , there were 446 vs 173 patients hospitalized with a secondary vs primary diagnosis , respectively . in specifically analyzing patients aged 65 years or older from atlantic canada , there was an even higher ratio , over five times , of secondary vs primary hospitalizations ( table 1 ) . overall , the majority of hospitalizations were related to cellulitis / erysipelas ( 32.8% to 63.4% ) followed by abscess ( 16.6% to 41.9% ) , and other subtypes ( 2.1% to 49.7% ; table 2 ) . cellulitis / erysipelas was the most frequent type of infection for patients with a primary diagnosis across all provinces ( 55.8% to 63.4% ) and for patients with a secondary diagnosis in ontario , alberta / british columbia , and the canadian prairie ( 47.3% to 58.0% ) . in all provinces , infections categorized as occurred more frequently among patients with a secondary diagnosis than among those with a primary diagnosis . in the total cohort , the majority of infections involved a limb ( 62.7% to 73.8% ) , followed by the trunk ( 16.6% to 26.8% ) . the most common comorbid conditions were diabetes mellitus ( 42.5% to 72.2% ) , renal disease ( 9.1% to 21.5% ) , and congestive heart failure ( 5.1% to 17.4% ) . the distribution of comorbidity burden differed slightly depending on whether the patient had a primary or secondary diagnosis . the majority of the patients across the samples had zero or one comorbidity ; however , among patients with a secondary diagnosis , a higher proportion of patients had two or more comorbidities . the number and percentage of patients with a primary or secondary diagnosis appeared to be relatively consistent from 2008 to 2014 ; the only exception was atlantic canada , where the proportion of patients with a secondary diagnosis decreased from 31.6% in 2008 to 5.8% in 2014 ( table 3 ) . the majority of hospitalizations were emergent ( 96.0% to 99.4% for patients with a primary diagnosis and 89.7% to 93.6% for patients with a secondary diagnosis ) ( table 3 ) . of the 365 patients with elective hospital admissions , a majority had a secondary diagnosis ranging from 79.1% in ontario to 88.9% in canadian prairie . more than 90% of the hospitalization costs were paid by the resident province ( table 3 ) . hospitalization outcomes for the entire study period ( 20082014 ) are shown in table 4 and for the most recent year ( 2014 ) are shown in table 5 . the mean los over the study period varied according to the geographic region , from 9 to 18 days for primary diagnosis and from 25 to 43 days for secondary diagnosis ( table 4 ) . in the final year of the study ( 2014 ) , los ranged from 7.7 to 13.4 days for a primary diagnosis and from 18.2 to 25.2 days for a secondary diagnosis . los in a specialty care facility varied by geographical region , ranging from 83 to 157 hours for patients with a primary diagnosis and from 153 to 203 hours for patients with a secondary diagnosis . over the entire study period , the majority of hospitalized patients were discharged to their residence ; rates of discharge to home were higher among patients with a primary diagnosis compared with patients with a secondary diagnosis ( 77.7% to 82.7% vs 56.8% to 64.5% , respectively ) . absssi due to mrsa as a secondary diagnosis was associated with higher in - hospital mortality rates ( 7.2% to 12.1% ) than infections identified as a primary diagnosis ( 0.4% to 1.6% ) . the proportion of patients receiving continuing care was also higher among patients with a secondary diagnosis ( 10.6% to 24.2% ) than those with a primary diagnosis ( 4.6% to 12.1% ; table 4 ) . the most common procedures overall and over the study period were drainage ( 15.6% to 43.3% ) , followed by surgical debridement ( 15.2% to 44.9% ) , incision ( 11.1% to 36.9% ) , amputation ( 2.3% to 25.3% ) , and excision of wound ( 2.3% to 4.9% ) . drainage was the most common surgical procedure in primary diagnosis patients in 2014 ( 20% to 42.4% ) and over the study period ( 34.5% to 43.3% ) , whereas among patients with a secondary diagnosis , surgical debridement was most common across all provinces ( 26.4% to 44.9% ) over the study period and in all provinces other than ontario in 2014 . amputations were more common among patients with absssi due to mrsa as a secondary diagnosis ( 21.0% to 25.3% ) , than patients with a primary diagnosis ( 2.3% to 10.2% ) . in patients with a primary diagnosis of absssi due to mrsa and who were hospitalized in either ontario or atlantic canada , there was a trend toward decreasing overall mean hospital los from 2008 ( 11.5 days in ontario , 14.0 days in atlantic canada ) to 2014 ( 7.7 days in ontario , 8.9 days in atlantic canada ) , with some variability in the intermittent years ( tables s2 and s3 ) . mean hospital los in the alberta / british columbia and canadian prairie regions was more consistent from 2008 to 2014 , with variability observed in the interim period ( tables s4 and s5 ) . in patients with a secondary diagnosis across all regions , there was a trend of decreasing mean hospital los from 2008 to 2014 , with variability observed in the intermittent years . there was a decrease of 13 days in ontario ( from 31.5 to 18.2 days ; table s2 ) , 12 days in alberta / british columbia ( from 37.1 to 25.0 days ; table s4 ) , 30 days in canadian prairie ( from 48.5 to 18.3 days ; table s5 ) , and 22 days in atlantic canada ( from 47.6 to 25.2 days ; table s3 ) . figure 1 shows the selection of patients from the database . among the 6,719 patients included in the study , 3,273 ( 48.7% ) had absssi due to mrsa as the primary reason for the hospital stay and 3,446 ( 51.3% ) had absssi due to mrsa as the secondary reason for the hospital stay . tables 13 summarize the demographic , clinical , and hospitalization characteristics of patients across all the geographic regions . the mean age of patients hospitalized with a primary diagnosis ranged from 49 to 55 years , while the mean age for patients with a secondary diagnosis ranged from 55 to 67 years , indicating that patients with a secondary diagnosis were older than those with a primary diagnosis ( table 1 ) . for all provinces except atlantic canada , patients aged 55 years and older were more likely to experience absssi as a result of mrsa as a secondary diagnosis . in atlantic canada , there were 446 vs 173 patients hospitalized with a secondary vs primary diagnosis , respectively . in specifically analyzing patients aged 65 years or older from atlantic canada , there was an even higher ratio , over five times , of secondary vs primary hospitalizations ( table 1 ) . overall , the majority of hospitalizations were related to cellulitis / erysipelas ( 32.8% to 63.4% ) followed by abscess ( 16.6% to 41.9% ) , and other subtypes ( 2.1% to 49.7% ; table 2 ) . cellulitis / erysipelas was the most frequent type of infection for patients with a primary diagnosis across all provinces ( 55.8% to 63.4% ) and for patients with a secondary diagnosis in ontario , alberta / british columbia , and the canadian prairie ( 47.3% to 58.0% ) . in all provinces , infections categorized as occurred more frequently among patients with a secondary diagnosis than among those with a primary diagnosis . in the total cohort , the majority of infections involved a limb ( 62.7% to 73.8% ) , followed by the trunk ( 16.6% to 26.8% ) . the most common comorbid conditions were diabetes mellitus ( 42.5% to 72.2% ) , renal disease ( 9.1% to 21.5% ) , and congestive heart failure ( 5.1% to 17.4% ) . the distribution of comorbidity burden differed slightly depending on whether the patient had a primary or secondary diagnosis . the majority of the patients across the samples had zero or one comorbidity ; however , among patients with a secondary diagnosis , a higher proportion of patients had two or more comorbidities . the number and percentage of patients with a primary or secondary diagnosis appeared to be relatively consistent from 2008 to 2014 ; the only exception was atlantic canada , where the proportion of patients with a secondary diagnosis decreased from 31.6% in 2008 to 5.8% in 2014 ( table 3 ) . the majority of hospitalizations were emergent ( 96.0% to 99.4% for patients with a primary diagnosis and 89.7% to 93.6% for patients with a secondary diagnosis ) ( table 3 ) . of the 365 patients with elective hospital admissions , a majority had a secondary diagnosis ranging from 79.1% in ontario to 88.9% in canadian prairie . more than 90% of the hospitalization costs were paid by the resident province ( table 3 ) . hospitalization outcomes for the entire study period ( 20082014 ) are shown in table 4 and for the most recent year ( 2014 ) are shown in table 5 . the mean los over the study period varied according to the geographic region , from 9 to 18 days for primary diagnosis and from 25 to 43 days for secondary diagnosis ( table 4 ) . in the final year of the study ( 2014 ) , los ranged from 7.7 to 13.4 days for a primary diagnosis and from 18.2 to 25.2 days for a secondary diagnosis . los in a specialty care facility varied by geographical region , ranging from 83 to 157 hours for patients with a primary diagnosis and from 153 to 203 hours for patients with a secondary diagnosis . over the entire study period , the majority of hospitalized patients were discharged to their residence ; rates of discharge to home were higher among patients with a primary diagnosis compared with patients with a secondary diagnosis ( 77.7% to 82.7% vs 56.8% to 64.5% , respectively ) . absssi due to mrsa as a secondary diagnosis was associated with higher in - hospital mortality rates ( 7.2% to 12.1% ) than infections identified as a primary diagnosis ( 0.4% to 1.6% ) . the proportion of patients receiving continuing care was also higher among patients with a secondary diagnosis ( 10.6% to 24.2% ) than those with a primary diagnosis ( 4.6% to 12.1% ; table 4 ) . the most common procedures overall and over the study period were drainage ( 15.6% to 43.3% ) , followed by surgical debridement ( 15.2% to 44.9% ) , incision ( 11.1% to 36.9% ) , amputation ( 2.3% to 25.3% ) , and excision of wound ( 2.3% to 4.9% ) . drainage was the most common surgical procedure in primary diagnosis patients in 2014 ( 20% to 42.4% ) and over the study period ( 34.5% to 43.3% ) , whereas among patients with a secondary diagnosis , surgical debridement was most common across all provinces ( 26.4% to 44.9% ) over the study period and in all provinces other than ontario in 2014 . amputations were more common among patients with absssi due to mrsa as a secondary diagnosis ( 21.0% to 25.3% ) , than patients with a primary diagnosis ( 2.3% to 10.2% ) . in patients with a primary diagnosis of absssi due to mrsa and who were hospitalized in either ontario or atlantic canada , there was a trend toward decreasing overall mean hospital los from 2008 ( 11.5 days in ontario , 14.0 days in atlantic canada ) to 2014 ( 7.7 days in ontario , 8.9 days in atlantic canada ) , with some variability in the intermittent years ( tables s2 and s3 ) . mean hospital los in the alberta / british columbia and canadian prairie regions was more consistent from 2008 to 2014 , with variability observed in the interim period ( tables s4 and s5 ) . in patients with a secondary diagnosis across all regions , there was a trend of decreasing mean hospital los from 2008 to 2014 , with variability observed in the intermittent years . there was a decrease of 13 days in ontario ( from 31.5 to 18.2 days ; table s2 ) , 12 days in alberta / british columbia ( from 37.1 to 25.0 days ; table s4 ) , 30 days in canadian prairie ( from 48.5 to 18.3 days ; table s5 ) , and 22 days in atlantic canada ( from 47.6 to 25.2 days ; table s3 ) . this is the first study to examine hospital los and time trends of hospital los , using data as recent as 2014 , for absssi due to mrsa across multiple geographic regions in canada , excluding quebec . in addition , this study is the first to use an algorithm to distinguish patients based on their primary reason for hospitalization and quantify results based on whether patients were hospitalized primarily for absssi due to mrsa or another condition . the majority of patients with a primary diagnosis of absssi caused by mrsa were younger ( 18 to 44 years ) and had fewer comorbid conditions than those with a secondary diagnosis . however , patients aged 65 years or older across all provinces were more likely to be hospitalized with absssi due to mrsa as a secondary diagnosis compared with a primary diagnosis . this could imply that those in the secondary diagnosis group might have acquired the infection during hospitalization or that other conditions might have motivated the admission , but absssi due to mrsa was also present at the time of admission . because medical chart data were not available to detail the sequence of events , this can not be confirmed using the dad database . regardless of the exact scenarios , hospital los for this patient group was likely influenced by comorbidities in addition to the absssi caused by mrsa diagnosis itself . however , we did observe that those with a primary diagnosis generally had a shorter los , lower mortality and need for continued care , higher discharge rates , and higher rates of discharge to home than patients with a secondary diagnosis . analysis across the regions showed that the most common infection types were cellulitis followed by abscess . common treatments vary by infection type and some may require more hospital resources than others . for example , an abscess requires incision and drainage , whereas cellulitis / erysipelas might require emergent surgical debridement in cases of severe infections in which a necrotizing process is suspected.33 these procedures were observed in this study , with incision and drainage occurring more frequently in patients with a primary infection and surgical debridement occurring more frequently in patients with a secondary infection . additional research regarding the association between infection type , specific resource utilization , and related micro - costing could be useful in evaluating the total cost of care . baibergenova et al evaluated factors associated with prolonged los in patients hospitalized for cellulitis as the primary diagnosis , defining prolonged los as greater than 7 days.32 as seen in this analysis , the los for the majority of the patients can be considered prolonged . using a multivariate analysis , baibergenova et al identified factors associated with prolonged admission as female sex ; patient age , where patients older than 65 years were at increased risk for longer los and patients older than 75 years incurred an even greater risk ; congestive heart failure as a comorbidity ; dermatology consultation ; and admission to or consultation by a surgical service . dermatology consultation was associated with the highest odds of longer los ( odds ratio : 4.5 ) . although not explicitly examined , it is interesting to consider how these factors may contribute to the longer los observed in our study . although the overall observation was that cellulitis and abscess comprised the majority of the infections for both primary and secondary diagnoses , patients with a secondary diagnosis in the atlantic canada region have a notably different demographic patterns with these infection types ; cellulitis and abscess made up just less than half of all infections , and nearly half of all infections were classified as in addition , over the study period , this region reported more than double the number of secondary diagnosis than primary diagnosis , whereas other regions had comparable numbers of patients with primary and secondary infections . since ~60% of the patients with secondary infection were 65 years or older in the atlantic canada province , this might be a contributing factor to the higher numbers of secondary infections reported . additional research is needed to fully elucidate the factors that might contribute to these findings . the data in this study show good consistency with data from another canadian study that reported a mean los of 7.1 days and 16.1 days for patients admitted for cellulitis as a primary condition and as a comorbidity , respectively.32 however , a key limitation of that study is the unknown proportion of patients with an mrsa infection in the cohort . the mean hospital los observed in this study is notably longer than that reported for admissions due to all causes in canadian hospitals . the organization for economic cooperation and development reported that the average canadian los for patients admitted for all causes was 7.6 to 7.7 days during the years 20082012.34 an evaluation by the cihi reported the average los as 7.2 days for all of canada , with an average los of 6.7 days for canada excluding quebec from 2004 to 2005.35 in addition , although the mean los reported herein ranged from 9 to 18 days for primary diagnosis and from 25 to 43 days for secondary diagnosis over the study period , the high end of the ranges and analysis of the 75th percentile of the interquartile range indicate substantial outliers at the higher end of the los spectrum . the canadian los data described in this study are similar to data reported in population - wide studies in the usa . los data obtained between 2002 and 2006 for cssti due to mrsa showed a mean of 12.6 ( sd : 18.9 ) days and a median los of 7.0 days.21 for ssti caused by s. aureus , mean hospital los based on information in the hcup database was 7.3 days in 2009 for hospitalized adult and pediatric patients ; los decreased from 9.9 days in 2001.7 similarly , in another study that evaluated hospitalization outcomes using the premier perspective database , the in - hospital mean and median los were 6.1 and 5 days , respectively , for a diagnosis of sssi due to s. aureus.30 a key limitation to these studies is the unknown proportion of patients with an mrsa infection in the cohort . compared with european hospital los data for cssti caused by mrsa as a primary diagnosis , representing a 1-year period between july 2010 and june 2011 , the mean los in canada in 2014 was lower ( 7.7 to 13.4 days ) ; the mean los in europe was 20.6 days with a range from 15.2 days in the uk to 25.0 days in portugal.28 this is consistent with clinical trial data that have shown a longer los for cssti caused by mrsa in europe than in other world regions.36 a secondary diagnosis of absssi due to mrsa generally entailed a longer los in both the hospital and the specialty care facility , with some exceptions . the longer los for patients with a secondary diagnosis is not unexpected because these patients were generally older and represented a sicker population , as determined by the higher comorbidity burden . in general , comorbidities might predict a longer los because of the more involved clinical management required to treat both the patient s infection and other conditions . patients with secondary diagnoses also underwent more complex surgical procedures ( surgical debridement and amputation ) , which might have contributed to longer hospital stays . consistent with what was observed in this analysis , other studies have linked longer hospital stays and increased mortality rate in hospitalized patients with skin infections to older age and comorbidities.23,30,37 in 2014 , the most recent year of the study , the interprovincial differences in los between primary and secondary diagnosis demonstrated notable variability , from 7.7 days in ontario to 13.4 days in canadian prairie for primary diagnosis and 18.2 days in ontario to 25.2 days in atlantic canada for secondary diagnosis . intraprovincial los variations in primary and secondary diagnosis were noted as well , with 1.5-fold to threefold differences in los noted for patients with a secondary diagnosis compared with patients with a primary diagnosis . based on an analysis of the special care facility data , two potential populations can be proposed from this data set : those who require hospitalization only and those who require further medical service at discharge , with medical care extended through the use of a special care facility . although a minority of patients required special care facility services , those who used the service required a mean of 10 to 25 days of additional care . a higher portion of patients with secondary infections required use of a special care facility , with the proportions of patients ranging from 1.7% to 5.2% for primary infections and 10.2% to 38.1% for secondary infections . this analysis did not investigate the demographics and potential factors associated with special care facility use of this resource - intense subgroup , but this could be the subject of future research . although the time trends showed variability over the years and lack of a linear decline , a general trend toward shorter mean los for patients with a secondary diagnosis between 2008 and 2014 was observed across all geographic regions ; the canadian prairie had the greatest reduction in los ( a reduction of 30 days from 48.5 days in 2008 to 18.3 days in 2014 ) . the time trend for primary diagnosis was less consistently defined across the regions , with variability over the years . however , a trend toward decreasing hospital los between 2008 and 2014 was observed in ontario and atlantic canada ; the alberta / british columbia and canadian prairie provinces had consistent hospital los . decreases in los may be due to the increased use of antibiotics in the outpatient setting , either due to the availability of oral medications or the implementation of outpatient - setting iv infusion centers or home - infusion capabilities . hospitals are the largest category of national health expenditure in canada . with hospitalization costs averaging us$1,000/day,3840 the cost of managing an episode of primary absssi due to mrsa can average more than us$7,000/stay . the observed longer los for patients diagnosed with a secondary infection , coupled with greater comorbid conditions and complex surgical procedures , might result in a higher average cost of secondary diagnosis compared with primary diagnosis . given the lower los and need for ancillary care and greater discharge rate , costs associated with primary absssi caused by mrsa diagnoses are minimized ; further cost reductions can be achieved by shifting care from an inpatient to an outpatient setting . , the accuracy of using diagnosis codes that appeared , on acute inpatient records , to identify a population of patients with absssi due to mrsa is not known . the accuracy of diagnosis codes to identify conditions varied depending on the type of diagnosis , and identifying the severity of infections in the context of patients who have underlying comorbidities is challenging . therefore , the study was likely subject to some misclassification bias as a result of coding errors or inclusion of codes to rule out specific diagnoses , such as diabetic foot infections . this study did not control for infection severity or distinguish whether the infection was widespread or localized . the evolving definitions of skin infection made collection of historical real - world data more challenging . because diabetic foot infection does not have a specific icd-10-ca code ( codes e10.70 and e10.71 refer to diabetes with foot ulcer and diabetes with foot ulcer with gangrene , respectively , but do not specify whether infection is present ) , it was not possible to exclude this code ; hence , some patients with diabetic foot infection might have been included in our sample . the general icd-10-ca code for diabetic foot ( e10.7 for diabetes with complications ) was not used as an exclusion criterion because we did not want to eliminate a proportion of patients . nevertheless , we believe that this study population is representative of the absssi due to mrsa population because the selected icd-10-ca diagnosis codes were consistent with those used in other absssi database studies and , most important , with codes that fall under the new fda - absssi definition . a further limitation was that it was not possible to examine hospitalization outcomes according to the specific antibiotic used because information related to the use of specific prescription medications and health services during the hospital stay was not available in the dad . since the study focused on outcomes experienced within the hospitalization episode , the results are limited by lack of potentially relevant patient information prior to hospitalization and after discharge . not all canadian provinces were included in the analysis because data for quebec were not available . in addition , all regions did not contribute data equally ; atlantic canada and the canadian prairie together contributed < 25% of patients , although these low numbers may reflect the actual burden of absssi due to mrsa . in conclusion , this study is the first to provide current real - world data on hospitalization patterns across canada for patients with a primary or secondary diagnosis of absssi due to mrsa . the mean los for hospitalized canadian patients ranged from 7.7 to 13.4 days across the geographic regions , which was shorter than that reported for europe but similar to that in the usa . these data can be used to inform best practices to optimize care with strategies such as medication management , which may facilitate hospital discharge or hospital avoidance altogether . diagnosis codes for identification of study population notes : if not otherwise specified , the coding definitions given in the table include all possible subsequent digits . superficial injury , infected : requires that codes for absssi and the superficial injury both be present . abbreviations : absssi , acute bacterial skin and skin structure infections ; mrsa , methicillin - resistant staphylococcus aureus . abbreviations : absssi , acute bacterial skin structure tissue infections ; los , length of stay ; mrsa , methicillin - resistant staphylococcus aureus . atlantic canada time trends abbreviations : absssi , acute bacterial skin and skin structure infections ; los , length of stay ; mrsa , methicillin - resistant staphylococcus aureus ; , no data . alberta and british columbia time trends abbreviations : absssi , acute bacterial skin and skin structure infections ; los , length of stay ; mrsa , methicillin - resistant staphylococcus aureus ; , no data . canadian prairie time trends abbreviations : absssi , acute bacterial skin and skin structure infections ; los , length of stay ; mrsa , methicillin - resistant staphylococcus aureus ; , no data .

purposeskin infections , particularly those caused by resistant pathogens , represent a clinical burden . hospitalization associated with acute bacterial skin and skin structure infections ( absssi ) caused by methicillin - resistant staphylococcus aureus ( mrsa ) is a major contributor to the economic burden of the disease . this study was conducted to provide current , real - world data on hospitalization patterns for patients with absssi caused by mrsa across multiple geographic regions in canada.patients and methodsthis retrospective cohort study evaluated length of stay ( los ) for hospitalized patients with absssi due to mrsa diagnosis across four canadian geographic regions using the discharge abstract database . patients with icd-10-ca diagnosis consistent with absssi caused by mrsa between january 2008 and december 2014 were selected and assigned a primary or secondary diagnosis based on a prespecified icd-10-ca code algorithm.resultsamong 6,719 patients , 3,273 ( 48.7% ) and 3,446 ( 51.3% ) had a primary and secondary diagnosis , respectively . among patients with a primary or secondary diagnosis , the cellulitis / erysipelas subtype was most common . the majority of patients presented with 0 or 1 comorbid condition ; the most common comorbidity was diabetes . the mean los over the study period varied by geographic region and year ; in 2014 ( the most recent year analyzed ) , los ranged from 7.7 days in ontario to 13.4 days in the canadian prairie for a primary diagnosis and from 18.2 days in ontario to 25.2 days in atlantic canada for a secondary diagnosis . a secondary diagnosis was associated with higher rates of continuing care compared with a primary diagnosis ( 10.6%24.2% vs 4.6%12.1%).conclusionthis study demonstrated that the mean los associated with absssi due to mrsa in canada was minimally 7 days . clinical management strategies , including medication management , which might facilitate hospital discharge , have the potential to reduce hospital los and related economic burden associated with absssi caused by mrsa .

Introduction Patients and methods Data source Patient selection Study measures and outcomes Data analysis Results Patient population Hospitalization outcomes Time trends: 20082014 Discussion Limitations Conclusion Supplementary materials

infections of the skin , particularly those caused by resistant pathogens , represent a clinical challenge to address . acute bacterial skin and skin structure infections ( absssi ) are a new classification of skin and skin structure infections ( sssis ) in accordance with health canada , the us food and drug administration , and the european medicines commission . sstis are most frequently caused by gram - positive pathogens , with methicillin - resistant staphylococcus aureus ( mrsa ) being increasingly the predominant pathogen in the usa and europe.25 linked to the growing prevalence of mrsa , rates of hospitalization for ssti have increased in the last decade in the usa.6,7 reports from canada also highlight the increasing prevalence of ssti due to mrsa in outpatients , emergency departments ( ed ) , and hospitals.813 in a study of visits to the ed for ssti , the prevalence of mrsa varied significantly across the canadian provinces ( 11%100% ) , with a higher prevalence in western canada.12 data from the canadian nosocomial infection surveillance program from 1995 to 2007 showed that hospitalization for ssti caused by mrsa increased from 24% to 37% between 1994 and 2007.13 surveillance reports have also documented increased isolation of mrsa from hospitalized patients with ssti in north america , although rates were lower in canada than in the usa.2,14 the involvement of mrsa in skin infections adversely affects patient outcomes . skin infections caused by mrsa are associated with increased instances of treatment failure,1517 disease recurrence,16,18 increased risk of invasive disease,19 and higher mortality.15,2022 mrsa - positive skin infections also have a significant economic impact because of increased durations of hospitalization15,22,23 and higher hospital costs.15 real - world data on cost drivers in patients with skin infections due to mrsa are important , so that health care can be delivered effectively and efficiently in an increasingly cost - constrained environment . hospitalization is the major cost driver associated with mrsa infection.24,25 hospital length of stay ( los ) is an important outcomes measure for patients with cssti due to mrsa , as the majority of medical costs for this condition are related to hospital ward and intensive care unit ( icu ) stays.26,27 although ssti are among the most common infections necessitating hospitalization,6 in general , there is limited countrywide information on hospitalizations for patients with skin infections caused by mrsa . a retrospective medical chart review of patients hospitalized with cssti between 2010 and 2011 in 12 european countries showed a mean hospital los of 20.6 days.28 in the usa , a retrospective multicenter observational registry of patients with mrsa skin infections treated with daptomycin showed median antibiotic - related los of 48 days , depending on age and comorbidities.29 another large us database study that used 4-year hospital discharge data from the solucient database documented mean and median los for cssti due to mrsa of 12.6 ( standard deviation [ sd ] : 18.9 ) days and 7.0 days , respectively.21 in patients hospitalized with cssti due to infection with s. aureus , healthcare cost and utilization project ( hcup ) data for 2009 showed an los of 7.3 days,7 and another us study using the premier perspective database reported an average hospital los of 6.1 days for the 20052006 period.30 canadian data on absssi due to mrsa are limited . in 2003 , a small study of 89 patients with ssti due to mrsa reported a mean hospital los of 28.9 ( sd : 29.8 ) days in patients treated with intravenous ( iv ) vancomycin.31 a large retrospective database study conducted over a 5-year period ( 2004 through 2008 inclusive ) in ~65,000 patients hospitalized for cellulitis ( not mrsa - specific ) in canada found a mean hospital los of 7.1 ( sd : 8.2 ) days ; 29.5% of patients admitted for cellulitis experienced a prolonged los in hospital ( > 7 days).32 to narrow the information gap for canada , we conducted a retrospective database analysis to characterize a cohort of patients hospitalized with absssi due to mrsa across multiple provinces ( alberta , british columbia , manitoba , newfoundland , new brunswick , nova scotia , ontario , prince edward island , and saskatchewan ) grouped into four geographic regions . patients were included if they were 18 years of age and older and were hospitalized between january 1 , 2008 and december 31 , 2014 , with icd-10-ca diagnosis codes consistent with absssi due to mrsa diagnosis ( see table s1 for absssi codes ) . patients were included if they were 18 years of age and older and were hospitalized between january 1 , 2008 and december 31 , 2014 , with icd-10-ca diagnosis codes consistent with absssi due to mrsa diagnosis ( see table s1 for absssi codes ) . among the 6,719 patients included in the study , 3,273 ( 48.7% ) had absssi due to mrsa as the primary reason for the hospital stay and 3,446 ( 51.3% ) had absssi due to mrsa as the secondary reason for the hospital stay . the mean age of patients hospitalized with a primary diagnosis ranged from 49 to 55 years , while the mean age for patients with a secondary diagnosis ranged from 55 to 67 years , indicating that patients with a secondary diagnosis were older than those with a primary diagnosis ( table 1 ) . cellulitis / erysipelas was the most frequent type of infection for patients with a primary diagnosis across all provinces ( 55.8% to 63.4% ) and for patients with a secondary diagnosis in ontario , alberta / british columbia , and the canadian prairie ( 47.3% to 58.0% ) . the majority of the patients across the samples had zero or one comorbidity ; however , among patients with a secondary diagnosis , a higher proportion of patients had two or more comorbidities . the number and percentage of patients with a primary or secondary diagnosis appeared to be relatively consistent from 2008 to 2014 ; the only exception was atlantic canada , where the proportion of patients with a secondary diagnosis decreased from 31.6% in 2008 to 5.8% in 2014 ( table 3 ) . the majority of hospitalizations were emergent ( 96.0% to 99.4% for patients with a primary diagnosis and 89.7% to 93.6% for patients with a secondary diagnosis ) ( table 3 ) . the mean los over the study period varied according to the geographic region , from 9 to 18 days for primary diagnosis and from 25 to 43 days for secondary diagnosis ( table 4 ) . in the final year of the study ( 2014 ) , los ranged from 7.7 to 13.4 days for a primary diagnosis and from 18.2 to 25.2 days for a secondary diagnosis . over the entire study period , the majority of hospitalized patients were discharged to their residence ; rates of discharge to home were higher among patients with a primary diagnosis compared with patients with a secondary diagnosis ( 77.7% to 82.7% vs 56.8% to 64.5% , respectively ) . absssi due to mrsa as a secondary diagnosis was associated with higher in - hospital mortality rates ( 7.2% to 12.1% ) than infections identified as a primary diagnosis ( 0.4% to 1.6% ) . the proportion of patients receiving continuing care was also higher among patients with a secondary diagnosis ( 10.6% to 24.2% ) than those with a primary diagnosis ( 4.6% to 12.1% ; table 4 ) . drainage was the most common surgical procedure in primary diagnosis patients in 2014 ( 20% to 42.4% ) and over the study period ( 34.5% to 43.3% ) , whereas among patients with a secondary diagnosis , surgical debridement was most common across all provinces ( 26.4% to 44.9% ) over the study period and in all provinces other than ontario in 2014 . amputations were more common among patients with absssi due to mrsa as a secondary diagnosis ( 21.0% to 25.3% ) , than patients with a primary diagnosis ( 2.3% to 10.2% ) . in patients with a primary diagnosis of absssi due to mrsa and who were hospitalized in either ontario or atlantic canada , there was a trend toward decreasing overall mean hospital los from 2008 ( 11.5 days in ontario , 14.0 days in atlantic canada ) to 2014 ( 7.7 days in ontario , 8.9 days in atlantic canada ) , with some variability in the intermittent years ( tables s2 and s3 ) . there was a decrease of 13 days in ontario ( from 31.5 to 18.2 days ; table s2 ) , 12 days in alberta / british columbia ( from 37.1 to 25.0 days ; table s4 ) , 30 days in canadian prairie ( from 48.5 to 18.3 days ; table s5 ) , and 22 days in atlantic canada ( from 47.6 to 25.2 days ; table s3 ) . among the 6,719 patients included in the study , 3,273 ( 48.7% ) had absssi due to mrsa as the primary reason for the hospital stay and 3,446 ( 51.3% ) had absssi due to mrsa as the secondary reason for the hospital stay . the mean age of patients hospitalized with a primary diagnosis ranged from 49 to 55 years , while the mean age for patients with a secondary diagnosis ranged from 55 to 67 years , indicating that patients with a secondary diagnosis were older than those with a primary diagnosis ( table 1 ) . cellulitis / erysipelas was the most frequent type of infection for patients with a primary diagnosis across all provinces ( 55.8% to 63.4% ) and for patients with a secondary diagnosis in ontario , alberta / british columbia , and the canadian prairie ( 47.3% to 58.0% ) . the majority of the patients across the samples had zero or one comorbidity ; however , among patients with a secondary diagnosis , a higher proportion of patients had two or more comorbidities . the number and percentage of patients with a primary or secondary diagnosis appeared to be relatively consistent from 2008 to 2014 ; the only exception was atlantic canada , where the proportion of patients with a secondary diagnosis decreased from 31.6% in 2008 to 5.8% in 2014 ( table 3 ) . the majority of hospitalizations were emergent ( 96.0% to 99.4% for patients with a primary diagnosis and 89.7% to 93.6% for patients with a secondary diagnosis ) ( table 3 ) . the mean los over the study period varied according to the geographic region , from 9 to 18 days for primary diagnosis and from 25 to 43 days for secondary diagnosis ( table 4 ) . in the final year of the study ( 2014 ) , los ranged from 7.7 to 13.4 days for a primary diagnosis and from 18.2 to 25.2 days for a secondary diagnosis . los in a specialty care facility varied by geographical region , ranging from 83 to 157 hours for patients with a primary diagnosis and from 153 to 203 hours for patients with a secondary diagnosis . over the entire study period , the majority of hospitalized patients were discharged to their residence ; rates of discharge to home were higher among patients with a primary diagnosis compared with patients with a secondary diagnosis ( 77.7% to 82.7% vs 56.8% to 64.5% , respectively ) . absssi due to mrsa as a secondary diagnosis was associated with higher in - hospital mortality rates ( 7.2% to 12.1% ) than infections identified as a primary diagnosis ( 0.4% to 1.6% ) . the proportion of patients receiving continuing care was also higher among patients with a secondary diagnosis ( 10.6% to 24.2% ) than those with a primary diagnosis ( 4.6% to 12.1% ; table 4 ) . drainage was the most common surgical procedure in primary diagnosis patients in 2014 ( 20% to 42.4% ) and over the study period ( 34.5% to 43.3% ) , whereas among patients with a secondary diagnosis , surgical debridement was most common across all provinces ( 26.4% to 44.9% ) over the study period and in all provinces other than ontario in 2014 . amputations were more common among patients with absssi due to mrsa as a secondary diagnosis ( 21.0% to 25.3% ) , than patients with a primary diagnosis ( 2.3% to 10.2% ) . in patients with a primary diagnosis of absssi due to mrsa and who were hospitalized in either ontario or atlantic canada , there was a trend toward decreasing overall mean hospital los from 2008 ( 11.5 days in ontario , 14.0 days in atlantic canada ) to 2014 ( 7.7 days in ontario , 8.9 days in atlantic canada ) , with some variability in the intermittent years ( tables s2 and s3 ) . there was a decrease of 13 days in ontario ( from 31.5 to 18.2 days ; table s2 ) , 12 days in alberta / british columbia ( from 37.1 to 25.0 days ; table s4 ) , 30 days in canadian prairie ( from 48.5 to 18.3 days ; table s5 ) , and 22 days in atlantic canada ( from 47.6 to 25.2 days ; table s3 ) . this is the first study to examine hospital los and time trends of hospital los , using data as recent as 2014 , for absssi due to mrsa across multiple geographic regions in canada , excluding quebec . the majority of patients with a primary diagnosis of absssi caused by mrsa were younger ( 18 to 44 years ) and had fewer comorbid conditions than those with a secondary diagnosis . however , patients aged 65 years or older across all provinces were more likely to be hospitalized with absssi due to mrsa as a secondary diagnosis compared with a primary diagnosis . although the overall observation was that cellulitis and abscess comprised the majority of the infections for both primary and secondary diagnoses , patients with a secondary diagnosis in the atlantic canada region have a notably different demographic patterns with these infection types ; cellulitis and abscess made up just less than half of all infections , and nearly half of all infections were classified as in addition , over the study period , this region reported more than double the number of secondary diagnosis than primary diagnosis , whereas other regions had comparable numbers of patients with primary and secondary infections . the organization for economic cooperation and development reported that the average canadian los for patients admitted for all causes was 7.6 to 7.7 days during the years 20082012.34 an evaluation by the cihi reported the average los as 7.2 days for all of canada , with an average los of 6.7 days for canada excluding quebec from 2004 to 2005.35 in addition , although the mean los reported herein ranged from 9 to 18 days for primary diagnosis and from 25 to 43 days for secondary diagnosis over the study period , the high end of the ranges and analysis of the 75th percentile of the interquartile range indicate substantial outliers at the higher end of the los spectrum . los data obtained between 2002 and 2006 for cssti due to mrsa showed a mean of 12.6 ( sd : 18.9 ) days and a median los of 7.0 days.21 for ssti caused by s. aureus , mean hospital los based on information in the hcup database was 7.3 days in 2009 for hospitalized adult and pediatric patients ; los decreased from 9.9 days in 2001.7 similarly , in another study that evaluated hospitalization outcomes using the premier perspective database , the in - hospital mean and median los were 6.1 and 5 days , respectively , for a diagnosis of sssi due to s. aureus.30 a key limitation to these studies is the unknown proportion of patients with an mrsa infection in the cohort . compared with european hospital los data for cssti caused by mrsa as a primary diagnosis , representing a 1-year period between july 2010 and june 2011 , the mean los in canada in 2014 was lower ( 7.7 to 13.4 days ) ; the mean los in europe was 20.6 days with a range from 15.2 days in the uk to 25.0 days in portugal.28 this is consistent with clinical trial data that have shown a longer los for cssti caused by mrsa in europe than in other world regions.36 a secondary diagnosis of absssi due to mrsa generally entailed a longer los in both the hospital and the specialty care facility , with some exceptions . consistent with what was observed in this analysis , other studies have linked longer hospital stays and increased mortality rate in hospitalized patients with skin infections to older age and comorbidities.23,30,37 in 2014 , the most recent year of the study , the interprovincial differences in los between primary and secondary diagnosis demonstrated notable variability , from 7.7 days in ontario to 13.4 days in canadian prairie for primary diagnosis and 18.2 days in ontario to 25.2 days in atlantic canada for secondary diagnosis . intraprovincial los variations in primary and secondary diagnosis were noted as well , with 1.5-fold to threefold differences in los noted for patients with a secondary diagnosis compared with patients with a primary diagnosis . although the time trends showed variability over the years and lack of a linear decline , a general trend toward shorter mean los for patients with a secondary diagnosis between 2008 and 2014 was observed across all geographic regions ; the canadian prairie had the greatest reduction in los ( a reduction of 30 days from 48.5 days in 2008 to 18.3 days in 2014 ) . in conclusion , this study is the first to provide current real - world data on hospitalization patterns across canada for patients with a primary or secondary diagnosis of absssi due to mrsa . the mean los for hospitalized canadian patients ranged from 7.7 to 13.4 days across the geographic regions , which was shorter than that reported for europe but similar to that in the usa . atlantic canada time trends abbreviations : absssi , acute bacterial skin and skin structure infections ; los , length of stay ; mrsa , methicillin - resistant staphylococcus aureus ; , no data . alberta and british columbia time trends abbreviations : absssi , acute bacterial skin and skin structure infections ; los , length of stay ; mrsa , methicillin - resistant staphylococcus aureus ; , no data . canadian prairie time trends abbreviations : absssi , acute bacterial skin and skin structure infections ; los , length of stay ; mrsa , methicillin - resistant staphylococcus aureus ; , no data .

the focus of policy , media and many academic discourses on drugs in sport has been upon cheating and negative health outcomes ( gleaves , 2010 ; simon , 2004 ) . these are the two main underlying reasons for the funding and implementation of anti - doping testing , and the subsequent legal and quasi - legal process aimed at punishing the cheats . the development of organised international anti - doping policy has been based on these central tenets since the 1960s ( beamish & ritchie , 2004 ; dimeo , 2007 ; hunt , dimeo , & jedlicka , 2012 ) . more recently , the world anti - doping agency ( wada ) included a third guideline known as the spirit of sport . for a substance or technique to be banned , it would have to contravene two of these three : fair play , health of the athlete and the spirit of sport ( wada , 2009 ) . previous research on doping in cycling has focused on the professional level ( mignon , 2003 ; ohl , fincoeur , lentillon - kaestner , defrance , & brissonneau , 2013 ; sefiha , 2012 ; waddington , 2000 ) , while largely neglecting the lower amateur levels of sport and masters competitors in higher age categories . in this study , we focus on the cases of all american cyclists , amateur and professional , who were caught doping and sanctioned during the period 2001 and summer 2014 . the range of cases includes athletes whose intentions to cheat others are not obvious , as athletes may have used recreational drugs like cannabis , used medicines that contained banned drugs without realising it , or bought and used nutritional supplements contaminated with banned substances . taking a broader view of us cycling that includes amateurs exposes patterns of doping behaviour beyond the narrow perspective of a small number of professionals whose motivations clearly lies with a desire for financial gain and celebrity status ( backhouse , mckenna , robinson , & atkin , 2007 ; laure & reinsberger , 1995 ) . lentillon - kaestner and carstairs ( 2010 ) noted doping may occur at any level of competition , asking what happens before cyclists become professional ? we add an exploration of what happens when cyclists are unlikely to ever become professional or when cyclists compete as older adults . in addition to elite cases , we describe and explain the doping culture that emerged in domestic us cycling among amateurs , those who compete at the lower categories of cycling , and semi - professionals , those who may win large races and prize purses but who do not have contracts with the largest professional teams . the critical approach adopted here aims to analyse and discuss the nature of policy in practice . we demonstrate the variety of doping situations and critically assess the outcomes of anti - doping against wada 's stated purpose and the general ideology underpinning anti - doping . the patterns that emerged starkly contrasted with the policy and media - led demonisation of doping athletes and with common assumptions of extrinsically motivated cheating . these cases point to possible trends in doping and raise new questions about the fairness of current anti - doping policies . the united states anti - doping agency ( usada ) was formed in 2000 as the trend globally was to move away from anti - doping being embedded within organisations with a vested interest in maintaining the integrity of their image , towards independent organisations that would be judged by their ability to protect thus , in parallel to the leadership shifting from the international olympic commission ( ioc ) to wada , the american re - organisation took responsibility away from the us olympic commission and established usada . similarly , the situation within professional cycling had dramatically altered after the 1998 festina scandal , an event that brought forward proposals for an independent global body . wada increased the pressure on the union cycliste internationale ( uci ) , not least because cycling at the top level had a reputation for allowing doping to occur and not punishing cyclists who did get caught with sufficient bans to act as a deterrent ( hoberman , 2003 ; mller , 2006 ; waddington , 2000 ) . there had been some precedence of doping behaviours in us cycling , such as the use of blood doping among the olympic team for the 1984 los angeles games ( gleaves , llewellyn , & lehrbach , 2014 ) . establishing the nature of the doping culture within us cycling in the 1990s is challenging . even in the extensive evidence presented against lance armstrong the point of origin for his doping decision is not entirely clear , but some evidence would suggest it was in 1995 with motorola ( hamilton & coyle , 2012 ) . however , matt dicanio and tyler hamilton both explained that their encounters with doping occurred only once they were established within the professional european scene . for hamilton , it was in 1997 he made the decision to dope , and he argues that for most professionals of that time period , the realisation that doping was a necessity would come in their second year and their own decision would have to be made in their third year ( hamilton & coyle , 2012 , p. 66 ) . we would propose , therefore , that the extent of doping within the domestic context was relatively low in the late 1990s on the basis that american professionals learned from european doctors , and as we detail below , evidence of a doping culture really emerges around the time that lance armstrong was winning his seven consecutive tour de france titles . it should be noted here that it is almost impossible to say with any certainty what the patterns of drug use were . evidence from testing and the eventual confessions of a small number of professional dopers can offer some indications . even when higher numbers of cyclists were caught , it may have been the result of the development or refinement of tests for specific drugs , rather than any specific pattern . many recent scholarly approaches to the study of doping in sport aim to support anti - doping through improving understanding of the contexts of athletes ' lives and training , psychological make - up and the processes through which they are educated ( backhouse & mckenna , 2012 ; morente - snchez & zabala , 2013 ; ntoumanis , ng , barkoukis , & backhouse , 2014 ) . there have recently been a significant number of approaches to modelling the doping decision , and statistically testing the relevant variables ( bilard , ninot , & hauw , 2011 ; ntoumanis , ng , barkoukis , & backhouse , 2013 ; sas - nowosielski & swiatkowska , 2008 ; tsorbatzoudis , rodafinos , spiliopoulou , barkoukis , & lazuras , 2009 ) . some of these studies have been funded by wada with the explicit remit to help the fight against drug misuse in sports . while these studies can be credited for improving the evidence base , there appears to be several unchallenged assumptions : that the decision to dope firstly is a rational , intentional and conscious decision to cheat ( hoff , 2012 ; overbye , knudsen , & pfister , 2013 ) that emerges from inherent psychological traits ( bilard et al . , 2011 ; petrczi & aidman , 2009 ) combined with environmental pressures ( smith et al . , 2010 ) , secondly is perceived as deviant by athletes and their entourage ( christiansen , 2005 ; pitsch , emrich , & klein , 2007 ) , and lastly that anti - doping is a legitimate and fair policy that functions to catch dopers and deter potential dopers ( martin , baron , & gold , 2006 ; mazanov , huybers , & connor , 2011 ) . however , it could be more critically argued among this wealth of recent empirical knowledge there has not yet been a study that shows the variety of doping situations and describes patterns of doping behaviour according to this variety , despite the availability of information on every sanctioned athlete ( yonamine , garcia , & de moraes moreau , 2004 ) . dopers are not homogenous and there is a significant conceptual and real difference between someone who systematically dopes over a period of time , and many innocuous doping situations ( see ramachandra et al . , 2012 ; strelan & boeckmann , 2006 ) . the social groupings affected by anti - doping are much more diverse than previous studies have considered where the focus has been on the extrinsically motivated elite and aspiring elite , male athletes and their use or potential use of peds ( bloodworth & mcnamee , 2010 ; christiansen , 2010 ; outram & stewart , 2015 ) . this narrow focus has left gaps in the existing research , overlooking patterns of behaviours , motivations and situations occurring outside the upper reaches of the sport . beyond the level of the individual and their immediate circumstances , some recent historical , sociological and policy analyses of anti - doping have driven towards a more critical deconstruction of ideological principles , common sense claims , self - governance of international agencies and the equity of policy decisions ( denham , 2011 ; hanstad & waddington , 2009 ; kayser & broers , 2012 ; stewart & smith , 2008 ; straubel , 2008 ; wiesing , 2011 ) . mller ( 2014 ) has used the term corrupt idealism to frame his criticism of anti - doping leaders who are willing to accept or indeed promote unethical behaviour in order to support the there are cases of athletes who likely did not intend to violate anti - doping regulations , such as those who unintentionally ingested a banned substance ( cox , 2014 ; pluim , 2008 ) . there may be others who failed to acquire a therapeutic use exemption ( tue ) but were following orders from their personal physician , risking a positive test and a sanction ( fitch , 2013 ; overbye & wagner , 2013 ) . however , under wada 's strict liability principle all athletes are held to account for the presence of any detected banned substance , regardless of their intent ( cox , 2014 ) . there are numerous examples from a range of sports where athletes have been punished for inadvertently breaking the rules . thus , it may be that the war on drugs in sport has been so heavily skewed towards the claimed outcomes of catching cheats that relatively innocent athletes who did not intend to enhance performance ( pluim , 2008 ) are swept up , in a sense becoming collateral damage the critical approach adopted here therefore aims to analyse and discuss the nature of policy in practice . we use a case study to demonstrate the variety of doping situations and critically assess the outcomes of anti - doping against wada 's stated purpose and the general ideology underpinning anti - doping . the social groupings affected by anti - doping are much more diverse than previous studies have considered where the focus has been on elite , male athletes . first , we utilised the on - line records of the national governing body , usa cycling , and the usada , the organisation that caught the us postal riders . we recorded details of all 88 sub - cases that resulted in a sanction . in our collection , we included the type of test ( in or out of competition ) , where the test was administered , the type and name of the substance the athlete tested positive for , and the length of the competition ban imposed . second , we used information available through usa cycling 's website regarding cyclists ' race results and competitive history , categories for racing and cycling discipline ( i.e. track , road ) . though we do not present this data here , it was the basis for identifying the range of case types we identify below . in some instances , the athlete challenged the outcome through the arbitration process . where this is so , the full details and decision are available through the usada website and were included in the analysis . third , we also recorded details from news reports of cases that attracted mainstream media attention . rather than ignore those sub - cases often left out of mass media coverage for lacking a newsworthy aspect , we sought to include these hidden in plain sight accounts in our analysis . to this end , we gathered information from online news sources , some of which were connected to print media and some that are exclusively web based . in most cases , these reports were found in american cycling magazines such as velopress and on websites reporting cycling news . we used google to search for each cyclists name . in the cases where the name search returned multiple or unrelated results , we entered a second search for the cyclists name with the word doping in each case this returned at least one relevant result other than the relevant usada press release . some cyclists had wikipedia pages , though information from these pages was used only where the information source was cited and the source could be confirmed . in cases outside of the widely covered elite doping scandals however , we agree with laurendeau and moroz ( 2012 ) that we might infer from the relatively small number of articles that the ideas that they produce and reproduce are so commonsense as to generate little controversy and , hence , little ( public ) discussion ( p. 387 ) yet are useful for analysis . many of these lesser - known cases contained details about cyclists motivations and circumstances that contradict those most commonly discussed in the literature on doping . as such , our findings provide correction to the myopic view of doping within us cycling . the news reports on each sub - case were collected and analysed for details of any response to the test result by the athlete , such as an admission or denial of intent , or for any alternative explanation for the positive result . often , the news reports functioned to provide background and context for several cases involving lesser or unknown amateur and master cyclists . there are factual elements of each sub - case such as the length of a sanction , drug type , location where the test was conducted , age of the cyclist , etc . , as well as descriptive and/or subjective elements , including statements by cyclists to sports journalists . we have analysed and presented the former type of data , then used the latter type to contextualise and highlight aspects of the patterns that emerged from the official records . we approached these more descriptive data with a sense of critical detachment , though inevitably used our judgment to select key quotes and interpretations to build an overarching argument . there are , of course , limitations to this form of data collection , not least the reliance on reporters and others to produce reliable accounts . especially with regard to the issue of drugs in sport , sportspersons create particular forms of self - presentation . dopers who have not been caught claim to be clean , those under suspicion divert blame elsewhere to distract attention , those who are caught usually only admit to the specific offence that has been proven , and even full confessions can not be trusted ( lamont - mills & christensen , 2008 ) . similarly , journalists and academic researchers who do access relevant sources are trying to create a version of events that fits dominant ideological and policy paradigms and expectations of readers ( denham , 2011 ; vetteniemi , 2010 ) . therefore , rather than accepting any one account as the singular truth of a case we cross - referenced journalistic reports with all available information from usa cycling , usada , and the cas and took note of any inconsistencies or contradictions in the journalistic accounts . additionally , we included only professional journalist accounts , and excluded blogs , forums , and other user - generated material , as well as opinion pieces pertaining to the sub - cases from the analysis . usada 's first tranche of tests in 2000 produced only three adverse analytical findings all of these for ephedrine and the length of bans were zero , one and three months . however , it would not be long before tests began to catch more serious cheats . through the years from 2002 to 2014 , there was a gradual increase in the number of cyclists who were caught trying to enhance their performance using testosterone , steroids , erythropoietin ( epo ) , growth hormone and blood doping . these cyclists may have been caught through blood or urine testing , but they may also have received sanctions resulting from a violation such as refusal to submit to a test or through their own admission . the total number of sanctions in a single year peaked at 15 in 2012 , the result of a long - term usada investigation into the us postal team . as the us postal team became more successful the doping prevalent in european cycling seeped into the american context , laying the foundation for a culture of doping to emerge ( brewer , 2002 ; lentillon - kaestner & carstairs , 2010 ; lentillon - kaestner , hagger , & hardcastle , 2012 ; usada , 2012 ) . it seems clear that elite american cycling had , in a broad sense , accepted the necessity of doping in order to compete in major races against european teams . this acceptance is evidenced by the extensive doping system detailed in usada 's reasoned decision against lance armstrong ( usada , 2012 ) . lance armstrong had won several of the world 's top races , including the tour de france ( 19992005 ) . usada have shown that he was using performance - enhancing drugs for all of those victories , and this is supported by the testimony of many other top american cyclists who also doped for part or all of this time ( usada , 2012 ) . yet of the 88 total sanctions from 2000 to 2014 , in all types of cycling , the majority of positive tests was for anabolic agents ( 42% ) . other forms of deliberate cheating were using peptide hormones such as epo ( 26% ) and blood doping ( 5% ) . the more ambivalent categories are stimulants ( 22% ) that may or may not have been for enhancing performance , and missing / refusing tests ( 11% ) . a number were sanctioned for non - analytical reasons , including the members of us postal who were subpoenaed by the federal investigators so had to confess . kayle leogrande 's case is more subtle : he told his team 's soigneur he was worried about being caught and she reported him to usada , suggesting that even the small number of known doping cases were not always the result of good anti - doping control mechanisms . the endurance - boosting drug epo had been popularised through the 1990s , due to the perceived performance benefit and lack of a test to detect usage ( lundby , achman - andersen , thomsen , norgaard , & robach , 2008 , for development and critique of test ) . once such a test was developed in 2004 , we could see that domestic cyclists were familiar with the drug : the first positive tests being adham sbeih ( 2004 ) , adam bergman ( 2005 ) , alvaro tardaguila ( 2006 ) and neal schubel ( 2006 ) . sbeih was a national level competitor ( usa cycling , 2004 ) , bergman was a member of the elite domestic team jelly belly ( knapp , 2004 ) , tardaguila was an amateur rider ( usa cycling , 2006 ) and schubel was a low achiever at masters level ( stokes , 2010 ) . the canadian woman who raced to a successful level in the usa , genevive jeanson , came to admit using epo for a period of 8 years , beginning when she was 16 years old , from 1997 to 2005 ( cbs sports , 2007 ) . since there was not a reliable test for epo use until 2004 , it is quite possible that other cyclists were using it before this time , and the lack of systematic testing makes it likely that these cases were the unfortunate ones who were caught rather than the only guilty parties . moreover , the total number of tests conducted relative to the increased number of competitive cyclists combined with evidence from those who used epo and other drugs without being caught , suggest the prevalence was significantly higher than the handful of positive cases . the emergence of doping behaviours the taking or using of prohibited substances or methods of performance enhancement depends upon suppliers , and in turn entrepreneurial business - like suppliers were responding to demand ( martin et al . , 2006 ; stewart & smith , 2008 ) . when joe papp first became part of this history , he was a 32-year - old moderately successful rider who tested positive for 6a - oh - androstenedione and was banned for 2 years . he was then given a lifetime ban in 2011 for distributing banned drugs ( ford , 2011 ) . investigators discovered that papp had a client list of 187 individuals , though not all of these were cyclists . the consequences of further investigations into papp 's customers led to a number of confessions and sanctions , and also provided some indication of wider doping among amateur and semi - professional teams . in 2007 , for example , chuck coyle was given a two - year ban when usada found a record of purchases for epo and insulin growth factor made on his credit card from papp 's website . however , coyle responded by claiming that some of his younger teammates had borrowed his credit card and laptop computer , which was a common practice in general , to buy the drugs ( rogers , 2010 ) . other cases seem to indicate a developing pattern of ped use among aspiring , non - elite cyclists . in 2001 , semi - professional rider duane dickey tested positive for three banned drugs : phentermine , boldenone and nandrolone ( usa cycling , 2010 ) . in 2002 , a former member of us postal , kirk o'bee , tested positive for elevated testosterone ( rogers , 2012 ) . female track cyclist , tammy thomas was given a lifetime ban for a second offence after testing positive for norbolethone in 2002 ( macur , 2004 ) . these and other similar cases demonstrate that drug use was indeed going on within the ranks of non - professional cycling . these cases are not directly linked to the 2012 us postal case but are a domestic sub - set of the doping culture that was disseminated from the elite context through the lower levels of the sport . kayle leogrande 's experience around the same time period helps to illustrate how strongly a doping culture had taken hold in american cycling , including among those cyclists who were not quite at the uppermost echelons of the sport . during the period 20042006 , other professionals he bought epo from joe papp in 2006 , and had to self - experiment with dosages . he joined the rock racing team in 2007 where he was introduced to former teammates of lance armstrong 's [ and ] began to dope more regularly there is no direct connection between leogrande and us postal , except that his eventual admission assisted the federal investigation led by jeff novitzky . later , travis tygart , chief executive of usada , would say that leogrande 's evidence played a significant role in piecing together the evidence against us postal , perhaps because leogrande speculated that doping would be necessary to compete successfully in the top european races ( ibid . ) . moreover , the former professional floyd landis gave evidence that the owner of the domestic team , rock racing , was involved in doping thus , we can see the emergence of doping as well as the failings of anti - doping to stop this culture from developing . within a few years of the first epo cases , doping had spread to amateur and masters levels such that organisers of mass participation events felt the requirement to fund and organise testing . in the new york gran fondo in 2012 , two masters - level riders tested positive for epo ( dreier , 2012 ) . yet , the authorities focused upon the top - level riders , in particular lance armstrong , and have not created an adequate system that would include regular , random in- and out - of - competition testing for the recreational and master levels of cycling . riders are disadvantaged and most seriously competitive riders would feel under pressure to take part in doping . level playing field is incongruous here , not least because a wide range of factors influence performance and that doping is an unknown contributing factor to race results . anti - doping policy did not serve to protect the fairness of sport or indeed the spirit of sport . as evidenced by the official doping cases , a sub - culture developed where amateur and semi - professional cyclists came to mimic the behaviours of their professional counterparts and indulged in doping . while it is hard to be certain about this , it may have been the case that a serious amateur competitor felt the need to participate in doping for reasons of identity , status and belonging within a social environment that respected and emulated those in the higher echelons of the sport . while such cases pointed to a sub - culture of deliberate , conscious cheating using the most serious of drugs , other cases showed the dilemmatic nature of policy : that not all dopers are created equally . anti - doping efforts aimed at detecting those who seek to cheat may result in bans for athletes who never intended such . for example , the three ephedrine cases in 2000 were given shorter bans , suggesting that the authorities did not deem them to be worthy of a full sanction due to the relatively innocuous nature of the substance and the likelihood that it was not intentionally consumed . these are indicative of a wider pattern wherein the intention to cheat is unsubstantiated yet competition bans still apply . the chuck coyle episode highlights this , and he was informed that the cost of appealing the decision was around $ 20,000 , so he had no choice but to accept it . thus , the legal process disadvantages amateur cyclists or lower paid semi - professionals . mller ( 2014 ) has recently argued that the creation of a doping - specific legal process is problematic , and does not give those charged with an offence the same legal protection or opportunities to defend their case in comparison to normal civic prosecutions . the use of the strict liability principle is designed to counter any athletes ' appeals and puts the onus fully of them to explain any substance found in their sample . american cases such as coyle and armstrong show how agencies can successfully pursue a charge on the basis of non - scientific evidence , such as coyle 's credit card purchases . it is questionable whether a similar case would be upheld under civic law when the defence has more scope to challenge the nature of the prosecution 's evidence . nonetheless , we can say that coyle 's unfortunate position of being unable to mount an appeal shows how discriminatory the legal process can be . however , as in the 2003 cases of mountain bikers kathi krause and gary houseman , the use of narcotics and cannabis during competition is explicitly banned by the anti - doping agencies . both were caught using cannabis and both were banned for a year . houseman was also stripped of his first place title in the uci world cup event ( albert , 2003 ) . krause was aged 41 and had only finished 15th in the national championships ( usada , 2003 ) . it is a different conceptual argument to propose that the use of drugs that are likely to inhibit rather than enhance performance is not a form of cheating or allow one competitor an advantage over another ( henne , koh , & mcdermott , 2013 ) . the social and moral value of this regulation has been debated among sports philosophers and sociologists , but wada insists that recreational drugs contravene the principle of the spirit of sport despite the problematic nature of that idea . bans for non - performance enhancing drugs do seem particularly harsh in the context of us cycling when an unknown quantity of riders doped to enhance performance without being caught , and when the majority of the us postal team was given reduced bans of six months because they provided additional information to assist the investigations . that is an unlikely option for cyclists unable to offer such information , which is likely to be the case for many at the amateur level . in total as mentioned above , they are generally accepted to be non - performance enhancing and the regulations an unnecessary intrusion into athletes ' private lives ( kayser , mauron , & miah , 2007 ) . perhaps more importantly , the science behind banning these drugs shows that cannabinoids can be present in urine long after it has been consumed ( varying depending upon consumption habits and amount used ) . wada changed the threshold from 150 to 15 ng in 2013 because it was recognised that this was a significant issue ( wada , 2013 ) . the rules state that consumption out - of - competition is allowable but not in - competition . under the pre-2013 rules , the presence of metabolites in urine during an event period might indicate usage occurring up to three weeks prior to the test ( huestis , mazzoni , & rabin , 2011 ) . thus , not only is the wider social context of banning recreational drug problematic , but also within wada 's very own rules . even the organisation 's first president richard pound said that an athlete who used marijuana a month before competition was likely to be detected , as was someone exposed to second - hand marijuana smoke two weeks before an event ( miceli , 2013 ) . as such , there clearly are inconsistencies and inequalities where innocent parties can end up sanctioned . there have also been several cases where the substance ingested appears to have come from a source unwittingly consumed by the athlete . a case in point here is that of 23-year - old amber neben who was found to have 19-norandrosterone in her sample in 2003 . she had the resources to take her appeal to the north american court of arbitration for sport ( nacas ) , and identified the source of the banned substance to be from a contaminated supplement . the panel agreed her positive test to be unintentional but still ruled that she be sanctioned with a 6-month ban . indeed , the transcription of neben 's arbitration decision shows the dilemmas faced when confronted with an inadvertent case of doping ( nacas , 2003 ) . the experts agreed that supplements were normal , such as electrolyte drinks and glucose gels . they even suggested not using supplements contravened the stated purpose of the fight against doping , to protect the health of athletes ( nacas , 2003 , p. 6 ) since these were needed to maintain health , avoid fatigue and mental lapses during races . ironically , they cite the example of lance armstrong to show that road racing is gruelling and all too often dangerous the demands placed on the body are greater than most world - class athletes in other sports . the source of neben 's banned substance was not fully explicated but it was proposed that it possibly came from a supplement bought from hammer nutrition by usa cycling coaches and distributed to the team . what neben 's appeal showed was potential negligence on the part of those who had responsibility to protect her from the risk of a positive test , and that all athletes are at risk of inadvertently taking contaminated supplements , which independent analyses have shown can contain banned substances ( cohen , 2009 ; cohen , travis , & venhuis , 2013 ) . though usa cycling was in the spotlight for this mistake and stood accused of not providing any real education to athletes about the dangers of supplement contamination ( nacas , 2003 , p. 10 ) , it remains the athlete who bears the brunt of the legal process including potentially career and reputation damaging sanctions ( amos , 2007 ) . taken together , these examples suggest that anti - doping regulations can have consequences for individuals whose offence is relatively innocuous , perhaps even completely unintentional , or even the result of negligence by their coaches or national governing bodies . yet there seems to be an acceptance among anti - doping policy decision - makers that there will be a degree of collateral damage in the pursuit of the real cheats . it seems ironic that lance armstrong negotiated his way around the anti - doping system for a period of 810 years , that his guilty team - mates received a light sanction and were allowed to return to the sport , yet others have faced consequences for behaviours that are completely different to the classic sense of doping as intentional cheating . perhaps it is not surprising when highly competitive riders wish to retain some level of their youthful competitive success . this appears to be the explanation for such cases as that of kenneth williams who was aged 42 when he tested positive for dhea ( weislo , 2009 ) . he was a well - known and popular racer who wished to maintain his performance levels . forty - nine - year - old todd robertson tested positive in 2011 in an out of competition test for oxygen - enhancing peptide hormone ( epo ) , and admitted using it for 2 years previously . , he was found to have the stimulant modafinil in his sample and thus was banned for 8 years at age 51 for this second offence ( usa cycling , 2014 ) . the second offence appears to have been accidental , since he learned from his first to be vigiliant , he said : i still do not know how or why a banned stimulant was found in my system ( usa cycling , 2014 ) . interestingly , robertson also admitted using supplements , which shows how even those over 50 years old seek performance - enhancing substances , whether legal or otherwise , to maintain their levels of competitiveness across their life - cycle . the new york gran fondo 2012 cases of david anthony ( aged 45 ) and gabriele guarini ( aged 49 ) brought this issue some national attention when they were reported in the new york times ( dreier , 2012 ) . however , anthony 's case presents a different scenario from life long competitors looking to maintain competitiveness . he was a relative latecomer to cycling but became by his own admission obsessed with improving his ability and performances . he had won the new york gran fondo for his age group , so had a degree of success , but was never going to become a professional or reap extrinsic rewards . however , he spoke of seeking relevance within his local cycling community ( ibid . ) . this raises questions about identity among non - elite and older sports men and women . rather than the rewards being related to prize - money , high salaries or sponsorship deals , their desire is for localised recognition and to belong to a specific sub - culture . thus , the increasing popularity of cycling in the usa , and the widening of race participation opportunities , has helped to create tribes of serious recreational riders who buy expensive equipment , learn about the science of training and preparation , and perhaps have a personal coach . within this culture doping is transmitted from older or more experienced cyclists to less experienced riders ( lentillon - kaestner & carstairs , 2010 ) , normalising doping as a rational part of higher level competition ( sefiha , 2012 ) . anthony , and no doubt others , took the next step towards the pseudo - professionalisation of their hobby by using doping drugs ( brewer , 2002 ) . though doping may have seeped into domestic semi - professional teams through connections and influences from elite riders , a further effect was to prompt amateurs to dope . the theme of older riders has another important dimension related to the notion of collateral damage : the overlap of anti - doping regulations and anti - ageing therapeutic drugs . an example here is that of 62-year - old david leduc who was caught and banned for 2 years in 2013 for using epo , testosterone and amphetamines . he did not manage to explain the epo , but the other drugs were prescribed by a doctor for age - related compensation and for attention deficit disorder . similarly , sloan teeple , aged 42 , was banned for 2 years for using testosterone prescribed by a doctor for therapeutic reasons , but he had not received a tue . other evidence points to the challenges associated with the overlap between anti - ageing therapies and older cyclists . a more publicly discussed case ( but not one that led to a sanction ) was that of jeff hammond ( beaudin , 2013 ) who was a low - level amateur category 4 cyclist , aged 58 . in order to treat hypogonadism and low bone density when he contacted usada to request a tue he was denied , in effect being told to either stop racing or stop medicating . a letter from usada informed him that his tue was denied because his use was to treat a functional disorder without demonstrating the specific root cause of his hypogonadism : justification for the use of testosterone must meet the standard of demonstrating an organic cause of androgen deficiency / male hypogonadism . a diagnosis based simply on a functional disorder does not meet this standard rather functional diagnoses often focus solely on low testosterone and generalized symptoms ( cited in beaudin , 2013 ) . though hammond 's testosterone therapy has successfully restored his energy levels to where he feels normal , the lack of a tue prevents him from racing . hammond 's analysis cut to the core of the anti - doping policy challenge here : they 're treating us like 20-year old olympians . something that 's considered a performance - enhancing drug for an 18-year - old may be a necessary life - saving medication for a senior athlete . it was reported at this time that usada had ( in 2012 ) received 409 requests for tues , of which 52 were for anabolic agents such as testosterone . the science director for usada , we 're seeing more athletes that are at masters level realising that they were perhaps taking a prohibited substance there is a certain irony to the risk of running afoul of a policy designed to protect health by using a substance that a doctor agrees is for health protection . another source of information on this subject is andrew tilin 's ( 2011 ) self - experiment that formed the basis of his 2011 book the doper next door : my strange and scandalous year on performance enhancing drugs . tilin had interviewed papp for velonews and subsequently sought other sources of doping products . as a male over 40 treatment even when he admitted to the advisors that he intended to use these to improve his cycling . he showed that the anti - ageing industry had grown to significant proportions valued in the billions of dollars annually , its leading proponents had become millionaires . it seems that the confluence of cycling 's popularity with anti - aging opportunities , and the pressures to promote clean sport , has created a perfect storm of older riders who wish to maintain youthful vigour finding themselves punished and marginalised ( hoberman , 2005 ; lopez , 2011 ) . of course , many other cyclists may take the view that they are unlikely to be podium finishers in events that have testing procedures so therefore may simply continue with their medical prescription regimes . to avoid such cases becoming further examples of collateral damage of anti - doping efforts , addressing doping requires a different approach to those presented in studies of high level professional sports . the motivation to take a banned substance may be purely medical , or part of strategy to reduce the effects of the ageing process . the deterrents may also be fewer as there is little risk of getting caught , and a two - year sanction for a cyclist over the age of 40 may not seem like a particularly onerous punishment , especially for those for whom cycling is merely a hobby . rather than the extrinsic motivations often provided as the main rationale for doping , the cases of amateur cyclists demonstrate that the reasons for using banned substances are varied and may be unrelated to any perceived sports reward . nonetheless , if doping behaviours are on the increase among a wider population with variable access to medical expertise , then a potentially significant public health issue may be emerging ( sjqvist , garle , & rane , 2008 ) . the prevalence of doping with amateur ranks and competitions may be an unknown quantity but there has been sufficient anecdotal evidence to prompt grassroots organisations to develop their own anti - doping programmes ( burns , 2014 ) . partly , the basis for this is the failure of usada to provide sufficient levels of testing to deter doping . however , usa cycling has committed funding of $ 270,000 to cover professional and amateur events . the latter is on the condition that local cycling associations can match fund the costs involved , for which the full costs are $ 3500 for a single day event and $ 7000 for two days . by january 2014 , 16 local organisations , comprising 70% of usa cycling 's membership had joined the programme ( burns , 2014 ) . by so doing , the responsibility for initiating , planning and raising funds lies with amateur cyclists whose desire is to protect their specific local sports environment from doping . working in collaboration with usa cycling to apply the wada rules creates this perhaps unique situation in which non - experts are collectively imposing the rules designed for professionals upon themselves and their compatriots . this represents an interesting innovation , that builds upon notions of discipline and ( self ) surveillance that are integral to anti - doping ( henning , 2013 ) , but where the power to decide who is observed and disciplined lies with a small group of well - organised cyclists who aim to promulgate the ideologies and practices of systematic anti - doping policy . these developments may be unique , we do not know of other similar initiatives , and may be a precursor of other forms of grassroots anti - doping . it raises questions about decision - making , transparency and trust , when those leading the anti - doping groups are potentially open to bias in the course of targeting specific races and riders . moreover , given some of the problematic cases outlined above , how would local organisational anti - doping movements be sustainable if they were largely catching inadvertent dopers , older riders using prescription anti - ageing drugs , and recreational drug users ? the commonly accepted assumption about and reasons for doping by professionals do not necessarily hold true at the lower levels of cycling . at these lower levels questions of intent , knowledge , accident and lifestyle , these cases call into question the basic tenets of health promotion , fair play and spirit of sport underpinning anti - doping . a broader and in - depth investigation into doping cases in cycling reveals that current anti - doping policies can have severe implications for non - professional cyclists . though the patterns of actual use are unclear , there are indications that banned substances function as different practices at different levels of the sport . current anti - doping test and ban efforts have had some success detecting a number of cyclists using banned substances for performance enhancement , as evidenced by the 2012 decision and sanctions against members of the us postal service team . however , the relatively few anti - doping victories have done little to prevent a doping culture from taking root in us cycling . professional cyclists competing on the european circuit likely had to choose between acquiescing to new performance demands by taking banned substances or to race at a disadvantage against their doping counterparts . during the same period anti - doping efforts were unable to stymie the spread of doping at the elite level and doping culture spread to lower levels of the sport . this culture of risk around doping ( bette , 2004 ) was normalised in this environment as a way to be competitive . instead of being viewed as a deviant practice doping might be viewed as a way to fit into cycling culture and demonstrate one 's competitive status . thus , by the early 2000s testing results and discoveries of distribution networks like the papp list reveal that doping was occurring at all levels of cycling , though the focus remained on the elite cases where intentional doping was likely . this analysis has shown that while some aspirational non - professionals did use peds with the intention of cheating , many infractions by non - professional cyclists are likely the result of recreational drug use not intended to aid performance , supplements containing banned substances , or doctor prescribed medications or anti - ageing products . anti - doping regulations have not made allowances for these substances despite the trends of decriminalisation and even legalisation of cannabis , expanding markets for supplement and anti - ageing products , and the increasing social acceptability of each within wider society . the strict liability principle leaves little room for explaining innocuous or incidental use of banned substances , as presence immediately equals guilt regardless of intent . however , the time and financial costs of appealing a ruling may act as a disincentive to challenging a positive test for many amateurs . the low number of appeals by non - professionals has resulted in a situation where professional riders engaged in intentional , systematic doping serve lighter bans than their amateur counterparts . as such , the unintentional or recreational use of a banned substance can result in a relatively innocent cyclist becoming an incidental casualty in the war on doping , a situation that seemingly runs afoul of the notions of fairness and spirit of sport that anti - doping policies are meant to foster . these problems are further compounded by the problematic tue system under which athletes may be denied a waiver to use a medically prescribed banned substance while actively competing . in glaring contradiction to the principle of health promotion , some athletes seeking to comply with anti - doping regulations are effectively told to decide between taking a substance that may be medically necessary or stop competing . it is perhaps unsurprising that riders may forgo the process altogether , taking their chances at being asked to submit for a test . one innovative outcome has been the emergence of localised anti - doping , which aims to focus on reducing cheating through use of performance - enhancing drugs . however , if the established expertise of ( inter)national anti - doping agencies can not address the extent of doping but creates innocent victims in pursuit of that failed ambition , what hope is there for community groups forced to follow wada guidelines ? there is perhaps an opportunity for implementing standards different from those aimed at professionals and that take local lifestyles and the variety of drug use into account . if such a model for volunteer anti - doping could be established , almost like a 2nd tier doping control , then groups in other sports and other countries may help forge a closer link between anti - doping idealism and athlete behaviour . rather than relying on the national - level usada and nacas to decide exemptions and hear appeals , localised anti - doping groups could take governance in their own hands . local groups could set up low or no - cost arbitration programs to allow athletes to appeal or explain specific situations , such as amber neben 's tainted supplement case or jeff hammond 's therapeutic testosterone use . carrying out these functions themselves , in tandem with localised testing , these grassroots anti - doping groups may allow them to correct some of the injustices that result from the current centralised anti - doping system . thus behind the headlines of us postal and lance armstrong may lie a form of anti - doping with the potential for changing policy and engaging all athletes in a realistic approach to clean sport .

abstractthe focus of researchers , media and policy on doping in cycling is often limited to the professional level of the sport . however , anti - doping test results since 2001 demonstrate that banned substances are also used by us cyclists at lower levels of the sport , necessitating a broader view of the patterns and motivations of substance use within the sport . in this article , we describe and explain the doping culture that has emerged in domestic us cycling among amateur and semi - professionals . through analysis of records from sports governing bodies and journalistic reports , we assess the range of violation types and discuss the detection and punishing of riders who were not proven to have intended to cheat but became collateral damage in the war on doping . we argue that the phenomenon of doping is more complex than what has been shown to occur in elite sport , as it includes a wider variety of behaviours , situations and motivations . we develop fresh insights by examining cases where doping has been accidental , intrinsically motivated , non - performance enhancing or the result of prescribed medical treatments banned by anti - doping authorities . such trends call into question the fairness of anti - doping measures , and we discuss the possibility of developing localised solutions to testing and sanctioning amateur athletes .

Introduction Background Evidence and methods Testing and doping culture: not just an elite problem Collateral damage: recreational and unintentional use of banned substances Masters: relevance, therapeutic use, and anti-ageing Localised anti-doping: testing's new frontier? Conclusions

the focus of policy , media and many academic discourses on drugs in sport has been upon cheating and negative health outcomes ( gleaves , 2010 ; simon , 2004 ) . these are the two main underlying reasons for the funding and implementation of anti - doping testing , and the subsequent legal and quasi - legal process aimed at punishing the cheats . the development of organised international anti - doping policy has been based on these central tenets since the 1960s ( beamish & ritchie , 2004 ; dimeo , 2007 ; hunt , dimeo , & jedlicka , 2012 ) . previous research on doping in cycling has focused on the professional level ( mignon , 2003 ; ohl , fincoeur , lentillon - kaestner , defrance , & brissonneau , 2013 ; sefiha , 2012 ; waddington , 2000 ) , while largely neglecting the lower amateur levels of sport and masters competitors in higher age categories . in this study , we focus on the cases of all american cyclists , amateur and professional , who were caught doping and sanctioned during the period 2001 and summer 2014 . the range of cases includes athletes whose intentions to cheat others are not obvious , as athletes may have used recreational drugs like cannabis , used medicines that contained banned drugs without realising it , or bought and used nutritional supplements contaminated with banned substances . taking a broader view of us cycling that includes amateurs exposes patterns of doping behaviour beyond the narrow perspective of a small number of professionals whose motivations clearly lies with a desire for financial gain and celebrity status ( backhouse , mckenna , robinson , & atkin , 2007 ; laure & reinsberger , 1995 ) . in addition to elite cases , we describe and explain the doping culture that emerged in domestic us cycling among amateurs , those who compete at the lower categories of cycling , and semi - professionals , those who may win large races and prize purses but who do not have contracts with the largest professional teams . the critical approach adopted here aims to analyse and discuss the nature of policy in practice . we demonstrate the variety of doping situations and critically assess the outcomes of anti - doping against wada 's stated purpose and the general ideology underpinning anti - doping . the patterns that emerged starkly contrasted with the policy and media - led demonisation of doping athletes and with common assumptions of extrinsically motivated cheating . these cases point to possible trends in doping and raise new questions about the fairness of current anti - doping policies . the united states anti - doping agency ( usada ) was formed in 2000 as the trend globally was to move away from anti - doping being embedded within organisations with a vested interest in maintaining the integrity of their image , towards independent organisations that would be judged by their ability to protect thus , in parallel to the leadership shifting from the international olympic commission ( ioc ) to wada , the american re - organisation took responsibility away from the us olympic commission and established usada . establishing the nature of the doping culture within us cycling in the 1990s is challenging . however , matt dicanio and tyler hamilton both explained that their encounters with doping occurred only once they were established within the professional european scene . we would propose , therefore , that the extent of doping within the domestic context was relatively low in the late 1990s on the basis that american professionals learned from european doctors , and as we detail below , evidence of a doping culture really emerges around the time that lance armstrong was winning his seven consecutive tour de france titles . even when higher numbers of cyclists were caught , it may have been the result of the development or refinement of tests for specific drugs , rather than any specific pattern . many recent scholarly approaches to the study of doping in sport aim to support anti - doping through improving understanding of the contexts of athletes ' lives and training , psychological make - up and the processes through which they are educated ( backhouse & mckenna , 2012 ; morente - snchez & zabala , 2013 ; ntoumanis , ng , barkoukis , & backhouse , 2014 ) . while these studies can be credited for improving the evidence base , there appears to be several unchallenged assumptions : that the decision to dope firstly is a rational , intentional and conscious decision to cheat ( hoff , 2012 ; overbye , knudsen , & pfister , 2013 ) that emerges from inherent psychological traits ( bilard et al . , 2010 ) , secondly is perceived as deviant by athletes and their entourage ( christiansen , 2005 ; pitsch , emrich , & klein , 2007 ) , and lastly that anti - doping is a legitimate and fair policy that functions to catch dopers and deter potential dopers ( martin , baron , & gold , 2006 ; mazanov , huybers , & connor , 2011 ) . however , it could be more critically argued among this wealth of recent empirical knowledge there has not yet been a study that shows the variety of doping situations and describes patterns of doping behaviour according to this variety , despite the availability of information on every sanctioned athlete ( yonamine , garcia , & de moraes moreau , 2004 ) . the social groupings affected by anti - doping are much more diverse than previous studies have considered where the focus has been on the extrinsically motivated elite and aspiring elite , male athletes and their use or potential use of peds ( bloodworth & mcnamee , 2010 ; christiansen , 2010 ; outram & stewart , 2015 ) . this narrow focus has left gaps in the existing research , overlooking patterns of behaviours , motivations and situations occurring outside the upper reaches of the sport . beyond the level of the individual and their immediate circumstances , some recent historical , sociological and policy analyses of anti - doping have driven towards a more critical deconstruction of ideological principles , common sense claims , self - governance of international agencies and the equity of policy decisions ( denham , 2011 ; hanstad & waddington , 2009 ; kayser & broers , 2012 ; stewart & smith , 2008 ; straubel , 2008 ; wiesing , 2011 ) . mller ( 2014 ) has used the term corrupt idealism to frame his criticism of anti - doping leaders who are willing to accept or indeed promote unethical behaviour in order to support the there are cases of athletes who likely did not intend to violate anti - doping regulations , such as those who unintentionally ingested a banned substance ( cox , 2014 ; pluim , 2008 ) . thus , it may be that the war on drugs in sport has been so heavily skewed towards the claimed outcomes of catching cheats that relatively innocent athletes who did not intend to enhance performance ( pluim , 2008 ) are swept up , in a sense becoming collateral damage the critical approach adopted here therefore aims to analyse and discuss the nature of policy in practice . we use a case study to demonstrate the variety of doping situations and critically assess the outcomes of anti - doping against wada 's stated purpose and the general ideology underpinning anti - doping . the social groupings affected by anti - doping are much more diverse than previous studies have considered where the focus has been on elite , male athletes . first , we utilised the on - line records of the national governing body , usa cycling , and the usada , the organisation that caught the us postal riders . in our collection , we included the type of test ( in or out of competition ) , where the test was administered , the type and name of the substance the athlete tested positive for , and the length of the competition ban imposed . in the cases where the name search returned multiple or unrelated results , we entered a second search for the cyclists name with the word doping in each case this returned at least one relevant result other than the relevant usada press release . in cases outside of the widely covered elite doping scandals however , we agree with laurendeau and moroz ( 2012 ) that we might infer from the relatively small number of articles that the ideas that they produce and reproduce are so commonsense as to generate little controversy and , hence , little ( public ) discussion ( p. 387 ) yet are useful for analysis . as such , our findings provide correction to the myopic view of doping within us cycling . dopers who have not been caught claim to be clean , those under suspicion divert blame elsewhere to distract attention , those who are caught usually only admit to the specific offence that has been proven , and even full confessions can not be trusted ( lamont - mills & christensen , 2008 ) . therefore , rather than accepting any one account as the singular truth of a case we cross - referenced journalistic reports with all available information from usa cycling , usada , and the cas and took note of any inconsistencies or contradictions in the journalistic accounts . additionally , we included only professional journalist accounts , and excluded blogs , forums , and other user - generated material , as well as opinion pieces pertaining to the sub - cases from the analysis . as the us postal team became more successful the doping prevalent in european cycling seeped into the american context , laying the foundation for a culture of doping to emerge ( brewer , 2002 ; lentillon - kaestner & carstairs , 2010 ; lentillon - kaestner , hagger , & hardcastle , 2012 ; usada , 2012 ) . it seems clear that elite american cycling had , in a broad sense , accepted the necessity of doping in order to compete in major races against european teams . a number were sanctioned for non - analytical reasons , including the members of us postal who were subpoenaed by the federal investigators so had to confess . kayle leogrande 's case is more subtle : he told his team 's soigneur he was worried about being caught and she reported him to usada , suggesting that even the small number of known doping cases were not always the result of good anti - doping control mechanisms . the consequences of further investigations into papp 's customers led to a number of confessions and sanctions , and also provided some indication of wider doping among amateur and semi - professional teams . other cases seem to indicate a developing pattern of ped use among aspiring , non - elite cyclists . these and other similar cases demonstrate that drug use was indeed going on within the ranks of non - professional cycling . these cases are not directly linked to the 2012 us postal case but are a domestic sub - set of the doping culture that was disseminated from the elite context through the lower levels of the sport . kayle leogrande 's experience around the same time period helps to illustrate how strongly a doping culture had taken hold in american cycling , including among those cyclists who were not quite at the uppermost echelons of the sport . moreover , the former professional floyd landis gave evidence that the owner of the domestic team , rock racing , was involved in doping thus , we can see the emergence of doping as well as the failings of anti - doping to stop this culture from developing . yet , the authorities focused upon the top - level riders , in particular lance armstrong , and have not created an adequate system that would include regular , random in- and out - of - competition testing for the recreational and master levels of cycling . anti - doping policy did not serve to protect the fairness of sport or indeed the spirit of sport . as evidenced by the official doping cases , a sub - culture developed where amateur and semi - professional cyclists came to mimic the behaviours of their professional counterparts and indulged in doping . while it is hard to be certain about this , it may have been the case that a serious amateur competitor felt the need to participate in doping for reasons of identity , status and belonging within a social environment that respected and emulated those in the higher echelons of the sport . anti - doping efforts aimed at detecting those who seek to cheat may result in bans for athletes who never intended such . for example , the three ephedrine cases in 2000 were given shorter bans , suggesting that the authorities did not deem them to be worthy of a full sanction due to the relatively innocuous nature of the substance and the likelihood that it was not intentionally consumed . these are indicative of a wider pattern wherein the intention to cheat is unsubstantiated yet competition bans still apply . the chuck coyle episode highlights this , and he was informed that the cost of appealing the decision was around $ 20,000 , so he had no choice but to accept it . mller ( 2014 ) has recently argued that the creation of a doping - specific legal process is problematic , and does not give those charged with an offence the same legal protection or opportunities to defend their case in comparison to normal civic prosecutions . however , as in the 2003 cases of mountain bikers kathi krause and gary houseman , the use of narcotics and cannabis during competition is explicitly banned by the anti - doping agencies . bans for non - performance enhancing drugs do seem particularly harsh in the context of us cycling when an unknown quantity of riders doped to enhance performance without being caught , and when the majority of the us postal team was given reduced bans of six months because they provided additional information to assist the investigations . in total as mentioned above , they are generally accepted to be non - performance enhancing and the regulations an unnecessary intrusion into athletes ' private lives ( kayser , mauron , & miah , 2007 ) . there have also been several cases where the substance ingested appears to have come from a source unwittingly consumed by the athlete . she had the resources to take her appeal to the north american court of arbitration for sport ( nacas ) , and identified the source of the banned substance to be from a contaminated supplement . what neben 's appeal showed was potential negligence on the part of those who had responsibility to protect her from the risk of a positive test , and that all athletes are at risk of inadvertently taking contaminated supplements , which independent analyses have shown can contain banned substances ( cohen , 2009 ; cohen , travis , & venhuis , 2013 ) . taken together , these examples suggest that anti - doping regulations can have consequences for individuals whose offence is relatively innocuous , perhaps even completely unintentional , or even the result of negligence by their coaches or national governing bodies . yet there seems to be an acceptance among anti - doping policy decision - makers that there will be a degree of collateral damage in the pursuit of the real cheats . it seems ironic that lance armstrong negotiated his way around the anti - doping system for a period of 810 years , that his guilty team - mates received a light sanction and were allowed to return to the sport , yet others have faced consequences for behaviours that are completely different to the classic sense of doping as intentional cheating . the second offence appears to have been accidental , since he learned from his first to be vigiliant , he said : i still do not know how or why a banned stimulant was found in my system ( usa cycling , 2014 ) . thus , the increasing popularity of cycling in the usa , and the widening of race participation opportunities , has helped to create tribes of serious recreational riders who buy expensive equipment , learn about the science of training and preparation , and perhaps have a personal coach . the theme of older riders has another important dimension related to the notion of collateral damage : the overlap of anti - doping regulations and anti - ageing therapeutic drugs . other evidence points to the challenges associated with the overlap between anti - ageing therapies and older cyclists . hammond 's analysis cut to the core of the anti - doping policy challenge here : they 're treating us like 20-year old olympians . he showed that the anti - ageing industry had grown to significant proportions valued in the billions of dollars annually , its leading proponents had become millionaires . it seems that the confluence of cycling 's popularity with anti - aging opportunities , and the pressures to promote clean sport , has created a perfect storm of older riders who wish to maintain youthful vigour finding themselves punished and marginalised ( hoberman , 2005 ; lopez , 2011 ) . to avoid such cases becoming further examples of collateral damage of anti - doping efforts , addressing doping requires a different approach to those presented in studies of high level professional sports . rather than the extrinsic motivations often provided as the main rationale for doping , the cases of amateur cyclists demonstrate that the reasons for using banned substances are varied and may be unrelated to any perceived sports reward . the prevalence of doping with amateur ranks and competitions may be an unknown quantity but there has been sufficient anecdotal evidence to prompt grassroots organisations to develop their own anti - doping programmes ( burns , 2014 ) . this represents an interesting innovation , that builds upon notions of discipline and ( self ) surveillance that are integral to anti - doping ( henning , 2013 ) , but where the power to decide who is observed and disciplined lies with a small group of well - organised cyclists who aim to promulgate the ideologies and practices of systematic anti - doping policy . these developments may be unique , we do not know of other similar initiatives , and may be a precursor of other forms of grassroots anti - doping . it raises questions about decision - making , transparency and trust , when those leading the anti - doping groups are potentially open to bias in the course of targeting specific races and riders . moreover , given some of the problematic cases outlined above , how would local organisational anti - doping movements be sustainable if they were largely catching inadvertent dopers , older riders using prescription anti - ageing drugs , and recreational drug users ? at these lower levels questions of intent , knowledge , accident and lifestyle , these cases call into question the basic tenets of health promotion , fair play and spirit of sport underpinning anti - doping . a broader and in - depth investigation into doping cases in cycling reveals that current anti - doping policies can have severe implications for non - professional cyclists . though the patterns of actual use are unclear , there are indications that banned substances function as different practices at different levels of the sport . current anti - doping test and ban efforts have had some success detecting a number of cyclists using banned substances for performance enhancement , as evidenced by the 2012 decision and sanctions against members of the us postal service team . however , the relatively few anti - doping victories have done little to prevent a doping culture from taking root in us cycling . during the same period anti - doping efforts were unable to stymie the spread of doping at the elite level and doping culture spread to lower levels of the sport . this analysis has shown that while some aspirational non - professionals did use peds with the intention of cheating , many infractions by non - professional cyclists are likely the result of recreational drug use not intended to aid performance , supplements containing banned substances , or doctor prescribed medications or anti - ageing products . anti - doping regulations have not made allowances for these substances despite the trends of decriminalisation and even legalisation of cannabis , expanding markets for supplement and anti - ageing products , and the increasing social acceptability of each within wider society . as such , the unintentional or recreational use of a banned substance can result in a relatively innocent cyclist becoming an incidental casualty in the war on doping , a situation that seemingly runs afoul of the notions of fairness and spirit of sport that anti - doping policies are meant to foster . in glaring contradiction to the principle of health promotion , some athletes seeking to comply with anti - doping regulations are effectively told to decide between taking a substance that may be medically necessary or stop competing . one innovative outcome has been the emergence of localised anti - doping , which aims to focus on reducing cheating through use of performance - enhancing drugs . however , if the established expertise of ( inter)national anti - doping agencies can not address the extent of doping but creates innocent victims in pursuit of that failed ambition , what hope is there for community groups forced to follow wada guidelines ? carrying out these functions themselves , in tandem with localised testing , these grassroots anti - doping groups may allow them to correct some of the injustices that result from the current centralised anti - doping system . thus behind the headlines of us postal and lance armstrong may lie a form of anti - doping with the potential for changing policy and engaging all athletes in a realistic approach to clean sport .

a great challenge in immunology is the discovery of new targets to the development of vaccines against even common infectious diseases . the choice of these targets depends on the identification of elements responsible for the stimulation of immune responses . evidence for the sharing of these elements was first provided by cross - reactivity studies ( 14 ) . cross - reactivity is defined by the ability of a given t - cell population to recognize different peptide : major histocompatibility complex ( mhc ) class i complexes ( pmhc - i ) . this phenomenon plays an important role in antiviral cellular immunity , and there are several described cases of heterologous immunity involving completely unrelated viruses . additionally , it has also been suggested that knowledge about the molecular features responsible for cross - reactivity can be applied to the development of a new generation of wide - spectrum viral vaccines ( 5 ) . classical studies use epitope sequence data for selection of targets to be subsequently used in in vivo experiments . however , these data lack information about elements that are crucial for the stimulation of an appropriate immunity , such as topology and charges distribution of the molecule . these targets ( peptides ) are presented to the immune system in the context of mhc - i molecules , and essential structural information can be obtained from crystals of these pmhc - i complexes . these structures are determined mainly by x - ray diffraction and nuclear magnetic resonance , providing a general view of the pmhc - i surface that interacts with the t - cell receptor ( tcr ) . such complexes can be used for comparison between different epitopes presented by the same mhc or the same epitope presented by different mhc alleles , and they can be applied on vaccinology studies , pathogenesis of autoimmune diseases , as well as for the selection of therapeutic targets for cancer treatment . however , the number of pmhc - i structures available at the protein data bank ( pdb ) is extremely low compared with the number of different pmhc - i complexes that can be generated by the combination of a given mhc - i allele and potential pathogen - derived immunogenic targets ( thousands in a single pathogen ) . until september 2012 , there were 430 pmhc - i structures available at pdb ( http://www.rcsb.org ) , including redundant complexes , and 6252 human mhc - i alleles described at the hla ( human leucocyte antigens ) nomenclature website ( http://hla.alleles.org/nomenclature/stats.html ) , encoding 4578 different proteins ( allotypes ) . additionally , the process of protein crystallization is highly costly and time - consuming for a large - scale prospecting study of therapeutic targets . an interesting alternative is the acquisition of such structures through in silico approaches ( molecular modelling ) , which are considerably faster and cheaper . there are already some crystal structures of pmhc complexes available , and there are many softwares and databases allowing reliable homology modelling of proteins , such as modeller ( 6 ) , modbase ( 7 ) , hhpred ( 8) , phyre ( 9 ) , i - tasser ( 10 ) and so forth . however , it is important to note that pmhc modelling is not a trivial homology modelling procedure , and there are no available servers able to generate reliable models of pmhc complexes . to understand the complexity of this topic mhc modelling and peptide modelling. the mhc 3d structure is conserved in terms of secondary structure and global arrangement of its heavy chain domains , which allow successful modelling of different alleles , even using as template an mhc allele with important differences in the cleft conformation . for instance , our group was able to perform the cross - modelling of two different murine mhc - i alleles , h2-d and h2-k , demonstrating that our modelling procedure with modeller software was able to reproduce all aspects of the mhc - i cleft , despite the known differences in the template ( 11 ) . on the other hand , modbase ( 7 ) , for instance , has tools to model small peptides , but here we are not discussing the natural folding of a given peptide . as previously discussed by our group , the conformation of the peptide inside the mhc - i cleft is not given by its amino acid sequence or its own properties , but it is rather imposed by the mhc - i cleft conformation and requirements ( 12 ) . even the very same peptide will adopt different conformations when presented by different mhc - i alleles ( 13 ) ; therefore , even if the same epitope is already available in a pmhc - i crystal structure , it might not be a good template for modelling . we have considered these issues to develop a reliable technique for building pmhc - i complexes ( d1-em - d2 ) ( 12 ) , which has been validated for human ( hla - b*27:05 and hla - a*02:01 ) and murine alleles ( h2-k and h2-d ) . some alternative in silico techniques to construct pmhc complexes have already been developed ( 1416 ) , but the question relying above all current available approaches is how reliable are these modelled structures , considering the importance of the information that they carry ? in immunology , it is not enough for a model to just meet the basic stereochemical conditions of molecular modelling . for instance , disagreements in the residues that contact the tcr could compromise approaches that attempt to explain subtle differences between epitopes presented by a given mhc - i allele ( 17 ) . differences among the available methods for pmhc structure prediction can be found in a recent review published by antunes et al . , we are ( i ) using a high - resolution x - ray structure from the mhc of interest , which we will call mhc donor , ( ii ) fitting the target epitope backbone to an allele - specific pattern conformation ( previously determined ) and ( iii ) using a reliable molecular docking software ( 19 ) to identify the best conformation for each epitope s side chain inside the cleft . furthermore , we also perform an energy minimization step of the entire pmhc structure to adjust the mhc donor cleft to this new epitope followed by a second round of molecular docking . the accuracy of our approach was confirmed by blind reproduction of crystal pmhc structures , presenting 8mers , 9mers and 10mers , including human and murine restricted peptides . forty - six crystal structures were successfully reproduced with root - mean - square deviation ( rmsd ) values of 1.754 0.4675 ( for all epitope atoms , not only backbone atoms ) ( 12 ) . besides to provide complexes with low - rmsd values when compared with the reproduced crystal structures , our approach was also able to reproduce the molecular characteristics of the tcr - interacting surfaces ( figure 2 ) . this approach was successfully applied to identify molecular features involved with immunogenicity variation among naturally occurring variants of an immunodominant hepatitis c virus ( hcv)-derived epitope , indicating a shared pattern of charges distribution among complexes that stimulate an immune response ( 20 ) . in addition , it is important to consider the difficulty to reproduce currently proposed approaches , and the limited access to the data already generated by them . considering these issues , the crosstope structural data bank was created : the first repository of 3d structures exclusively of peptide : mhc complexes , including curated data on immunogenicity , similarity relationships and cross - reactivity . the structure of a given pmhc complex not yet determined by experimental methods can be modelled through the sequential use of docking energy minimization docking ( d1-em - d2 ) . the linear amino acid sequence of this peptide can be used as input for a pymol script ( a ) that generates a coordinate file ( pdb ) of the target epitope , fitting this backbone conformation to an allele - specific pattern . epitope 3d are used as input for a molecular docking with autodock vina ( b ) , generating a new pmhc . in this step , only the side chains of the epitope remain flexible , whereas the epitope backbone and the entire mhc structure will be kept rigid . to adjust the mhc cleft to this new epitope , an energy minimization ( em ) step of the complete pmhc complex is performed with gromacs package ( c ) , followed by a second docking ( d2 ) ( d ) , which will generate the optimized final structure of the desired pmhc complex . three complexes obtained through d1-em - d2 approach ( a c ) were compared with crystal structures presenting the same epitopes in the context of the same human mhc allele ( d f ) . these crystal structures were included in the crosstope db under the ids a0201_0095 , a0201_0101 and a0201_0102 , respectively . the modelled structures are similar to the reference crystal structure , especially regarding the charges distribution pattern over the tcr - interacting surface . minor topology differences , mainly in side chain orientations , are observed because of random differences in the crystallization process . the 2v2w crystal was used as mhc donor to build the three modelled complexes . the structure of a given pmhc complex not yet determined by experimental methods can be modelled through the sequential use of docking energy minimization docking ( d1-em - d2 ) . the linear amino acid sequence of this peptide can be used as input for a pymol script ( a ) that generates a coordinate file ( pdb ) of the target epitope , fitting this backbone conformation to an allele - specific pattern . epitope 3d are used as input for a molecular docking with autodock vina ( b ) , generating a new pmhc . in this step , only the side chains of the epitope remain flexible , whereas the epitope backbone and the entire mhc structure will be kept rigid . to adjust the mhc cleft to this new epitope , an energy minimization ( em ) step of the complete pmhc complex is performed with gromacs package ( c ) , followed by a second docking ( d2 ) ( d ) , which will generate the optimized final structure of the desired pmhc complex . three complexes obtained through d1-em - d2 approach ( a c ) were compared with crystal structures presenting the same epitopes in the context of the same human mhc allele ( d f ) . these crystal structures were included in the crosstope db under the ids a0201_0095 , a0201_0101 and a0201_0102 , respectively . the modelled structures are similar to the reference crystal structure , especially regarding the charges distribution pattern over the tcr - interacting surface . minor topology differences , mainly in side chain orientations , are observed because of random differences in the crystallization process . the 2v2w crystal was used as mhc donor to build the three modelled complexes . crosstope database consists of structures built through our previously commented approach and by crystallographic structures obtained from the protein data bank . in the first case , new pmhc complexes are continuously included through manual curation of updated literature , searching for immunogenic epitopes that will pass through our modelling process ( figure 3a ) . immunogenic epitopes included in the crosstope database are supposed to elicit an in vitro and/or in vivo ctl immune response . , there is a reference that presents the tests that testify the epitope immunogenicity , as well a link to the immune epitope database ( epitope i d by iedb ) , where such information will also be found ( in a more detailed way ) . this same literature revision criterion , searching for experimental evidence , was used to retrieve cross - reactive epitopes . crystallographic structures are included by the search of mhc crystals also complexed with immunogenic epitopes ( figure 3b ) . when the search for crystals recover two structures from the same peptide : mhc complex ( considered as redundant structures ) , only the best resolution structure is included in crosstope database . information regarding the inclusion of each complex is available on the main page of each complex , specifically in the item structure type , section complex information. structure that was obtained by the modelling process is indicated as model ( d1-em - d2) and crystal structures as crystal , with its respective pdb i d , which is linked to the pdb structure into the rcsb protein data bank . it is important to note that the crosstope database contains specially models , and the crystals are the minority . as previously commented , the crystals inclusion criteria follow a manual curated process , searching for structures containing immunogenic epitopes . thus , it is possible that some crystals already available have not yet been included . ( a ) new pmhc complexes are continuously included through manual curation of updated literature , searching for immunogenic epitopes that will pass through our modelling process ( d1-em - d2 ) . ( b ) structural databanks are continuously searched for pmhcs carrying immunogenic epitopes . in this process , pmhc / tcr complexes and complexes presenting mutations on mhc -chain are excluded . when the search recovers two structures from the same pmhc ( considered as redundant structures ) , only the best resolution structure is included in crosstope database . ( a ) new pmhc complexes are continuously included through manual curation of updated literature , searching for immunogenic epitopes that will pass through our modelling process ( d1-em - d2 ) . ( b ) structural databanks are continuously searched for pmhcs carrying immunogenic epitopes . in this process , pmhc / tcr complexes and complexes presenting mutations on mhc -chain are excluded . when the search recovers two structures from the same pmhc ( considered as redundant structures ) , only the best resolution structure is included in crosstope database . at the time of writing crosstope contained 182 non - redundant structures deposited ( 169 models and 13 crystals ) that belong to two different human alleles ( hla - a*02:01 and hla - b*27:05 ) and two murine alleles ( h2-d and h2-k ) . as aforementioned , we have included only crystals with immunogenic , non - redundant epitopes , i.e. crystals of the same mhc complexed with different epitopes . our crystal sample is inferior to the total number of pmhc - i structures available in pdb . the search for structures can be done by several ways : by mhc allele ( this option also presents the image of the structural pattern adopted by the epitope for the respective allele ) ; by peptide sequence ; by source protein ; and by source organism . the search returns an immunogenic target list , except when the search is done for a specific sequence of the peptide . by clicking the plus icon in a specific epitope from the list , an output containing a complete description ( manually curated ) of that target is generated ( figure 4 ) : complex code , source protein and source organism , epitope position , immunological background ( cross - reactivity data and immunogenicity degree presented by the epitopes ) and original reference of the epitopes , as well as links to the major databases in the area ( ncbi : http://www.ncbi.nlm.nih.gov/ ; uniprot : http://www.uniprot.org/ ; and iedb : http://www.iedb.org/ ) . at the bottom of the page , there is a java - integrated molecular viewer , jmol ( 21 ) , where it is possible to perform the initial visual inspection of the complexes . below , it is possible to download structural coordinates in .pdb format file , as well as topology and charges distribution files ( 5/+5 and 10/+10 kiloteslas ) in .jpg format , which can also be viewed online at the top of the page . it is also possible to select two epitopes from the list and compare them via the compare option . this function facilitates the comparison of the topology and charges distribution of tcr - interacting surfaces from two pmhc - i complexes , to inspect putative targets for stimulation of cross - reactivity against different epitopes ( see the example later in the text ) . ( a ) partial list of recovered complexes in a search by mhc ( hla - a*02:01 ) . ( b ) clicking the plus icon , the user can access information regarding both the epitope ( manually curated ) and the pmhc complex . links to iedb and uniprot are also provided , as well to download coordinate and charges distribution files . ( a ) partial list of recovered complexes in a search by mhc ( hla - a*02:01 ) . ( b ) clicking the plus icon , the user can access information regarding both the epitope ( manually curated ) and the pmhc complex . links to iedb and uniprot are also provided , as well to download coordinate and charges distribution files . a well - known case of cross - recognition involving the epitopes iv - m15866 ( gilgfvftl ) and hiv - gag7785 ( slyntiavl ) was investigated through the surface analysis , using the compare option . the visual inspection revealed a striking similarity between them , evidencing the reliability of this kind of investigation . here , we extended a previous study ( 20 ) and analysed 60 unrelated pmhc - i complexes presenting virus - derived peptides , in the context of the most frequent human mhc allele ( hla - a*02:01 ) . these complexes , 5 crystal structures and 55 in silico predicted structures , images of the tcr - interacting surface of these complexes , presenting the electrostatic potential distribution ( supplementary figure s1 ) , were used to extract the rgb ( red - green - blue ) colour histograms of seven selected regions . these regions were selected considering the spots of variation in charges distribution over the pmhc - i surface , and they are placed within an area corresponding to the already described footprints of public tcrs ( 22 ) . values of mean and standard deviation of the three rgb components , for each one of these selected areas , were used as input for multivariate statistical methods , to predict possible targets of cross - reactivity . our dataset included some peptides with already known cross - reactivity , and the hierarchical cluster analysis ( hca ) results were in agreement with the experimental background ( supplementary figure s2 ) . for instance , we included 10 variants of the wild - type immunodominant epitope hcv - ns31073 ( cv / ingvcwtv ) . the wild - type and all the cross - reactive variants ( genotypes 4 , 5 and 6 ) fall in the same group , whereas the non cross - reactive variant ( genotype 3 ) falls in a completely unrelated group . both complexes containing the epitopes iv - m15866 ( gilgfvftl ) and hiv - gag7785 ( slyntvatl ) grouped together . interestingly , this cluster also contained the cross - reactive variants of hcv - ns31073 epitope . cross - reactivity between this hcv immunodominant epitope and the hiv - gag7785 peptide has not been described so far . there is yet other two complexes included in the same cluster , presenting the llwtlvvll and the nlvpmvatv peptides , from the human herpes virus 4 ( lmp2329 ) and 5 ( pp65485 ) , respectively . it is important to note that the former peptide does not share even a single amino acid with the target peptide ( cv / ingvcwtv ) and , nevertheless , presented almost the same topology and charges distribution when presented in the context of hla - a*02:01 . the crosstope structural data bank opens a way for the exploration of an additional level of complexity of immunogenic epitopes , the comparison at the molecular level , hitherto confined to analysis of scarce pmhc - i complexes . for now , our approach is restricted to mhc alleles containing a sufficient number of different epitopes in pmhc - i crystals , so that the allele - specific structural pattern of them could be inferred ( 12 ) . the alleles already available include two murine mhcs largely used for in vitro / in vivo assays of immunogenicity and cross - reactivity , and also two key human mhcs . hla - a*02:01 is one of the most frequent human mhc alleles ( http://www.allelefrequencies.net/ ) , and hla - b*27:05 has important roles in autoimmunity ( 23,24 ) and also in viral control of hcv and hiv ( 2527 ) . additionally , our expectation is that we can perform the continuous inclusion of new complexes ( including new mhc alleles ) and the development of automated tools ( clustering cross - reactive targets ) . moreover , epitopes restricted to mhc - ii present a variable number of amino acids , and the structural patterns are not as conspicuous as in mhc - i epitopes . this could be explained by the differential nature ( feature ) of the mhc - i cleft , which presents closed extremities , forcing in a more explicit manner the epitopes to adopt more stringent structural patterns . moreover , for mhc - ii epitopes , we would need to define which core region would be located inside the cleft , and this is more difficult to define , even for sequence predictors . considering that these pmhc - i complexes are the putative carriers of the immunogenic signals in cytotoxic stimulation , and that structural features of the pmhc complexes , especially regarding to charges distribution over the tcr - interacting surface , are key elements for cross - reactivity and heterologous immunity the crosstope database was developed to give support to researchers interested in exploring such elements . in this context , crosstope provides images of the charges distribution over the tcr - interacting surface of each pmhc - i available in the databank for cross - reactivity prediction ( as in the example provided earlier in the text ) . we choose two different colouring spectra to represent the electrostatic potential ( 5 to + 5 kt and 10 to + 10 kt ) , which will be depicted as a gradient from dark red ( negative charges ) to dark blue ( positive charges ) . the pdb file for each complex is also provided , allowing the users to generate their own charges distribution file through the grasp2 program ( 28 ) , as well as to perform other structural analysis . it is important to note that the most important regions for predicting cross - reactivity in one subset might vary according to the mhc allele and even the t - cell population that is being considered . therefore , here we just provided one example of how these surface images can be used to make predictions about cross - reactivity . other researchers might want to define their own selected regions for analysis . in any case , the regions contacted by the tcr will be represented within the tcr - interacting surface , which are represented in the images available in our database . our ultimate goal is to provide a platform that allows scientists to perform the prospection of new cross - reactive targets , or even to identify the molecular basis for triggering an adequate immune response , envisaging a new generation of vaccines . cnpq , capes ( process no 23038.035722/2008 - 19 ) and a grant from bill & melinda gates foundation through the grand challenges exploration initiative ( grant i d 53049 ) . funding for open access charge : programa de ps - graduao em gentica e biologia molecular ( ppgbm / ufrgs ) .

the crosstope is a highly curate repository of three - dimensional structures of peptide : major histocompatibility complex ( mhc ) class i complexes ( pmhc - i ) . the complexes hosted by this databank were obtained in protein databases and by large - scale in silico construction of pmhc - i structures , using a new approach developed by our group . at this moment , the database contains 182 non - redundant pmhc - i complexes from two human and two murine alleles . a web server provides interface for database query . the user can download ( i ) structure coordinate files and ( ii ) topological and charges distribution maps images from the t - cell receptor - interacting surface of pmhc - i complexes . the retrieved structures and maps can be used to cluster similar epitopes in cross - reactivity approaches , to analyse viral escape mutations in a structural level or even to improve the immunogenicity of tumour antigens.database url : http://www.crosstope.com.br

Introduction Structures construction and obtention, query and retrieval Exampleuse of multivariate statistical methods for structural virtual screening of cross-reactive targets Discussion Supplementary Data Funding

the choice of these targets depends on the identification of elements responsible for the stimulation of immune responses . evidence for the sharing of these elements was first provided by cross - reactivity studies ( 14 ) . cross - reactivity is defined by the ability of a given t - cell population to recognize different peptide : major histocompatibility complex ( mhc ) class i complexes ( pmhc - i ) . additionally , it has also been suggested that knowledge about the molecular features responsible for cross - reactivity can be applied to the development of a new generation of wide - spectrum viral vaccines ( 5 ) . however , these data lack information about elements that are crucial for the stimulation of an appropriate immunity , such as topology and charges distribution of the molecule . these targets ( peptides ) are presented to the immune system in the context of mhc - i molecules , and essential structural information can be obtained from crystals of these pmhc - i complexes . these structures are determined mainly by x - ray diffraction and nuclear magnetic resonance , providing a general view of the pmhc - i surface that interacts with the t - cell receptor ( tcr ) . such complexes can be used for comparison between different epitopes presented by the same mhc or the same epitope presented by different mhc alleles , and they can be applied on vaccinology studies , pathogenesis of autoimmune diseases , as well as for the selection of therapeutic targets for cancer treatment . however , the number of pmhc - i structures available at the protein data bank ( pdb ) is extremely low compared with the number of different pmhc - i complexes that can be generated by the combination of a given mhc - i allele and potential pathogen - derived immunogenic targets ( thousands in a single pathogen ) . until september 2012 , there were 430 pmhc - i structures available at pdb ( http://www.rcsb.org ) , including redundant complexes , and 6252 human mhc - i alleles described at the hla ( human leucocyte antigens ) nomenclature website ( http://hla.alleles.org/nomenclature/stats.html ) , encoding 4578 different proteins ( allotypes ) . additionally , the process of protein crystallization is highly costly and time - consuming for a large - scale prospecting study of therapeutic targets . an interesting alternative is the acquisition of such structures through in silico approaches ( molecular modelling ) , which are considerably faster and cheaper . there are already some crystal structures of pmhc complexes available , and there are many softwares and databases allowing reliable homology modelling of proteins , such as modeller ( 6 ) , modbase ( 7 ) , hhpred ( 8) , phyre ( 9 ) , i - tasser ( 10 ) and so forth . however , it is important to note that pmhc modelling is not a trivial homology modelling procedure , and there are no available servers able to generate reliable models of pmhc complexes . the mhc 3d structure is conserved in terms of secondary structure and global arrangement of its heavy chain domains , which allow successful modelling of different alleles , even using as template an mhc allele with important differences in the cleft conformation . for instance , our group was able to perform the cross - modelling of two different murine mhc - i alleles , h2-d and h2-k , demonstrating that our modelling procedure with modeller software was able to reproduce all aspects of the mhc - i cleft , despite the known differences in the template ( 11 ) . as previously discussed by our group , the conformation of the peptide inside the mhc - i cleft is not given by its amino acid sequence or its own properties , but it is rather imposed by the mhc - i cleft conformation and requirements ( 12 ) . even the very same peptide will adopt different conformations when presented by different mhc - i alleles ( 13 ) ; therefore , even if the same epitope is already available in a pmhc - i crystal structure , it might not be a good template for modelling . we have considered these issues to develop a reliable technique for building pmhc - i complexes ( d1-em - d2 ) ( 12 ) , which has been validated for human ( hla - b*27:05 and hla - a*02:01 ) and murine alleles ( h2-k and h2-d ) . some alternative in silico techniques to construct pmhc complexes have already been developed ( 1416 ) , but the question relying above all current available approaches is how reliable are these modelled structures , considering the importance of the information that they carry ? for instance , disagreements in the residues that contact the tcr could compromise approaches that attempt to explain subtle differences between epitopes presented by a given mhc - i allele ( 17 ) . differences among the available methods for pmhc structure prediction can be found in a recent review published by antunes et al . , we are ( i ) using a high - resolution x - ray structure from the mhc of interest , which we will call mhc donor , ( ii ) fitting the target epitope backbone to an allele - specific pattern conformation ( previously determined ) and ( iii ) using a reliable molecular docking software ( 19 ) to identify the best conformation for each epitope s side chain inside the cleft . the accuracy of our approach was confirmed by blind reproduction of crystal pmhc structures , presenting 8mers , 9mers and 10mers , including human and murine restricted peptides . besides to provide complexes with low - rmsd values when compared with the reproduced crystal structures , our approach was also able to reproduce the molecular characteristics of the tcr - interacting surfaces ( figure 2 ) . this approach was successfully applied to identify molecular features involved with immunogenicity variation among naturally occurring variants of an immunodominant hepatitis c virus ( hcv)-derived epitope , indicating a shared pattern of charges distribution among complexes that stimulate an immune response ( 20 ) . in addition , it is important to consider the difficulty to reproduce currently proposed approaches , and the limited access to the data already generated by them . considering these issues , the crosstope structural data bank was created : the first repository of 3d structures exclusively of peptide : mhc complexes , including curated data on immunogenicity , similarity relationships and cross - reactivity . the structure of a given pmhc complex not yet determined by experimental methods can be modelled through the sequential use of docking energy minimization docking ( d1-em - d2 ) . the linear amino acid sequence of this peptide can be used as input for a pymol script ( a ) that generates a coordinate file ( pdb ) of the target epitope , fitting this backbone conformation to an allele - specific pattern . epitope 3d are used as input for a molecular docking with autodock vina ( b ) , generating a new pmhc . in this step , only the side chains of the epitope remain flexible , whereas the epitope backbone and the entire mhc structure will be kept rigid . three complexes obtained through d1-em - d2 approach ( a c ) were compared with crystal structures presenting the same epitopes in the context of the same human mhc allele ( d f ) . these crystal structures were included in the crosstope db under the ids a0201_0095 , a0201_0101 and a0201_0102 , respectively . the modelled structures are similar to the reference crystal structure , especially regarding the charges distribution pattern over the tcr - interacting surface . minor topology differences , mainly in side chain orientations , are observed because of random differences in the crystallization process . the 2v2w crystal was used as mhc donor to build the three modelled complexes . the structure of a given pmhc complex not yet determined by experimental methods can be modelled through the sequential use of docking energy minimization docking ( d1-em - d2 ) . the linear amino acid sequence of this peptide can be used as input for a pymol script ( a ) that generates a coordinate file ( pdb ) of the target epitope , fitting this backbone conformation to an allele - specific pattern . epitope 3d are used as input for a molecular docking with autodock vina ( b ) , generating a new pmhc . in this step , only the side chains of the epitope remain flexible , whereas the epitope backbone and the entire mhc structure will be kept rigid . three complexes obtained through d1-em - d2 approach ( a c ) were compared with crystal structures presenting the same epitopes in the context of the same human mhc allele ( d f ) . these crystal structures were included in the crosstope db under the ids a0201_0095 , a0201_0101 and a0201_0102 , respectively . the modelled structures are similar to the reference crystal structure , especially regarding the charges distribution pattern over the tcr - interacting surface . crosstope database consists of structures built through our previously commented approach and by crystallographic structures obtained from the protein data bank . immunogenic epitopes included in the crosstope database are supposed to elicit an in vitro and/or in vivo ctl immune response . , there is a reference that presents the tests that testify the epitope immunogenicity , as well a link to the immune epitope database ( epitope i d by iedb ) , where such information will also be found ( in a more detailed way ) . this same literature revision criterion , searching for experimental evidence , was used to retrieve cross - reactive epitopes . when the search for crystals recover two structures from the same peptide : mhc complex ( considered as redundant structures ) , only the best resolution structure is included in crosstope database . information regarding the inclusion of each complex is available on the main page of each complex , specifically in the item structure type , section complex information. it is important to note that the crosstope database contains specially models , and the crystals are the minority . as previously commented , the crystals inclusion criteria follow a manual curated process , searching for structures containing immunogenic epitopes . thus , it is possible that some crystals already available have not yet been included . in this process , pmhc / tcr complexes and complexes presenting mutations on mhc -chain are excluded . when the search recovers two structures from the same pmhc ( considered as redundant structures ) , only the best resolution structure is included in crosstope database . ( b ) structural databanks are continuously searched for pmhcs carrying immunogenic epitopes . in this process , pmhc / tcr complexes and complexes presenting mutations on mhc -chain are excluded . when the search recovers two structures from the same pmhc ( considered as redundant structures ) , only the best resolution structure is included in crosstope database . at the time of writing crosstope contained 182 non - redundant structures deposited ( 169 models and 13 crystals ) that belong to two different human alleles ( hla - a*02:01 and hla - b*27:05 ) and two murine alleles ( h2-d and h2-k ) . as aforementioned , we have included only crystals with immunogenic , non - redundant epitopes , i.e. our crystal sample is inferior to the total number of pmhc - i structures available in pdb . the search for structures can be done by several ways : by mhc allele ( this option also presents the image of the structural pattern adopted by the epitope for the respective allele ) ; by peptide sequence ; by source protein ; and by source organism . by clicking the plus icon in a specific epitope from the list , an output containing a complete description ( manually curated ) of that target is generated ( figure 4 ) : complex code , source protein and source organism , epitope position , immunological background ( cross - reactivity data and immunogenicity degree presented by the epitopes ) and original reference of the epitopes , as well as links to the major databases in the area ( ncbi : http://www.ncbi.nlm.nih.gov/ ; uniprot : http://www.uniprot.org/ ; and iedb : http://www.iedb.org/ ) . at the bottom of the page , there is a java - integrated molecular viewer , jmol ( 21 ) , where it is possible to perform the initial visual inspection of the complexes . below , it is possible to download structural coordinates in .pdb format file , as well as topology and charges distribution files ( 5/+5 and 10/+10 kiloteslas ) in .jpg format , which can also be viewed online at the top of the page . it is also possible to select two epitopes from the list and compare them via the compare option . this function facilitates the comparison of the topology and charges distribution of tcr - interacting surfaces from two pmhc - i complexes , to inspect putative targets for stimulation of cross - reactivity against different epitopes ( see the example later in the text ) . ( a ) partial list of recovered complexes in a search by mhc ( hla - a*02:01 ) . ( b ) clicking the plus icon , the user can access information regarding both the epitope ( manually curated ) and the pmhc complex . links to iedb and uniprot are also provided , as well to download coordinate and charges distribution files . ( a ) partial list of recovered complexes in a search by mhc ( hla - a*02:01 ) . ( b ) clicking the plus icon , the user can access information regarding both the epitope ( manually curated ) and the pmhc complex . links to iedb and uniprot are also provided , as well to download coordinate and charges distribution files . a well - known case of cross - recognition involving the epitopes iv - m15866 ( gilgfvftl ) and hiv - gag7785 ( slyntiavl ) was investigated through the surface analysis , using the compare option . here , we extended a previous study ( 20 ) and analysed 60 unrelated pmhc - i complexes presenting virus - derived peptides , in the context of the most frequent human mhc allele ( hla - a*02:01 ) . these complexes , 5 crystal structures and 55 in silico predicted structures , images of the tcr - interacting surface of these complexes , presenting the electrostatic potential distribution ( supplementary figure s1 ) , were used to extract the rgb ( red - green - blue ) colour histograms of seven selected regions . these regions were selected considering the spots of variation in charges distribution over the pmhc - i surface , and they are placed within an area corresponding to the already described footprints of public tcrs ( 22 ) . values of mean and standard deviation of the three rgb components , for each one of these selected areas , were used as input for multivariate statistical methods , to predict possible targets of cross - reactivity . our dataset included some peptides with already known cross - reactivity , and the hierarchical cluster analysis ( hca ) results were in agreement with the experimental background ( supplementary figure s2 ) . the wild - type and all the cross - reactive variants ( genotypes 4 , 5 and 6 ) fall in the same group , whereas the non cross - reactive variant ( genotype 3 ) falls in a completely unrelated group . both complexes containing the epitopes iv - m15866 ( gilgfvftl ) and hiv - gag7785 ( slyntvatl ) grouped together . interestingly , this cluster also contained the cross - reactive variants of hcv - ns31073 epitope . cross - reactivity between this hcv immunodominant epitope and the hiv - gag7785 peptide has not been described so far . there is yet other two complexes included in the same cluster , presenting the llwtlvvll and the nlvpmvatv peptides , from the human herpes virus 4 ( lmp2329 ) and 5 ( pp65485 ) , respectively . it is important to note that the former peptide does not share even a single amino acid with the target peptide ( cv / ingvcwtv ) and , nevertheless , presented almost the same topology and charges distribution when presented in the context of hla - a*02:01 . the crosstope structural data bank opens a way for the exploration of an additional level of complexity of immunogenic epitopes , the comparison at the molecular level , hitherto confined to analysis of scarce pmhc - i complexes . for now , our approach is restricted to mhc alleles containing a sufficient number of different epitopes in pmhc - i crystals , so that the allele - specific structural pattern of them could be inferred ( 12 ) . the alleles already available include two murine mhcs largely used for in vitro / in vivo assays of immunogenicity and cross - reactivity , and also two key human mhcs . additionally , our expectation is that we can perform the continuous inclusion of new complexes ( including new mhc alleles ) and the development of automated tools ( clustering cross - reactive targets ) . moreover , epitopes restricted to mhc - ii present a variable number of amino acids , and the structural patterns are not as conspicuous as in mhc - i epitopes . this could be explained by the differential nature ( feature ) of the mhc - i cleft , which presents closed extremities , forcing in a more explicit manner the epitopes to adopt more stringent structural patterns . considering that these pmhc - i complexes are the putative carriers of the immunogenic signals in cytotoxic stimulation , and that structural features of the pmhc complexes , especially regarding to charges distribution over the tcr - interacting surface , are key elements for cross - reactivity and heterologous immunity the crosstope database was developed to give support to researchers interested in exploring such elements . in this context , crosstope provides images of the charges distribution over the tcr - interacting surface of each pmhc - i available in the databank for cross - reactivity prediction ( as in the example provided earlier in the text ) . the pdb file for each complex is also provided , allowing the users to generate their own charges distribution file through the grasp2 program ( 28 ) , as well as to perform other structural analysis . it is important to note that the most important regions for predicting cross - reactivity in one subset might vary according to the mhc allele and even the t - cell population that is being considered . therefore , here we just provided one example of how these surface images can be used to make predictions about cross - reactivity . in any case , the regions contacted by the tcr will be represented within the tcr - interacting surface , which are represented in the images available in our database . our ultimate goal is to provide a platform that allows scientists to perform the prospection of new cross - reactive targets , or even to identify the molecular basis for triggering an adequate immune response , envisaging a new generation of vaccines . cnpq , capes ( process no 23038.035722/2008 - 19 ) and a grant from bill & melinda gates foundation through the grand challenges exploration initiative ( grant i d 53049 ) .

adjuvant radiotherapy ( rt ) after conservative surgery for early stage breast cancer is a standard of care that is well tolerated by the patients in terms of compliance and risk of adverse effects . in most cases , breast cancer patients have a long life expectancy with a risk of late normal tissue complications that may continue to accumulate years after treatment concludes . for this reason , late effects remain important health concerns for long - term survivors . common late toxicity manifestations in the breast include fibrosis , cutaneous atrophy and skin telangiectasia . in particular , fibrosis is the development of excess fibrous connective tissue due to fibroblasts proliferation because of tissue injury . there is considerable interindividual variability in the development of late toxicity , which depends on rt parameters such as total dose , dose per fraction , irradiated volume , and dose inhomogeneity , as well as on patient related factors such as age and life style factors . in the last decade , several studies have suggested that development of late normal tissue complications in the breast after rt is at least in part genetically determined . currently , clinical radiosensitivity is nowadays regarded as a complex polygenic trait resulting from the combined effects of multiple factors , each with relatively modest effects . therefore , an approach based on the combination of multiple genetic factors into a genetic risk score ( grs ) may be an attractive strategy for prediction of adverse rt effects . dna methylation is an epigenetic mechanism established and maintained by a family of dna methyltransferase ( dnmt ) enzymes that catalyze the addition of a methyl group to cytosine residues , using s - adenosylmethionine as the methyl group donor . dna methylation generally occurs on cytosine residues located in cpg dinucleotides within the promoter region , resulting in chromatin compaction and gene silencing . three active dnmts have been described in mammals : dnmt1 is required for maintenance of methylation through generations , while dnmt3a and dnmt3b are mainly involved in establishment of de novo dna methylation patterns . in vitro and in vivo experimental evidence suggests that enzymes of the methylation machinery play a role in fibrogenesis and radiation response . for instance , upregulation of dnmt1 has been detected in the fibrotic tissue of the skin , kidneys , lungs , and liver , whereas activation of myofibroblasts or hepatic stellate cells can be reversed via inhibition of dnmt1 by dna - demethylating drugs or by specific sirna shutdown . in addition , reduction of global methylation levels has been reported after irradiation , probably due to decreased expression of dnmt1 , dnmt3a , and dnmt3b . despite evidence of involvement of dna methylating enzymes in fibrogenesis and radiation response , no information is currently available regarding whether common genetic variants of dnmts genes contribute to the development of radiation - induced fibrosis in cancer patients . however , recent in vitro studies suggest that mitochondria are the primary loci of rt effects , and that mitochondrial dna haplogroups differently affect mrna expression of dnmt1 , dnmt3a , and dnmt3b , as well as global dna methylation levels . in the present study , we assessed the role of four single nucleotide polymorphisms ( snps ) of dnmt genes ( dnmt1 rs2228611 , dnmt3a rs1550117 , dnmt3a rs7581217 , and dnmt3b rs2424908 ) as risk factors for subcutaneous fibrosis in a cohort of italian breast cancer patients who received rt after breast conserving surgery . in addition to dnmt snps , we evaluated the predictive role of xrcc1 rs2682585 , which was previously reported to be associated with the standardized total average toxicity ( stat ) score , an index of overall toxicity combining skin toxicities and fibrosis of the breast . we also assessed the ability of the aforementioned snps to improve prediction accuracy when combined with mitochondrial haplogroup h , which we recently found to be independently associated with a lower hazard of radiationinduced fibrosis in breast cancer patients . this study included 286 caucasian patients affected by histologically confirmed breast cancer who underwent conservative surgery and adjuvant rt from 1989 to 2010 at our department of radiotherapy . briefly , rt consisted of two opposite tangential wedged beams , followed by a boost on the tumor bed . patients underwent whole breast rt with conventional fractionation to a total dose of 50 gy followed by boost dose on the tumor bed in cases of invasive tumors . at the time of patient recruitment , a peripheral blood sample was taken and stored at 4c until analysis . during annual follow - up visits ( last update on january 2015 ) , radiation oncologists evaluated the appearance of subcutaneous and cutaneous late toxicities , with particular attention to the onset of fibrosis toxicity was scored according to the late effects of normal tissue - subjective objective management analytical ( lent - soma ) scale . patients with moderate to severe fibrosis ( grade 2 ) were referred to as the " radiosensitive group " and compared to patients with no or minimal fibrotic reactions ( grade 0 - 1 , control group ) . this study was approved by the local ethics committees of our university hospital and met the requirements of the declaration of helsinki . determination of snps was conducted on genomic dna by real - time polymerase chain reaction ( pcr ) using the following taqman pre - designed snp genotyping assays ( applied biosystems , milan , italy ) : c_27838930_10 ( dnmt1 rs2228611 ) ; c_8722920_10 ( dnmt3a rs1550117 ) ; c_7863728_10 ( dnmt3a rs7581217 ) ; c_16013055_10 ( dnmt3b rs2424908 ) ; and c_16269889_10 ( xrcc1 rs2682585 ) . real - time pcr amplification and detection was performed in 96-well pcr plates using a cfx connect real - time pcr detection system ( bio - rad , milan , italy ) . each polymorphism was tested for deviation from the hardy - weinberg equilibrium ( hwe ) by use of pearson s chisquared test as implemented in finetti s program ( http://ihg.gsf.de/cgi-bin/hw/hwa1.pl ) . for the selected polymorphisms , we considered the co - dominant , dominant , and recessive modes of inheritance . the time to event end - point ( grade 2 fibrosis ) was calculated from the first session of rt , and patients not experiencing the end - point were censored at the last follow - up performed . the cumulative incidence of grade 2 fibrosis was calculated by the kaplan - meier method and comparisons between genotype groups were performed using the log - rank test . univariate cox regression analyses were performed to calculate the hazard ratio ( hr ) and the 95% confidence interval ( ci ) to evaluate the influence of genotypes on grade 2 fibrosis risk . multivariate adjustments were made for the mitochondrial haplogroup ( h vs. non - h ) and clinical variables having a cut - off p - value of < 0.20 in univariate cox analysis ( body mass index , breast diameter , adjuvant treatment , dose per fraction , radiation quality , acute skin toxicity , and postsurgical complications ) . to avoid the risk of over - fitting biases , only significant genetic factors were considered for construction of a grs . to accomplish this , the cox proportional hazard regression coefficient of each genetic factor was converted into an integer risk score by rounding the quotient and dividing the regression coefficient by a single constant . discrimination capabilities of genetic models were assessed using the area under the receiver operating characteristic curve ( roc ) . the area under the roc curve and comparisons between roc curves were calculated using the method described by delong et al . . because of the exploratory nature of this study , we reported nominal statistical associations ( p < 0.05 ) . adjusted p - values based on the bonferroni correction were also considered to avoid chance findings due to multiple testing of five snps , and the significance was lowered to p < 0.01 . this study included 286 caucasian patients affected by histologically confirmed breast cancer who underwent conservative surgery and adjuvant rt from 1989 to 2010 at our department of radiotherapy . briefly , rt consisted of two opposite tangential wedged beams , followed by a boost on the tumor bed . patients underwent whole breast rt with conventional fractionation to a total dose of 50 gy followed by boost dose on the tumor bed in cases of invasive tumors . at the time of patient recruitment , a peripheral blood sample was taken and stored at 4c until analysis . during annual follow - up visits ( last update on january 2015 ) , radiation oncologists evaluated the appearance of subcutaneous and cutaneous late toxicities , with particular attention to the onset of fibrosis toxicity was scored according to the late effects of normal tissue - subjective objective management analytical ( lent - soma ) scale . patients with moderate to severe fibrosis ( grade 2 ) were referred to as the " radiosensitive group " and compared to patients with no or minimal fibrotic reactions ( grade 0 - 1 , control group ) . this study was approved by the local ethics committees of our university hospital and met the requirements of the declaration of helsinki . determination of snps was conducted on genomic dna by real - time polymerase chain reaction ( pcr ) using the following taqman pre - designed snp genotyping assays ( applied biosystems , milan , italy ) : c_27838930_10 ( dnmt1 rs2228611 ) ; c_8722920_10 ( dnmt3a rs1550117 ) ; c_7863728_10 ( dnmt3a rs7581217 ) ; c_16013055_10 ( dnmt3b rs2424908 ) ; and c_16269889_10 ( xrcc1 rs2682585 ) . real - time pcr amplification and detection was performed in 96-well pcr plates using a cfx connect real - time pcr detection system ( bio - rad , milan , italy ) . each polymorphism was tested for deviation from the hardy - weinberg equilibrium ( hwe ) by use of pearson s chisquared test as implemented in finetti s program ( http://ihg.gsf.de/cgi-bin/hw/hwa1.pl ) . for the selected polymorphisms , we considered the co - dominant , dominant , and recessive modes of inheritance . the time to event end - point ( grade 2 fibrosis ) was calculated from the first session of rt , and patients not experiencing the end - point were censored at the last follow - up performed . the cumulative incidence of grade 2 fibrosis was calculated by the kaplan - meier method and comparisons between genotype groups were performed using the log - rank test . univariate cox regression analyses were performed to calculate the hazard ratio ( hr ) and the 95% confidence interval ( ci ) to evaluate the influence of genotypes on grade 2 fibrosis risk . multivariate adjustments were made for the mitochondrial haplogroup ( h vs. non - h ) and clinical variables having a cut - off p - value of < 0.20 in univariate cox analysis ( body mass index , breast diameter , adjuvant treatment , dose per fraction , radiation quality , acute skin toxicity , and postsurgical complications ) . to avoid the risk of over - fitting biases , only significant genetic factors were considered for construction of a grs . to accomplish this , the cox proportional hazard regression coefficient of each genetic factor was converted into an integer risk score by rounding the quotient and dividing the regression coefficient by a single constant . discrimination capabilities of genetic models were assessed using the area under the receiver operating characteristic curve ( roc ) . the area under the roc curve and comparisons between roc curves were calculated using the method described by delong et al . . 13.3.3 ( medcalc software , mariakerke , belgium ) software . because of the exploratory nature of this study , we reported nominal statistical associations ( p < 0.05 ) . adjusted p - values based on the bonferroni correction were also considered to avoid chance findings due to multiple testing of five snps , and the significance was lowered to p < 0.01 . overall , 51 of the 286 participants ( 17.8% ) experienced moderate to severe fibrosis ( lent - soma grade 2 ) , while 235 patients ( 82.2% ) had no or minimal fibrosis ( lentsoma grade 0 - 1 ) . detailed demographic and clinical data in the whole cohort of breast cancer patients and following stratification according to their radiosensitive status have been reported elsewhere . the distributions of genotype frequencies for the polymorphisms analyzed were in hwe ( all p > 0.05 ) . for the entire set of breast cancer patients , frequencies of minor variant alleles were 0.48 ( dnmt1 rs2228611 g ) , 0.09 ( dnmt3a rs1550117a ) , 0.35 ( dnmt3a rs7581217 t ) , 0.14 ( dnmt3b rs2424908 t ) , and 0.22 ( xrcc1 rs2682585 ) . kaplan - meier curves for dnmt1 rs2228611 showed differences among genotypes in the cumulative incidence of grade 2 subcutaneous fibrosis ( log - rank test p - value=0.018 ) ( fig . univariate cox regression analysis revealed that dnmt1 rs2228611 was associated with a lower risk of grade 2 fibrosis under either the codominant ( gg vs. aa : hr , 0.26 ; 95% ci , 0.10 to 0.70 ; p=0.007 ) or the recessive contrast ( gg vs. aa+ag : hr , 0.31 ; 95% ci , 0.12 to 0.78 ; p=0.013 ) ( table 1 ) . multivariate cox regression analysis adjusted for mitochondrial haplogroup ( h vs. non - h ) and clinical confounding factors revealed dnmt1 rs2228611 as an independent protective factor for moderate to severe radiation - induced fibrosis ( gg vs. aa : hr , 0.26 ; 95% ci , 0.10 to 0.71 ; p=0.009 ; gg vs. aa+ag : hr , 0.29 ; 95% ci , 0.12 to 0.75 ; p=0.011 ) ( table 1 ) . conversely , none of the other dnmt snps ( table 1 ) , nor xrcc1 rs2682585 ( table 2 ) were associated with radiation - induced fibrosis of the breast upon both univariate or multivariate cox regression analysis . it is worth noting that mitochondrial haplogroup h emerged as a significant protective factor in these multivariate models when dnmt1 rs2228611 was considered under the codominant ( hr , 0.48 ; 95% ci , 0.26 to 0.88 ; p=0.018 ) or the recessive mode of inheritance ( hr , 0.48 ; 95% ci , 0.26 to 0.89 ; p=0.019 ) . to understand the joint effects of genetic markers on individual risk of grade 2 fibrosis , we built a grs for grade 2 fibrosis based on different combinations of dnmt1 rs2228611 and/or mitochondrial haplogroups according to five cox regression models , as shown in table 3 . model 1 included mitochondrial haplogroups only ( h vs. non - h ) , model 2 and model 3 comprised the codominant or the recessive contrast of rs2228611 , respectively , model 4 combined mitochondrial haplogroup h with the codominant contrast of dnmt1 rs2228611 and model 5 incorporated mitochondrial haplogroup h with the recessive contrast of dnmt1 rs2228611 . we then performed roc curve analyses to determine the performance of the five models at discriminating patients with or without grade 2 fibrosis ( fig . the lowest discrimination accuracy , quantified by area under the receiver operating characteristic curves ( auc ) , was obtained for models 2 and 3 , which were based on the two contrasts of rs2228611 ( both auc , 0.587 ; 95% ci , 0.28 to 0.645 ) , and the highest was observed for model 5 , which included the combination of mitochondrial haplogroup h with the recessive contrast of rs2228611 ( auc , 0.655 ; 95% ci , 0.597 to 0.710 ) . 2 , adding the recessive contrast of dnmt1 rs2228611 to haplogroup h determined an increase of the discrimination accuracy from 0.595 ( 95% ci , 0.536 to 0.653 ) to 0.655 ( 95% ci , 0.597 to 0.710 ) . when applying the score developed in model 5 , the proportion of patients with grade 2 radiation - induced fibrosis for each score group showed an increasing trend from lower to higher sum risk scores : 3.4% ( score 0 ) , 10.0% ( score 1 ) , 14.0% ( score 2 ) , and 27.3% ( score 2 ) . the kaplan - meier analysis of model 5 revealed that patients with an increasing sum risk score showed a trend towards higher frequencies of grade 2 fibrosis ( p trend=0.0005 ) ( fig . overall , 51 of the 286 participants ( 17.8% ) experienced moderate to severe fibrosis ( lent - soma grade 2 ) , while 235 patients ( 82.2% ) had no or minimal fibrosis ( lentsoma grade 0 - 1 ) . detailed demographic and clinical data in the whole cohort of breast cancer patients and following stratification according to their radiosensitive status have been reported elsewhere . the distributions of genotype frequencies for the polymorphisms analyzed were in hwe ( all p > 0.05 ) . for the entire set of breast cancer patients , frequencies of minor variant alleles were 0.48 ( dnmt1 rs2228611 g ) , 0.09 ( dnmt3a rs1550117a ) , 0.35 ( dnmt3a rs7581217 t ) , 0.14 ( dnmt3b rs2424908 t ) , and 0.22 ( xrcc1 rs2682585 ) . kaplan - meier curves for dnmt1 rs2228611 showed differences among genotypes in the cumulative incidence of grade 2 subcutaneous fibrosis ( log - rank test p - value=0.018 ) ( fig . univariate cox regression analysis revealed that dnmt1 rs2228611 was associated with a lower risk of grade 2 fibrosis under either the codominant ( gg vs. aa : hr , 0.26 ; 95% ci , 0.10 to 0.70 ; p=0.007 ) or the recessive contrast ( gg vs. aa+ag : hr , 0.31 ; 95% ci , 0.12 to 0.78 ; p=0.013 ) ( table 1 ) . multivariate cox regression analysis adjusted for mitochondrial haplogroup ( h vs. non - h ) and clinical confounding factors revealed dnmt1 rs2228611 as an independent protective factor for moderate to severe radiation - induced fibrosis ( gg vs. aa : hr , 0.26 ; 95% ci , 0.10 to 0.71 ; p=0.009 ; gg vs. aa+ag : hr , 0.29 ; 95% ci , 0.12 to 0.75 ; p=0.011 ) ( table 1 ) . conversely , none of the other dnmt snps ( table 1 ) , nor xrcc1 rs2682585 ( table 2 ) were associated with radiation - induced fibrosis of the breast upon both univariate or multivariate cox regression analysis . it is worth noting that mitochondrial haplogroup h emerged as a significant protective factor in these multivariate models when dnmt1 rs2228611 was considered under the codominant ( hr , 0.48 ; 95% ci , 0.26 to 0.88 ; p=0.018 ) or the recessive mode of inheritance ( hr , 0.48 ; 95% ci , 0.26 to 0.89 ; p=0.019 ) . to understand the joint effects of genetic markers on individual risk of grade 2 fibrosis , we built a grs for grade 2 fibrosis based on different combinations of dnmt1 rs2228611 and/or mitochondrial haplogroups according to five cox regression models , as shown in table 3 . model 1 included mitochondrial haplogroups only ( h vs. non - h ) , model 2 and model 3 comprised the codominant or the recessive contrast of rs2228611 , respectively , model 4 combined mitochondrial haplogroup h with the codominant contrast of dnmt1 rs2228611 and model 5 incorporated mitochondrial haplogroup h with the recessive contrast of dnmt1 rs2228611 . we then performed roc curve analyses to determine the performance of the five models at discriminating patients with or without grade 2 fibrosis ( fig . the lowest discrimination accuracy , quantified by area under the receiver operating characteristic curves ( auc ) , was obtained for models 2 and 3 , which were based on the two contrasts of rs2228611 ( both auc , 0.587 ; 95% ci , 0.28 to 0.645 ) , and the highest was observed for model 5 , which included the combination of mitochondrial haplogroup h with the recessive contrast of rs2228611 ( auc , 0.655 ; 95% ci , 0.597 to 0.710 ) . 2 , adding the recessive contrast of dnmt1 rs2228611 to haplogroup h determined an increase of the discrimination accuracy from 0.595 ( 95% ci , 0.536 to 0.653 ) to 0.655 ( 95% ci , 0.597 to 0.710 ) . when applying the score developed in model 5 , the proportion of patients with grade 2 radiation - induced fibrosis for each score group showed an increasing trend from lower to higher sum risk scores : 3.4% ( score 0 ) , 10.0% ( score 1 ) , 14.0% ( score 2 ) , and 27.3% the kaplan - meier analysis of model 5 revealed that patients with an increasing sum risk score showed a trend towards higher frequencies of grade 2 fibrosis ( p trend=0.0005 ) ( fig . in the present study , we tested whether the occurrence of radiation - induced fibrosis in breast cancer patients could be at least partially explained by genetic variability in dnmt genes , which encode for epigenetic enzymes known to be involved in fibrogenesis and radiation response . to accomplish this , we investigated the association between common polymorphisms in dnmt1 , dnmt3a , and dnmt3b genes and the risk of subcutaneous fibrosis in a cohort of breast cancer survivors who received rt after breast - conserving surgery . multivariate cox analysis revealed dnmt1 rs2228611gg genotype as an independent predictor of lower risk for radiation - induced skin fibrosis . to the best of our knowledge , this is the first study to suggest that genetic variability at the dnmt1 locus contributes to the development of late normal tissue complications after radiation therapy , which further supports the importance of dnmt1 on fibrogenesis and wound healing after radiation exposure . it is worth noting that xrcc1 rs2682585 , previously reported to be associated with an overall toxicity score combining skin toxicities and fibrosis of the breast , was not found to be correlated with fibrosis alone in the present study . snps in dnmts genes have primarily been investigated as risk factors for cancer susceptibility given that aberrant dna methylation is one of the earliest molecular events during carcinogenesis . in addition , since snps in dnmts genes influence dna methylation at global and gene - specific levels , they have also been also investigated as susceptibility factors for common complex diseases other than cancer . among the snps investigated in the present study , dnmt3a rs1550117 and dnmt3b rs2424908 have been associated with a lower cancer risk , as shown by recent meta - analyses . with regard to dnmt1 rs2228611 , the dnmt1 rs2228611gg genotype has been reported to be associated with a higher risk of ovarian cancer . although dnmt1 rs2228611 is a synonymous snp located in exon 17 , it has a possible splice regulatory function with a > g resulting in gaining of three exonic splicing enhancer binding motifs . in addition , dnmt1 rs2228611 has significant modifying effects on the inverse relationship between line-1 methylation , a marker of global methylation , and cadmium exposure in argentinean women , suggesting a possible role in regulating the methylation level in response to environmental cues . reactive oxygen species ( ros ) caused by radiation could be a common trigger mechanism in both fibrosis and radiation response . in oxygenated tumor and normal cells , mitochondria play a major role in the generation of ros and appear to be the primary loci of rt effects . conversely , mitochondrial polymorphisms defining major haplogroups have been suggested to alter oxidative phosphorylation coupling as well as ros production . in addition , cybrids containing mitochondrial haplogroup h have lower levels of dnmt1 , dnmt3a , and dnmt3b and global methylation than those with mitochondrial haplogroup j . based on our previous findings highlighting a protective role of mitochondrial haplogroup h on radiation - induced fibrosis , we built a grs based on its combination with dnmt1 rs2228611 . in line with the concern recently raised for high risk of over - fitting and false positive results of multifactorial genetic models , for the grs construction we only considered dnmt snps significantly associated with radiationinduced fibrosis , rather than using the total number of putative risk alleles . our results provided evidence of an increased discrimination accuracy when dnmt1 rs2228611 was combined with mitochondrial haplogroup h , suggesting potential usefulness of rs2228611 in genetic - based models for prediction of radiation - induced skin fibrosis . in addition , the method used for weighting predictors , which was based on cox hazard regression coefficients , can be easily applied for inclusion of clinical factors as well . unfortunately , none of the clinical or dosimetric variables analyzed emerged as independent predictors for grade 2 fibrosis , as we reported in our previous analysis . first , the lack of a second cohort of breast cancer patients precluded the possibility of validating dnmt1 rs2228611 as a predictive factor for radiation - induced fibrosis . thus , our results need to be interpreted cautiously because of the small sample size and risk of false positive findings given that very few radiogenomic studies with a candidate gene approach have been replicated in independent cohorts . second , in the present study we did not evaluate gene polymorphisms involved in enzymatic dna demethylation , such as the ten - eleven translocation ( tet ) protein family , which are known to play a role in fibrogenesis . therefore , a more comprehensive investigation of genes encoding epigenetic enzymes should be conducted to develop and validate a clinically useful geneticbased model for prediction of radiation - induced skin fibrosis . third , the predictive performance of the genetic model , based solely on rs2228611 and haplogroup h , is rather modest . however , this is expected for complex polygenic traits such as clinical radiosensitivity , which is probably the result of the combined effects of multiple polymorphic loci . in addition , further investigation in cellular and in vivo animal models is still required to provide mechanistic insights into the protective role of rs2228611gg on radiation - induced fibrosis . our findings support a protective effect of the dnmt1 rs2228611gg genotype on the risk of radiation - induced fibrosis in breast cancer patients . in addition , we provide evidence that inclusion of dnmt1 rs2228611 in a genetic - based model has the potential to increase prediction accuracy for radiation - induced fibrosis . however , further investigation to provide mechanistic insights into the role of dnmt1 rs2228611 in fibrogenesis and radiation response is warranted . in addition , a more comprehensive investigation of the role of germline polymorphisms should be conducted to develop and validate a clinically useful genetic - based model for prediction of radiation - induced fibrosis after treatment of breast cancer .

purposethis study was conducted to investigate the role of four polymorphic variants of dna methyltransferase genes as risk factors for radiation - induced fibrosis in breast cancer patients . we also assessed their ability to improve prediction accuracy when combined with mitochondrial haplogroup h , which we previously found to be independently associated with a lower hazard of radiation - induced fibrosis.materials and methodsdnmt1 rs2228611,dnmt3a rs1550117,dnmt3a rs7581217 , and dnmt3b rs2424908 were genotyped by real - time polymerase chain reaction in 286 italian breast cancer patients who received radiotherapy after breast conserving surgery . subcutaneous fibrosis was scored according to the late effects of normal tissue subjective objective management analytical ( lent - soma ) scale . the discriminative accuracy of genetic models was assessed by the area under the receiver operating characteristic curves ( auc).resultskaplan - meier curves showed significant differences among dnmt1 rs2228611 genotypes in the cumulative incidence of grade 2 subcutaneous fibrosis ( log - rank test p - value= 0.018 ) . multivariate cox regression analysis revealed dnmt1 rs2228611 as an independent protective factor for moderate to severe radiation - induced fibrosis ( gg vs. aa ; hazard ratio , 0.26 ; 95% confidence interval [ ci ] , 0.10 to 0.71 ; p=0.009 ) . adding dnmt1 rs2228611 to haplogroup h increased the discrimination accuracy ( auc ) of the model from 0.595 ( 95% ci , 0.536 to 0.653 ) to 0.655 ( 95% ci , 0.597 to 0.710).conclusiondnmt1 rs2228611 may represent a determinant of radiation - induced fibrosis in breast cancer patients with promise for clinical usefulness in genetic - based predictive models .

Introduction Materials and Methods 1. Study subject and data collection 2. Genotyping 3. Statistical analysis Results 1. Single locus analysis 2. GRS analysis Discussion Conclusion

in most cases , breast cancer patients have a long life expectancy with a risk of late normal tissue complications that may continue to accumulate years after treatment concludes . there is considerable interindividual variability in the development of late toxicity , which depends on rt parameters such as total dose , dose per fraction , irradiated volume , and dose inhomogeneity , as well as on patient related factors such as age and life style factors . for instance , upregulation of dnmt1 has been detected in the fibrotic tissue of the skin , kidneys , lungs , and liver , whereas activation of myofibroblasts or hepatic stellate cells can be reversed via inhibition of dnmt1 by dna - demethylating drugs or by specific sirna shutdown . despite evidence of involvement of dna methylating enzymes in fibrogenesis and radiation response , no information is currently available regarding whether common genetic variants of dnmts genes contribute to the development of radiation - induced fibrosis in cancer patients . in the present study , we assessed the role of four single nucleotide polymorphisms ( snps ) of dnmt genes ( dnmt1 rs2228611 , dnmt3a rs1550117 , dnmt3a rs7581217 , and dnmt3b rs2424908 ) as risk factors for subcutaneous fibrosis in a cohort of italian breast cancer patients who received rt after breast conserving surgery . in addition to dnmt snps , we evaluated the predictive role of xrcc1 rs2682585 , which was previously reported to be associated with the standardized total average toxicity ( stat ) score , an index of overall toxicity combining skin toxicities and fibrosis of the breast . we also assessed the ability of the aforementioned snps to improve prediction accuracy when combined with mitochondrial haplogroup h , which we recently found to be independently associated with a lower hazard of radiationinduced fibrosis in breast cancer patients . during annual follow - up visits ( last update on january 2015 ) , radiation oncologists evaluated the appearance of subcutaneous and cutaneous late toxicities , with particular attention to the onset of fibrosis toxicity was scored according to the late effects of normal tissue - subjective objective management analytical ( lent - soma ) scale . patients with moderate to severe fibrosis ( grade 2 ) were referred to as the " radiosensitive group " and compared to patients with no or minimal fibrotic reactions ( grade 0 - 1 , control group ) . this study was approved by the local ethics committees of our university hospital and met the requirements of the declaration of helsinki . determination of snps was conducted on genomic dna by real - time polymerase chain reaction ( pcr ) using the following taqman pre - designed snp genotyping assays ( applied biosystems , milan , italy ) : c_27838930_10 ( dnmt1 rs2228611 ) ; c_8722920_10 ( dnmt3a rs1550117 ) ; c_7863728_10 ( dnmt3a rs7581217 ) ; c_16013055_10 ( dnmt3b rs2424908 ) ; and c_16269889_10 ( xrcc1 rs2682585 ) . the cumulative incidence of grade 2 fibrosis was calculated by the kaplan - meier method and comparisons between genotype groups were performed using the log - rank test . univariate cox regression analyses were performed to calculate the hazard ratio ( hr ) and the 95% confidence interval ( ci ) to evaluate the influence of genotypes on grade 2 fibrosis risk . multivariate adjustments were made for the mitochondrial haplogroup ( h vs. non - h ) and clinical variables having a cut - off p - value of < 0.20 in univariate cox analysis ( body mass index , breast diameter , adjuvant treatment , dose per fraction , radiation quality , acute skin toxicity , and postsurgical complications ) . discrimination capabilities of genetic models were assessed using the area under the receiver operating characteristic curve ( roc ) . the area under the roc curve and comparisons between roc curves were calculated using the method described by delong et al . during annual follow - up visits ( last update on january 2015 ) , radiation oncologists evaluated the appearance of subcutaneous and cutaneous late toxicities , with particular attention to the onset of fibrosis toxicity was scored according to the late effects of normal tissue - subjective objective management analytical ( lent - soma ) scale . patients with moderate to severe fibrosis ( grade 2 ) were referred to as the " radiosensitive group " and compared to patients with no or minimal fibrotic reactions ( grade 0 - 1 , control group ) . this study was approved by the local ethics committees of our university hospital and met the requirements of the declaration of helsinki . determination of snps was conducted on genomic dna by real - time polymerase chain reaction ( pcr ) using the following taqman pre - designed snp genotyping assays ( applied biosystems , milan , italy ) : c_27838930_10 ( dnmt1 rs2228611 ) ; c_8722920_10 ( dnmt3a rs1550117 ) ; c_7863728_10 ( dnmt3a rs7581217 ) ; c_16013055_10 ( dnmt3b rs2424908 ) ; and c_16269889_10 ( xrcc1 rs2682585 ) . the cumulative incidence of grade 2 fibrosis was calculated by the kaplan - meier method and comparisons between genotype groups were performed using the log - rank test . univariate cox regression analyses were performed to calculate the hazard ratio ( hr ) and the 95% confidence interval ( ci ) to evaluate the influence of genotypes on grade 2 fibrosis risk . discrimination capabilities of genetic models were assessed using the area under the receiver operating characteristic curve ( roc ) . the area under the roc curve and comparisons between roc curves were calculated using the method described by delong et al . overall , 51 of the 286 participants ( 17.8% ) experienced moderate to severe fibrosis ( lent - soma grade 2 ) , while 235 patients ( 82.2% ) had no or minimal fibrosis ( lentsoma grade 0 - 1 ) . detailed demographic and clinical data in the whole cohort of breast cancer patients and following stratification according to their radiosensitive status have been reported elsewhere . for the entire set of breast cancer patients , frequencies of minor variant alleles were 0.48 ( dnmt1 rs2228611 g ) , 0.09 ( dnmt3a rs1550117a ) , 0.35 ( dnmt3a rs7581217 t ) , 0.14 ( dnmt3b rs2424908 t ) , and 0.22 ( xrcc1 rs2682585 ) . kaplan - meier curves for dnmt1 rs2228611 showed differences among genotypes in the cumulative incidence of grade 2 subcutaneous fibrosis ( log - rank test p - value=0.018 ) ( fig . univariate cox regression analysis revealed that dnmt1 rs2228611 was associated with a lower risk of grade 2 fibrosis under either the codominant ( gg vs. aa : hr , 0.26 ; 95% ci , 0.10 to 0.70 ; p=0.007 ) or the recessive contrast ( gg vs. aa+ag : hr , 0.31 ; 95% ci , 0.12 to 0.78 ; p=0.013 ) ( table 1 ) . multivariate cox regression analysis adjusted for mitochondrial haplogroup ( h vs. non - h ) and clinical confounding factors revealed dnmt1 rs2228611 as an independent protective factor for moderate to severe radiation - induced fibrosis ( gg vs. aa : hr , 0.26 ; 95% ci , 0.10 to 0.71 ; p=0.009 ; gg vs. aa+ag : hr , 0.29 ; 95% ci , 0.12 to 0.75 ; p=0.011 ) ( table 1 ) . conversely , none of the other dnmt snps ( table 1 ) , nor xrcc1 rs2682585 ( table 2 ) were associated with radiation - induced fibrosis of the breast upon both univariate or multivariate cox regression analysis . it is worth noting that mitochondrial haplogroup h emerged as a significant protective factor in these multivariate models when dnmt1 rs2228611 was considered under the codominant ( hr , 0.48 ; 95% ci , 0.26 to 0.88 ; p=0.018 ) or the recessive mode of inheritance ( hr , 0.48 ; 95% ci , 0.26 to 0.89 ; p=0.019 ) . to understand the joint effects of genetic markers on individual risk of grade 2 fibrosis , we built a grs for grade 2 fibrosis based on different combinations of dnmt1 rs2228611 and/or mitochondrial haplogroups according to five cox regression models , as shown in table 3 . we then performed roc curve analyses to determine the performance of the five models at discriminating patients with or without grade 2 fibrosis ( fig . the lowest discrimination accuracy , quantified by area under the receiver operating characteristic curves ( auc ) , was obtained for models 2 and 3 , which were based on the two contrasts of rs2228611 ( both auc , 0.587 ; 95% ci , 0.28 to 0.645 ) , and the highest was observed for model 5 , which included the combination of mitochondrial haplogroup h with the recessive contrast of rs2228611 ( auc , 0.655 ; 95% ci , 0.597 to 0.710 ) . 2 , adding the recessive contrast of dnmt1 rs2228611 to haplogroup h determined an increase of the discrimination accuracy from 0.595 ( 95% ci , 0.536 to 0.653 ) to 0.655 ( 95% ci , 0.597 to 0.710 ) . when applying the score developed in model 5 , the proportion of patients with grade 2 radiation - induced fibrosis for each score group showed an increasing trend from lower to higher sum risk scores : 3.4% ( score 0 ) , 10.0% ( score 1 ) , 14.0% ( score 2 ) , and 27.3% ( score 2 ) . the kaplan - meier analysis of model 5 revealed that patients with an increasing sum risk score showed a trend towards higher frequencies of grade 2 fibrosis ( p trend=0.0005 ) ( fig . overall , 51 of the 286 participants ( 17.8% ) experienced moderate to severe fibrosis ( lent - soma grade 2 ) , while 235 patients ( 82.2% ) had no or minimal fibrosis ( lentsoma grade 0 - 1 ) . detailed demographic and clinical data in the whole cohort of breast cancer patients and following stratification according to their radiosensitive status have been reported elsewhere . for the entire set of breast cancer patients , frequencies of minor variant alleles were 0.48 ( dnmt1 rs2228611 g ) , 0.09 ( dnmt3a rs1550117a ) , 0.35 ( dnmt3a rs7581217 t ) , 0.14 ( dnmt3b rs2424908 t ) , and 0.22 ( xrcc1 rs2682585 ) . kaplan - meier curves for dnmt1 rs2228611 showed differences among genotypes in the cumulative incidence of grade 2 subcutaneous fibrosis ( log - rank test p - value=0.018 ) ( fig . univariate cox regression analysis revealed that dnmt1 rs2228611 was associated with a lower risk of grade 2 fibrosis under either the codominant ( gg vs. aa : hr , 0.26 ; 95% ci , 0.10 to 0.70 ; p=0.007 ) or the recessive contrast ( gg vs. aa+ag : hr , 0.31 ; 95% ci , 0.12 to 0.78 ; p=0.013 ) ( table 1 ) . multivariate cox regression analysis adjusted for mitochondrial haplogroup ( h vs. non - h ) and clinical confounding factors revealed dnmt1 rs2228611 as an independent protective factor for moderate to severe radiation - induced fibrosis ( gg vs. aa : hr , 0.26 ; 95% ci , 0.10 to 0.71 ; p=0.009 ; gg vs. aa+ag : hr , 0.29 ; 95% ci , 0.12 to 0.75 ; p=0.011 ) ( table 1 ) . conversely , none of the other dnmt snps ( table 1 ) , nor xrcc1 rs2682585 ( table 2 ) were associated with radiation - induced fibrosis of the breast upon both univariate or multivariate cox regression analysis . it is worth noting that mitochondrial haplogroup h emerged as a significant protective factor in these multivariate models when dnmt1 rs2228611 was considered under the codominant ( hr , 0.48 ; 95% ci , 0.26 to 0.88 ; p=0.018 ) or the recessive mode of inheritance ( hr , 0.48 ; 95% ci , 0.26 to 0.89 ; p=0.019 ) . to understand the joint effects of genetic markers on individual risk of grade 2 fibrosis , we built a grs for grade 2 fibrosis based on different combinations of dnmt1 rs2228611 and/or mitochondrial haplogroups according to five cox regression models , as shown in table 3 . model 1 included mitochondrial haplogroups only ( h vs. non - h ) , model 2 and model 3 comprised the codominant or the recessive contrast of rs2228611 , respectively , model 4 combined mitochondrial haplogroup h with the codominant contrast of dnmt1 rs2228611 and model 5 incorporated mitochondrial haplogroup h with the recessive contrast of dnmt1 rs2228611 . we then performed roc curve analyses to determine the performance of the five models at discriminating patients with or without grade 2 fibrosis ( fig . the lowest discrimination accuracy , quantified by area under the receiver operating characteristic curves ( auc ) , was obtained for models 2 and 3 , which were based on the two contrasts of rs2228611 ( both auc , 0.587 ; 95% ci , 0.28 to 0.645 ) , and the highest was observed for model 5 , which included the combination of mitochondrial haplogroup h with the recessive contrast of rs2228611 ( auc , 0.655 ; 95% ci , 0.597 to 0.710 ) . 2 , adding the recessive contrast of dnmt1 rs2228611 to haplogroup h determined an increase of the discrimination accuracy from 0.595 ( 95% ci , 0.536 to 0.653 ) to 0.655 ( 95% ci , 0.597 to 0.710 ) . when applying the score developed in model 5 , the proportion of patients with grade 2 radiation - induced fibrosis for each score group showed an increasing trend from lower to higher sum risk scores : 3.4% ( score 0 ) , 10.0% ( score 1 ) , 14.0% ( score 2 ) , and 27.3% the kaplan - meier analysis of model 5 revealed that patients with an increasing sum risk score showed a trend towards higher frequencies of grade 2 fibrosis ( p trend=0.0005 ) ( fig . in the present study , we tested whether the occurrence of radiation - induced fibrosis in breast cancer patients could be at least partially explained by genetic variability in dnmt genes , which encode for epigenetic enzymes known to be involved in fibrogenesis and radiation response . to accomplish this , we investigated the association between common polymorphisms in dnmt1 , dnmt3a , and dnmt3b genes and the risk of subcutaneous fibrosis in a cohort of breast cancer survivors who received rt after breast - conserving surgery . multivariate cox analysis revealed dnmt1 rs2228611gg genotype as an independent predictor of lower risk for radiation - induced skin fibrosis . it is worth noting that xrcc1 rs2682585 , previously reported to be associated with an overall toxicity score combining skin toxicities and fibrosis of the breast , was not found to be correlated with fibrosis alone in the present study . snps in dnmts genes have primarily been investigated as risk factors for cancer susceptibility given that aberrant dna methylation is one of the earliest molecular events during carcinogenesis . among the snps investigated in the present study , dnmt3a rs1550117 and dnmt3b rs2424908 have been associated with a lower cancer risk , as shown by recent meta - analyses . with regard to dnmt1 rs2228611 , the dnmt1 rs2228611gg genotype has been reported to be associated with a higher risk of ovarian cancer . in addition , cybrids containing mitochondrial haplogroup h have lower levels of dnmt1 , dnmt3a , and dnmt3b and global methylation than those with mitochondrial haplogroup j . based on our previous findings highlighting a protective role of mitochondrial haplogroup h on radiation - induced fibrosis , we built a grs based on its combination with dnmt1 rs2228611 . our results provided evidence of an increased discrimination accuracy when dnmt1 rs2228611 was combined with mitochondrial haplogroup h , suggesting potential usefulness of rs2228611 in genetic - based models for prediction of radiation - induced skin fibrosis . first , the lack of a second cohort of breast cancer patients precluded the possibility of validating dnmt1 rs2228611 as a predictive factor for radiation - induced fibrosis . thus , our results need to be interpreted cautiously because of the small sample size and risk of false positive findings given that very few radiogenomic studies with a candidate gene approach have been replicated in independent cohorts . therefore , a more comprehensive investigation of genes encoding epigenetic enzymes should be conducted to develop and validate a clinically useful geneticbased model for prediction of radiation - induced skin fibrosis . however , this is expected for complex polygenic traits such as clinical radiosensitivity , which is probably the result of the combined effects of multiple polymorphic loci . in addition , further investigation in cellular and in vivo animal models is still required to provide mechanistic insights into the protective role of rs2228611gg on radiation - induced fibrosis . our findings support a protective effect of the dnmt1 rs2228611gg genotype on the risk of radiation - induced fibrosis in breast cancer patients . in addition , we provide evidence that inclusion of dnmt1 rs2228611 in a genetic - based model has the potential to increase prediction accuracy for radiation - induced fibrosis . in addition , a more comprehensive investigation of the role of germline polymorphisms should be conducted to develop and validate a clinically useful genetic - based model for prediction of radiation - induced fibrosis after treatment of breast cancer .

in mammals , dna methylation contributes to the establishment and maintenance of cell - type - specific gene expression programmes , imprinting , x - chromosome inactivation and genome stability ( bird , 2002 ) . the majority of genomic methylation occurs at cytosine residues within cpg dinucleotides and is catalysed by the dna methyltransferases ( dnmt ) 1 , 3a and 3b . dnmt1 is responsible for maintaining genomic methylation , whereas dnmt3a and dnmt3b are mainly involved in de novo establishment of methylation patterns during cellular differentiation ( leonhardt et al , 1992 ; li et al , 1992 ; lei et al , 1996 ; okano et al , 1999 ; spada et al , 2007 ) . nuclear protein of 95 kda ( np95 ; also known as uhrf1 ) has recently been identified as an essential co - factor for maintaining genomic methylation ( bostick et al , 2007 ; sharif et al , 2007 ; achour et al , 2008 ) . dnmt1 and np95 embryonic stem cells ( escs ) and embryos have similar reduced levels of dna methylation . in addition , np95 interacts with dnmt1 , binds hemi - methylated cpg sites through its set and ring associated ( sra ) domain and both np95 and dnmt1 accumulate at replication sites ( uemura et al , 2000 ; bostick et al , 2007 ; papait et al , 2007 ; arita et al , 2008 ; avvakumov et al , 2008 ; hashimoto et al , 2008 ) . thus , it has been proposed that np95 mediates maintenance of genomic methylation by recruiting dnmt1 to hemi - methylated cpg sites generated during replication . here , we investigated a possible involvement of np95 in epigenetic regulation beyond its role in dnmt1-mediated maintenance of dna methylation . we found that np95 interacts with the de novo methyltransferases , dnmt3a and dnmt3b , and mediates promoter silencing before dna methylation is detected . immunoprecipitation experiments showed that different isoforms of both de novo methyltransferases dnmt3a and dnmt3b interact with np95 in wild - type ( wt ) escs , including the more abundant dnmt3a2 and dnmt3b1 ( fig 1a ) . furthermore , using a green fluorescent protein ( gfp ) trap ( rothbauer et al , 2008 ) , we co - immunoprecipitated endogenous dnmt1 and isoforms of dnmt3a and dnmt3b with a gfp np95 fusion construct transiently expressed in np95 escs and , vice versa , endogenous np95 co - immunoprecipitated with gfp dnmt3a or gfp dnmt3b1 fusions in dnmt3a and 3b double knockout ( dko ) escs ( supplementary fig s1a , b online ) . in addition , we observed co - immunoprecipitation of endogenous dnmt3b and inverted ccaat box binding protein of 90 kda the human homologue of np95from human embryonic kidney 293 t ( hek293 t ) cell extracts ( supplementary fig s1c online ) . we confirmed the interaction of np95 with dnmt3a / b by using a recently developed fluorescent two hybrid assay ( f2h ; zolghadr et al , 2008 ) . dnmt3 fusion constructs were used as bait by tethering them to a lac operator array present in baby hamster kidney ( bhk ) cells , so that the array was visible as a distinct nuclear spot of enriched gfp fluorescence ( fig 1b ) . a cherry np95 fusion ( prey ) accumulated at this spot only when gfp fusions of full - length dnmt3a and dnmt3b1 or their amino - terminal regions were used as bait and not when their isolated carboxy - terminal catalytic domains were used . we further mapped the interaction of np95 with dnmt3a / b through co - immunoprecipitation of deletion constructs and isolated domains transiently expressed in hek293 t cells ( supplementary fig s2 online ) . the results were consistent with those produced by f2h : the n - terminal regions of dnmt3a and dnmt3b1 , but not their c - terminal catalytic domains , interacted with np95 . deletion of the phd or pwwp domains of dnmt3a and dnmt3b did not eliminate the interaction with np95 . we found that the sra domain and the n - terminal 298 amino acids of np95 , which include the ubiquitin - like domain , interacted with dnmt3a and dnmt3b1 , whereas the phd domain and the c - terminal 132 amino acids , including the ring domain , did not . furthermore , we observed co - immunoprecipitation of endogenous np95 with gfp dnmt3a and gfp dnmt3b transiently expressed in dnmt1 escs , indicating that dnmt3a and dnmt3b interact with np95 independently of dnmt1 ( supplementary fig s1d online ) . to compare the relative association between endogenous np95 and dnmts , we re - probed the blot in fig 1a with a dnmt1 antibody and observed a substantially weaker signal for the co - immunoprecipitated dnmt1 relative to the input than in the case of dnmt3a2 and dnmt3b1 ( supplementary fig s3a online ) . to compare further the stability of np95 interactions with the dnmts , we transiently co - expressed np95-his with gfp dnmt3a or gfp dnmt3b1 in hek293 t cells and immunoprecipitated with the gfp trap in the presence of different salt concentrations ( fig 1c ; supplementary fig s3b online ) . interestingly , under high salt conditions , the interaction between np95-his and gfp dnmt1 was lost , whereas co - immunoprecipitation of gfp these data clearly indicate that np95 interacts more strongly with the de novo methyltransferases , dnmt3a and dnmt3b , than with dnmt1 . as dna methylation has a central role in epigenetic silencing , we investigated the requirement of dna methyltransferases and np95 for promoter silencing in escs . we found that , on transient transfection of wt escs , constructs driven by the cytomegalovirus ( cmv ) promoter were rapidly silenced , as opposed to longer - lasting expression of constructs driven by the chimeric cmv early enhancer / chicken actin ( cag ) promoter ( fig 2 ) , which is consistent with the popularity of the cag promoter for stable transgene expression in escs and mice . escs were co - transfected with two distinct plasmids , one expressing monomeric red fluorescent protein ( mrfp ) under the cmv promoter , the other expressing gfp driven by the cag promoter . mrfp and gfp expression was monitored after transfection for up to ten days by using automated image acquisition and quantification of fluorescent signals ( supplementary fig s4a online ) . the ratio between mrfp and gfp expression declined steadily in wt escs , reflecting preferential silencing of the cmv promoter ( fig 2 ) . by contrast , dko escs and escs lacking all three major dna methyltransferases ( dnmt1 , 3a and 3b triple knockout ) showed no preferential silencing of the cmv promoter . surprisingly , np95 escs were also unable to silence the cmv promoter , whereas dnmt1 escs showed only partly reduced silencing under these conditions . similar results were obtained on swapping gfp and mrfp reporter sequences , ruling out potential artefacts due to differences in their coding sequences or stability of the reporter proteins ( supplementary fig s4b online ) . thus , despite expressing a full complement of dna methyltransferases , escs lacking np95 are as deficient in promoter silencing activity as escs lacking all three major dnmts . interestingly , promoter methylation was detected only ten days after transfection and was lower in np95 than in wt escs , whereas none of the dnmt mutant escs showed appreciable dna methylation ( fig 3 ; supplementary fig s5a online ) . at the same time no obvious methylation was detected in any of the esc lines within the cpg island of the cag promoter construct ( supplementary fig s5b online ) . thus , cmv promoter silencing depends on the presence of both np95 and de novo dnmts , but ensues well before de novo methylation of the promoter is detected . this prompted us to investigate the involvement of repressive histone methylation as a possible mechanism for the observed silencing . we found that in the absence of histone h3 lysine 9 methyltransferases ( h3k9mts ) , g9a or suv39h1/2 , silencing of the cmv promoter was completely abolished or reduced , respectively , indicating that g9a and , in part , suv39h1/2 are also required for silencing ( fig 2b ) . the results shown here indicate that np95 interacts with dnmt3a and dnmt3b and mediates silencing of the cmv promoter by mechanisms that are , at least initially , independent of de novo dna methylation . importantly , our data also show the involvement of h3k9mts , g9a and suv39h1/2 in cmv promoter silencing . h3k9mts were reported to associate with de novo dnmts , and major satellite repeats were found to be hypomethylated in escs lacking either suv39h1/2 or dnmt3 enzymes . however , major satellite transcript levels were altered only in suv39h1/2-deficient cells and not in dnmt3-deficient cells ( fuks et al , 2003 ; lehnertz et al , 2003 ; martens et al , 2005 ) . a recent study showed that g9a , dnmt1 , dnmt3a and dnmt3b are required for normal methylation at long terminal repeats of endogenous retrotransposable elements , although transcription of these elements was increased in dnmt - deficient escs , but not g9a - deficient escs ( dong et al , 2008 ) . furthermore , recent studies have shown that g9a interacts with dnmt3a and dnmt3b and mediates de novo methylation of the oct4 , nanog and dnmt3l promoters on retinoic - acid - induced differentiation of escs ( feldman et al , 2006 ; li et al , 2007 ; epsztejn - litman et al , 2008 ) . however , two of these studies showed that neither g9a nor de novo dnmts are required to silence the oct4 promoter , and g9a was also found to be dispensable for silencing the nanog and dnmt3l promoters ( feldman et al , 2006 ; epsztejn - litman et al , 2008 ) . in the third study , nanog , but not oct4 , was shown to be silenced in differentiating escs lacking both dnmt3a and dnmt3b ( li et al , 2007 ) . we found that silencing of oct4 during embryoid body differentiation is largely independent from the presence of np95 as well as all three major dnmts , and occurs in the absence of dna methylation ( d.m . these data , together with our findings on silencing of the cmv promoter in escs , indicate that dnmts , np95 and h3k9mts mediate silencing through many mechanisms that do not necessarily involve dna methylation and might depend on the presence of different cis elements and an intricate interplay with other epigenetic and transcription factors . interestingly , np95 was recently shown to interact with g9a ( kim et al , 2009 ) and here we show that silencing of the cmv promoter in escs strictly depends on np95 and on de novo dnmts as well as g9a . taken together these observations suggest that np95 , de novo dnmts and g9a might be involved in a common silencing pathway . in summary , our data clearly support a crucial role of np95 in epigenetic silencing mediated by de novo dna and histone methyltransferases , and make np95 an attractive target for epigenetic reprogramming strategies . immunoprecipitation experiments showed that different isoforms of both de novo methyltransferases dnmt3a and dnmt3b interact with np95 in wild - type ( wt ) escs , including the more abundant dnmt3a2 and dnmt3b1 ( fig 1a ) . furthermore , using a green fluorescent protein ( gfp ) trap ( rothbauer et al , 2008 ) , we co - immunoprecipitated endogenous dnmt1 and isoforms of dnmt3a and dnmt3b with a gfp np95 fusion construct transiently expressed in np95 escs and , vice versa , endogenous np95 co - immunoprecipitated with gfp dnmt3a or gfp dnmt3b1 fusions in dnmt3a and 3b double knockout ( dko ) escs ( supplementary fig s1a , b online ) . in addition , we observed co - immunoprecipitation of endogenous dnmt3b and inverted ccaat box binding protein of 90 kda the human homologue of np95from human embryonic kidney 293 t ( hek293 t ) cell extracts ( supplementary fig s1c online ) . we confirmed the interaction of np95 with dnmt3a / b by using a recently developed fluorescent two hybrid assay ( f2h ; zolghadr et al , 2008 ) . dnmt3 fusion constructs were used as bait by tethering them to a lac operator array present in baby hamster kidney ( bhk ) cells , so that the array was visible as a distinct nuclear spot of enriched gfp fluorescence ( fig 1b ) . a cherry np95 fusion ( prey ) accumulated at this spot only when gfp fusions of full - length dnmt3a and dnmt3b1 or their amino - terminal regions were used as bait and not when their isolated carboxy - terminal catalytic domains were used . we further mapped the interaction of np95 with dnmt3a / b through co - immunoprecipitation of deletion constructs and isolated domains transiently expressed in hek293 t cells ( supplementary fig s2 online ) . the results were consistent with those produced by f2h : the n - terminal regions of dnmt3a and dnmt3b1 , but not their c - terminal catalytic domains , interacted with np95 . deletion of the phd or pwwp domains of dnmt3a and dnmt3b did not eliminate the interaction with np95 . we found that the sra domain and the n - terminal 298 amino acids of np95 , which include the ubiquitin - like domain , interacted with dnmt3a and dnmt3b1 , whereas the phd domain and the c - terminal 132 amino acids , including the ring domain , did not . furthermore , we observed co - immunoprecipitation of endogenous np95 with gfp dnmt3a and gfp dnmt3b transiently expressed in dnmt1 escs , indicating that dnmt3a and dnmt3b interact with np95 independently of dnmt1 ( supplementary fig s1d online ) . to compare the relative association between endogenous np95 and dnmts , we re - probed the blot in fig 1a with a dnmt1 antibody and observed a substantially weaker signal for the co - immunoprecipitated dnmt1 relative to the input than in the case of dnmt3a2 and dnmt3b1 ( supplementary fig s3a online ) . to compare further the stability of np95 interactions with the dnmts , we transiently co - expressed np95-his with gfp dnmt3a or gfp dnmt3b1 in hek293 t cells and immunoprecipitated with the gfp trap in the presence of different salt concentrations ( fig 1c ; supplementary fig s3b online ) . interestingly , under high salt conditions , the interaction between np95-his and gfp dnmt1 was lost , whereas co - immunoprecipitation of gfp these data clearly indicate that np95 interacts more strongly with the de novo methyltransferases , dnmt3a and dnmt3b , than with dnmt1 . as dna methylation has a central role in epigenetic silencing , we investigated the requirement of dna methyltransferases and np95 for promoter silencing in escs . we found that , on transient transfection of wt escs , constructs driven by the cytomegalovirus ( cmv ) promoter were rapidly silenced , as opposed to longer - lasting expression of constructs driven by the chimeric cmv early enhancer / chicken actin ( cag ) promoter ( fig 2 ) , which is consistent with the popularity of the cag promoter for stable transgene expression in escs and mice . escs were co - transfected with two distinct plasmids , one expressing monomeric red fluorescent protein ( mrfp ) under the cmv promoter , the other expressing gfp driven by the cag promoter . mrfp and gfp expression was monitored after transfection for up to ten days by using automated image acquisition and quantification of fluorescent signals ( supplementary fig s4a online ) . the ratio between mrfp and gfp expression declined steadily in wt escs , reflecting preferential silencing of the cmv promoter ( fig 2 ) . by contrast , dko escs and escs lacking all three major dna methyltransferases ( dnmt1 , 3a and 3b triple knockout ) showed no preferential silencing of the cmv promoter . surprisingly , np95 escs were also unable to silence the cmv promoter , whereas dnmt1 escs showed only partly reduced silencing under these conditions . similar results were obtained on swapping gfp and mrfp reporter sequences , ruling out potential artefacts due to differences in their coding sequences or stability of the reporter proteins ( supplementary fig s4b online ) . thus , despite expressing a full complement of dna methyltransferases , escs lacking np95 are as deficient in promoter silencing activity as escs lacking all three major dnmts . interestingly , promoter methylation was detected only ten days after transfection and was lower in np95 than in wt escs , whereas none of the dnmt mutant escs showed appreciable dna methylation ( fig 3 ; supplementary fig s5a online ) . at the same time no obvious methylation was detected in any of the esc lines within the cpg island of the cag promoter construct ( supplementary fig s5b online ) . thus , cmv promoter silencing depends on the presence of both np95 and de novo dnmts , but ensues well before de novo methylation of the promoter is detected . this prompted us to investigate the involvement of repressive histone methylation as a possible mechanism for the observed silencing . we found that in the absence of histone h3 lysine 9 methyltransferases ( h3k9mts ) , g9a or suv39h1/2 , silencing of the cmv promoter was completely abolished or reduced , respectively , indicating that g9a and , in part , suv39h1/2 are also required for silencing ( fig 2b ) . the results shown here indicate that np95 interacts with dnmt3a and dnmt3b and mediates silencing of the cmv promoter by mechanisms that are , at least initially , independent of de novo dna methylation . importantly , our data also show the involvement of h3k9mts , g9a and suv39h1/2 in cmv promoter silencing . h3k9mts were reported to associate with de novo dnmts , and major satellite repeats were found to be hypomethylated in escs lacking either suv39h1/2 or dnmt3 enzymes . however , major satellite transcript levels were altered only in suv39h1/2-deficient cells and not in dnmt3-deficient cells ( fuks et al , 2003 ; lehnertz et al , 2003 ; martens et al , 2005 ) . a recent study showed that g9a , dnmt1 , dnmt3a and dnmt3b are required for normal methylation at long terminal repeats of endogenous retrotransposable elements , although transcription of these elements was increased in dnmt - deficient escs , but not g9a - deficient escs ( dong et al , 2008 ) . furthermore , recent studies have shown that g9a interacts with dnmt3a and dnmt3b and mediates de novo methylation of the oct4 , nanog and dnmt3l promoters on retinoic - acid - induced differentiation of escs ( feldman et al , 2006 ; li et al , 2007 ; epsztejn - litman et al , 2008 ) . however , two of these studies showed that neither g9a nor de novo dnmts are required to silence the oct4 promoter , and g9a was also found to be dispensable for silencing the nanog and dnmt3l promoters ( feldman et al , 2006 ; epsztejn - litman et al , 2008 ) . in the third study , nanog , but not oct4 , was shown to be silenced in differentiating escs lacking both dnmt3a and dnmt3b ( li et al , 2007 ) . we found that silencing of oct4 during embryoid body differentiation is largely independent from the presence of np95 as well as all three major dnmts , and occurs in the absence of dna methylation ( d.m . these data , together with our findings on silencing of the cmv promoter in escs , indicate that dnmts , np95 and h3k9mts mediate silencing through many mechanisms that do not necessarily involve dna methylation and might depend on the presence of different cis elements and an intricate interplay with other epigenetic and transcription factors . interestingly , np95 was recently shown to interact with g9a ( kim et al , 2009 ) and here we show that silencing of the cmv promoter in escs strictly depends on np95 and on de novo dnmts as well as g9a . taken together these observations suggest that np95 , de novo dnmts and g9a might be involved in a common silencing pathway . in summary , our data clearly support a crucial role of np95 in epigenetic silencing mediated by de novo dna and histone methyltransferases , and make np95 an attractive target for epigenetic reprogramming strategies . hek293 t cells , bhk cells and escs were cultured and transfected as described by schermelleh et al ( 2007 ) , except fugene hd ( roche , mannheim , germany ) was used for transfection of escs . the dnmt1 j1 escs used in this study were homozygous for the c allele ( lei et al , 1996 ) . bhk cells were co - transfected on glass coverslips with gfp dnmt3 and cherry np95 constructs using transfectin ( bio - rad , munich , germany ) according to the manufacturer 's instructions . cell fixation and microscopy were carried out as described by zolghadr et al ( 2008 ) . escs and hek293 t cell extracts were prepared in lysis buffer ( 20 mm tris hcl ( ph 7.5 ) , 0.5 mm edta , 2 mm phenylmethyl sulphonyl fluoride and 0.5% np40 ) containing 150 or 300 mm nacl ( high - salt condition ) and diluted with lysis buffer without np40 . gfp trap ( rothbauer et al , 2008 ) and a specific rabbit antiserum ( citterio et al , 2004 ) were used for immunoprecipitation of gfp fusions and endogenous np95 , respectively . gfp trap and protein g beads ( sigma , taufkirchen , germany ) were washed with dilution buffer containing increasing salt concentrations ( 150 and 300 mm , or 300 and 500 mm nacl for the high - salt condition ) and re - suspended in sds the following mouse monoclonal antibodies were used for immunoblotting : anti - his ( c - terminal , invitrogen , karlsruhe , germany ) , anti - dnmt3a ( clone 64b1446 , imgenex , san diego , ca , usa ) and anti - dnmt3b ( clone 52a1018 , abcam , cambridge , uk ) . np95 was detected with the same antiserum used for immunoprecipitation and a rabbit antiserum was used for detection of dnmt1 ( grohmann et al , 2005 ) . horseradish peroxidase - conjugated rabbit anti - mouse or goat anti - rabbit secondary antibodies ( sigma ) and ecl plus reagent ( ge healthcare , munich , germany ) were used for detection . escs were co - transfected with pcag - egfp - ib and pcmv - mrfp as described above and images from live cells were acquired at the indicated time points with an incell analyser 1000 ( ge healthcare ) using a 20 air objective ( na=0.45 ) and standard filter settings for gfp and rfp . a total of 90150 images were acquired for each channel , using the same exposure time throughout the time course . pictures were processed using a gaussian blur algorithm ( radius ( sigma)=2 ) , and a threshold for maximal signal and minimal background coverage was adjusted and applied to each channel ( supplementary fig s4a online ) . the threshold was converted into area selection and the total size of the selected area was measured . escs were transfected as in the silencing assay with pcag - egfp - ib and pcmv - mrfp , and gfp - positive cells were sequentially sorted with a facsvantage or facsaria ii ( becton dickinson , heidelberg , germany ) at days 2 , 6 and 10 after transfection . after each sorting , total dna was isolated using the qiamp dna mini kit ( qiagen , hilden , germany ) and bisulphite treated with the ez dna methylation - gold kit ( zymo research , orange , ca , usa ) . the following primers were used for pcr amplification : cmv - forward tgggatttttttatttggtagt ; cmv - reverse atgggagtttgttttggtatta ; cag - forward ggagaggtgaggaggtagttaattaga and cag - reverse ccccaaacccctcaaaactt .

recent studies have indicated that nuclear protein of 95 kda ( np95 ) is essential for maintaining genomic methylation by recruiting dna methyltransferase ( dnmt ) 1 to hemi - methylated sites . here , we show that np95 interacts more strongly with regulatory domains of the de novo methyltransferases dnmt3a and dnmt3b . to investigate possible functions , we developed an epigenetic silencing assay using fluorescent reporters in embryonic stem cells ( escs ) . interestingly , silencing of the cytomegalovirus promoter in escs preceded dna methylation and was strictly dependent on the presence of either np95 , histone h3 methyltransferase g9a or dnmt3a and dnmt3b . our results indicate a regulatory role for np95 , dnmt3a and dnmt3b in mediating epigenetic silencing through histone modification followed by dna methylation .

Introduction Results And Discussion Np95 interacts with Dnmt3a and Dnmt3b Np95, Dnmt3a/3b and G9a mediate epigenetic silencing Methods Supplementary Material

the majority of genomic methylation occurs at cytosine residues within cpg dinucleotides and is catalysed by the dna methyltransferases ( dnmt ) 1 , 3a and 3b . dnmt1 is responsible for maintaining genomic methylation , whereas dnmt3a and dnmt3b are mainly involved in de novo establishment of methylation patterns during cellular differentiation ( leonhardt et al , 1992 ; li et al , 1992 ; lei et al , 1996 ; okano et al , 1999 ; spada et al , 2007 ) . nuclear protein of 95 kda ( np95 ; also known as uhrf1 ) has recently been identified as an essential co - factor for maintaining genomic methylation ( bostick et al , 2007 ; sharif et al , 2007 ; achour et al , 2008 ) . dnmt1 and np95 embryonic stem cells ( escs ) and embryos have similar reduced levels of dna methylation . in addition , np95 interacts with dnmt1 , binds hemi - methylated cpg sites through its set and ring associated ( sra ) domain and both np95 and dnmt1 accumulate at replication sites ( uemura et al , 2000 ; bostick et al , 2007 ; papait et al , 2007 ; arita et al , 2008 ; avvakumov et al , 2008 ; hashimoto et al , 2008 ) . thus , it has been proposed that np95 mediates maintenance of genomic methylation by recruiting dnmt1 to hemi - methylated cpg sites generated during replication . here , we investigated a possible involvement of np95 in epigenetic regulation beyond its role in dnmt1-mediated maintenance of dna methylation . we found that np95 interacts with the de novo methyltransferases , dnmt3a and dnmt3b , and mediates promoter silencing before dna methylation is detected . immunoprecipitation experiments showed that different isoforms of both de novo methyltransferases dnmt3a and dnmt3b interact with np95 in wild - type ( wt ) escs , including the more abundant dnmt3a2 and dnmt3b1 ( fig 1a ) . furthermore , using a green fluorescent protein ( gfp ) trap ( rothbauer et al , 2008 ) , we co - immunoprecipitated endogenous dnmt1 and isoforms of dnmt3a and dnmt3b with a gfp np95 fusion construct transiently expressed in np95 escs and , vice versa , endogenous np95 co - immunoprecipitated with gfp dnmt3a or gfp dnmt3b1 fusions in dnmt3a and 3b double knockout ( dko ) escs ( supplementary fig s1a , b online ) . in addition , we observed co - immunoprecipitation of endogenous dnmt3b and inverted ccaat box binding protein of 90 kda the human homologue of np95from human embryonic kidney 293 t ( hek293 t ) cell extracts ( supplementary fig s1c online ) . a cherry np95 fusion ( prey ) accumulated at this spot only when gfp fusions of full - length dnmt3a and dnmt3b1 or their amino - terminal regions were used as bait and not when their isolated carboxy - terminal catalytic domains were used . we further mapped the interaction of np95 with dnmt3a / b through co - immunoprecipitation of deletion constructs and isolated domains transiently expressed in hek293 t cells ( supplementary fig s2 online ) . deletion of the phd or pwwp domains of dnmt3a and dnmt3b did not eliminate the interaction with np95 . we found that the sra domain and the n - terminal 298 amino acids of np95 , which include the ubiquitin - like domain , interacted with dnmt3a and dnmt3b1 , whereas the phd domain and the c - terminal 132 amino acids , including the ring domain , did not . furthermore , we observed co - immunoprecipitation of endogenous np95 with gfp dnmt3a and gfp dnmt3b transiently expressed in dnmt1 escs , indicating that dnmt3a and dnmt3b interact with np95 independently of dnmt1 ( supplementary fig s1d online ) . to compare further the stability of np95 interactions with the dnmts , we transiently co - expressed np95-his with gfp dnmt3a or gfp dnmt3b1 in hek293 t cells and immunoprecipitated with the gfp trap in the presence of different salt concentrations ( fig 1c ; supplementary fig s3b online ) . interestingly , under high salt conditions , the interaction between np95-his and gfp dnmt1 was lost , whereas co - immunoprecipitation of gfp these data clearly indicate that np95 interacts more strongly with the de novo methyltransferases , dnmt3a and dnmt3b , than with dnmt1 . as dna methylation has a central role in epigenetic silencing , we investigated the requirement of dna methyltransferases and np95 for promoter silencing in escs . we found that , on transient transfection of wt escs , constructs driven by the cytomegalovirus ( cmv ) promoter were rapidly silenced , as opposed to longer - lasting expression of constructs driven by the chimeric cmv early enhancer / chicken actin ( cag ) promoter ( fig 2 ) , which is consistent with the popularity of the cag promoter for stable transgene expression in escs and mice . the ratio between mrfp and gfp expression declined steadily in wt escs , reflecting preferential silencing of the cmv promoter ( fig 2 ) . by contrast , dko escs and escs lacking all three major dna methyltransferases ( dnmt1 , 3a and 3b triple knockout ) showed no preferential silencing of the cmv promoter . interestingly , promoter methylation was detected only ten days after transfection and was lower in np95 than in wt escs , whereas none of the dnmt mutant escs showed appreciable dna methylation ( fig 3 ; supplementary fig s5a online ) . at the same time no obvious methylation was detected in any of the esc lines within the cpg island of the cag promoter construct ( supplementary fig s5b online ) . thus , cmv promoter silencing depends on the presence of both np95 and de novo dnmts , but ensues well before de novo methylation of the promoter is detected . this prompted us to investigate the involvement of repressive histone methylation as a possible mechanism for the observed silencing . we found that in the absence of histone h3 lysine 9 methyltransferases ( h3k9mts ) , g9a or suv39h1/2 , silencing of the cmv promoter was completely abolished or reduced , respectively , indicating that g9a and , in part , suv39h1/2 are also required for silencing ( fig 2b ) . the results shown here indicate that np95 interacts with dnmt3a and dnmt3b and mediates silencing of the cmv promoter by mechanisms that are , at least initially , independent of de novo dna methylation . h3k9mts were reported to associate with de novo dnmts , and major satellite repeats were found to be hypomethylated in escs lacking either suv39h1/2 or dnmt3 enzymes . however , major satellite transcript levels were altered only in suv39h1/2-deficient cells and not in dnmt3-deficient cells ( fuks et al , 2003 ; lehnertz et al , 2003 ; martens et al , 2005 ) . a recent study showed that g9a , dnmt1 , dnmt3a and dnmt3b are required for normal methylation at long terminal repeats of endogenous retrotransposable elements , although transcription of these elements was increased in dnmt - deficient escs , but not g9a - deficient escs ( dong et al , 2008 ) . furthermore , recent studies have shown that g9a interacts with dnmt3a and dnmt3b and mediates de novo methylation of the oct4 , nanog and dnmt3l promoters on retinoic - acid - induced differentiation of escs ( feldman et al , 2006 ; li et al , 2007 ; epsztejn - litman et al , 2008 ) . in the third study , nanog , but not oct4 , was shown to be silenced in differentiating escs lacking both dnmt3a and dnmt3b ( li et al , 2007 ) . we found that silencing of oct4 during embryoid body differentiation is largely independent from the presence of np95 as well as all three major dnmts , and occurs in the absence of dna methylation ( d.m . these data , together with our findings on silencing of the cmv promoter in escs , indicate that dnmts , np95 and h3k9mts mediate silencing through many mechanisms that do not necessarily involve dna methylation and might depend on the presence of different cis elements and an intricate interplay with other epigenetic and transcription factors . interestingly , np95 was recently shown to interact with g9a ( kim et al , 2009 ) and here we show that silencing of the cmv promoter in escs strictly depends on np95 and on de novo dnmts as well as g9a . taken together these observations suggest that np95 , de novo dnmts and g9a might be involved in a common silencing pathway . in summary , our data clearly support a crucial role of np95 in epigenetic silencing mediated by de novo dna and histone methyltransferases , and make np95 an attractive target for epigenetic reprogramming strategies . immunoprecipitation experiments showed that different isoforms of both de novo methyltransferases dnmt3a and dnmt3b interact with np95 in wild - type ( wt ) escs , including the more abundant dnmt3a2 and dnmt3b1 ( fig 1a ) . furthermore , using a green fluorescent protein ( gfp ) trap ( rothbauer et al , 2008 ) , we co - immunoprecipitated endogenous dnmt1 and isoforms of dnmt3a and dnmt3b with a gfp np95 fusion construct transiently expressed in np95 escs and , vice versa , endogenous np95 co - immunoprecipitated with gfp dnmt3a or gfp dnmt3b1 fusions in dnmt3a and 3b double knockout ( dko ) escs ( supplementary fig s1a , b online ) . in addition , we observed co - immunoprecipitation of endogenous dnmt3b and inverted ccaat box binding protein of 90 kda the human homologue of np95from human embryonic kidney 293 t ( hek293 t ) cell extracts ( supplementary fig s1c online ) . a cherry np95 fusion ( prey ) accumulated at this spot only when gfp fusions of full - length dnmt3a and dnmt3b1 or their amino - terminal regions were used as bait and not when their isolated carboxy - terminal catalytic domains were used . the results were consistent with those produced by f2h : the n - terminal regions of dnmt3a and dnmt3b1 , but not their c - terminal catalytic domains , interacted with np95 . deletion of the phd or pwwp domains of dnmt3a and dnmt3b did not eliminate the interaction with np95 . we found that the sra domain and the n - terminal 298 amino acids of np95 , which include the ubiquitin - like domain , interacted with dnmt3a and dnmt3b1 , whereas the phd domain and the c - terminal 132 amino acids , including the ring domain , did not . furthermore , we observed co - immunoprecipitation of endogenous np95 with gfp dnmt3a and gfp dnmt3b transiently expressed in dnmt1 escs , indicating that dnmt3a and dnmt3b interact with np95 independently of dnmt1 ( supplementary fig s1d online ) . to compare further the stability of np95 interactions with the dnmts , we transiently co - expressed np95-his with gfp dnmt3a or gfp dnmt3b1 in hek293 t cells and immunoprecipitated with the gfp trap in the presence of different salt concentrations ( fig 1c ; supplementary fig s3b online ) . interestingly , under high salt conditions , the interaction between np95-his and gfp dnmt1 was lost , whereas co - immunoprecipitation of gfp these data clearly indicate that np95 interacts more strongly with the de novo methyltransferases , dnmt3a and dnmt3b , than with dnmt1 . as dna methylation has a central role in epigenetic silencing , we investigated the requirement of dna methyltransferases and np95 for promoter silencing in escs . we found that , on transient transfection of wt escs , constructs driven by the cytomegalovirus ( cmv ) promoter were rapidly silenced , as opposed to longer - lasting expression of constructs driven by the chimeric cmv early enhancer / chicken actin ( cag ) promoter ( fig 2 ) , which is consistent with the popularity of the cag promoter for stable transgene expression in escs and mice . the ratio between mrfp and gfp expression declined steadily in wt escs , reflecting preferential silencing of the cmv promoter ( fig 2 ) . by contrast , dko escs and escs lacking all three major dna methyltransferases ( dnmt1 , 3a and 3b triple knockout ) showed no preferential silencing of the cmv promoter . similar results were obtained on swapping gfp and mrfp reporter sequences , ruling out potential artefacts due to differences in their coding sequences or stability of the reporter proteins ( supplementary fig s4b online ) . interestingly , promoter methylation was detected only ten days after transfection and was lower in np95 than in wt escs , whereas none of the dnmt mutant escs showed appreciable dna methylation ( fig 3 ; supplementary fig s5a online ) . at the same time no obvious methylation was detected in any of the esc lines within the cpg island of the cag promoter construct ( supplementary fig s5b online ) . thus , cmv promoter silencing depends on the presence of both np95 and de novo dnmts , but ensues well before de novo methylation of the promoter is detected . this prompted us to investigate the involvement of repressive histone methylation as a possible mechanism for the observed silencing . we found that in the absence of histone h3 lysine 9 methyltransferases ( h3k9mts ) , g9a or suv39h1/2 , silencing of the cmv promoter was completely abolished or reduced , respectively , indicating that g9a and , in part , suv39h1/2 are also required for silencing ( fig 2b ) . the results shown here indicate that np95 interacts with dnmt3a and dnmt3b and mediates silencing of the cmv promoter by mechanisms that are , at least initially , independent of de novo dna methylation . h3k9mts were reported to associate with de novo dnmts , and major satellite repeats were found to be hypomethylated in escs lacking either suv39h1/2 or dnmt3 enzymes . however , major satellite transcript levels were altered only in suv39h1/2-deficient cells and not in dnmt3-deficient cells ( fuks et al , 2003 ; lehnertz et al , 2003 ; martens et al , 2005 ) . a recent study showed that g9a , dnmt1 , dnmt3a and dnmt3b are required for normal methylation at long terminal repeats of endogenous retrotransposable elements , although transcription of these elements was increased in dnmt - deficient escs , but not g9a - deficient escs ( dong et al , 2008 ) . furthermore , recent studies have shown that g9a interacts with dnmt3a and dnmt3b and mediates de novo methylation of the oct4 , nanog and dnmt3l promoters on retinoic - acid - induced differentiation of escs ( feldman et al , 2006 ; li et al , 2007 ; epsztejn - litman et al , 2008 ) . in the third study , nanog , but not oct4 , was shown to be silenced in differentiating escs lacking both dnmt3a and dnmt3b ( li et al , 2007 ) . we found that silencing of oct4 during embryoid body differentiation is largely independent from the presence of np95 as well as all three major dnmts , and occurs in the absence of dna methylation ( d.m . these data , together with our findings on silencing of the cmv promoter in escs , indicate that dnmts , np95 and h3k9mts mediate silencing through many mechanisms that do not necessarily involve dna methylation and might depend on the presence of different cis elements and an intricate interplay with other epigenetic and transcription factors . interestingly , np95 was recently shown to interact with g9a ( kim et al , 2009 ) and here we show that silencing of the cmv promoter in escs strictly depends on np95 and on de novo dnmts as well as g9a . taken together these observations suggest that np95 , de novo dnmts and g9a might be involved in a common silencing pathway . in summary , our data clearly support a crucial role of np95 in epigenetic silencing mediated by de novo dna and histone methyltransferases , and make np95 an attractive target for epigenetic reprogramming strategies . gfp trap ( rothbauer et al , 2008 ) and a specific rabbit antiserum ( citterio et al , 2004 ) were used for immunoprecipitation of gfp fusions and endogenous np95 , respectively . the threshold was converted into area selection and the total size of the selected area was measured . escs were transfected as in the silencing assay with pcag - egfp - ib and pcmv - mrfp , and gfp - positive cells were sequentially sorted with a facsvantage or facsaria ii ( becton dickinson , heidelberg , germany ) at days 2 , 6 and 10 after transfection .

crs has been classified broadly as crs with nasal polyps ( crswnp ) , crs without nasal polyps , and allergic fungal rhinosinusitis ( afrs ) . in particular , crswnp may have a complex pathogenesis and is thought to arise from multiple factors including allergy . there is recent evidence suggesting that 1 ) staphylococcus aureus enterotoxins , 2 ) type i hypersensitivity to fungus , and 3 ) non - immunoglobulin e ( ige)-mediated hypersensitivity to fungus may play a role in the pathogenesis of eosinophilic inflammation.1 however , the role of the microorganisms , particularly fungal pathogens , in the etiology of crs remains largely unknown . conflicting with the prevailing belief that fungi were responsible for crs in a selected group of patients with distinct pathophysiology , ponikau et al.2 and braun et al.3 observed that fungus is a ubiquitous intranasal presence , identified in close to 100% of both crs patients and controls . the former group also detected fungi along with eosinophil and eosinophil - degraded products with mucus . shin et al.4 exposed peripheral blood mononuclear cells to fungal antigens in vitro and reported increased interleukin ( il)5 and il-13 production in 89% of crs patients but not in controls . these observations formed the basis of the fungal hypothesis of crs . as further evidence , nasal mucus or tissue from crs patients triggered eosinophil migration,5 and alternaria fungus in particular can directly induce eosinophil degranulation mediated by protease - activated receptor ( par ) activation.6 however , other investigators reported the absence of a universal hyper - responsiveness to fungal antigens in crs patients.7,8 furthermore , a multicenter , randomized clinical trial of topical antifungal agents for crs eventually failed to show any evidence of efficacy,9 and a meta - analysis did not support the routine use of topical antifungals for crs.10 thus , the precise roles of fungi in the etiopathology of crs remain unknown . the present study was conducted to detect and identify fungal species from the nasal polyp tissues of eosinophilic and noneosinophilic crs using grocott methanamine silver staining and polymerase chain reaction ( pcr ) methods . moreover , the effects of fungal extracts identified in the nasal polyps were examined by the ex vivo cellular responses of dispersed nasal polyp cells ( dnpcs ) . thirty - five patients with crs with nasal polyps ( 21 males and 14 females , ranging in age from 2377 years , mean age of 49 years ) were consecutively recruited from the department of otorhinolaryngology of juntendo university hospital from april 2011 to march 2012 . crs with nasal polyps was diagnosed based on the criteria of the european position paper.11 none of the patients was treated with antibiotics , systemic or topical corticosteroids , or other immune - modulating drugs for at least 1 month before the surgery . the criteria of afrs of two positive findings , 1 ) specific ige antibodies against fungi , and 2 ) the presence of fungi in the sinus effusion using grocott methanamine cytological silver staining or microbiological examination . serum fungus - specific ige concentrations against alternaria , aspergillus , candida , penicillium , mucor , cladosporium , and pityrosporium were measured . patients with crswnp associated with current signs of purulent nasal discharge , chronic obstructive pulmonary disease , diffuse panbronchiolitis , fungal sinus disease , congenital mucociliary disease , or cystic fibrosis were excluded from this study . the control group consisted of 15 patients with pituitary tumor surgery ( four males and 11 females , age range from 36 to 73 years , mean age of 55 years ) . the study was approved by the ethics committee of the juntendo university faculty of medicine . surgically removed human nasal polyps located in the middle meatus were obtained from the patients with crswnp , and the mucosa of the sphenoid sinus as a control were procured from patients with pituitary tumor . they were treated with 70% ethanol and physiologic saline to eliminate microorganisms outside of the nasal polyps . the samples were placed immediately in a 50% glycerol , then transferred to a 80c freezer for storage until dna extraction could be performed . at the same time , some of these samples were fixed in 10% formalin , embedded in paraffin wax , processed routinely , and stained with hematoxylin - eosin . according to our previous studies,12,13 the eosinophilic and noneosinophilic groups were defined as eosinophil counts of the nasal polyps pf more and less 100/microacopic field ( magnification 400 ) using three fields , respectively . the total number of eosinophils present with a 10 10-mm reticulate present in the eyepiece was determined as the count per high - power field . after the nasal polyps were disrupted with glass beads and 100 l of tris - edta ( 10 mm tris - cl , 1 mm ethylenediaminetetraacetic acid ph 8.0 ) using a homogenizer ( biomasher ) ( takara bio inc . , otsu , japan ) , and 1 l of homogenized polyp samples were spread onto 5% sheep blood agar , chocolate agar , columbia anaerobic blood agar , drigalski agar , and sabouraud agar , and incubated for 2 weeks at 35c . all specimens were stained with grocott methanamine silver staining for confirmation of the existence of fungal organisms . pcr amplification of fungal specific ribosomal dna was performed using its-1f and its-4r primer , the pcr product covering the end of the 18srdna gene to the start of 26srdna gene and nl-1 and nl-4 primer , the pcr product covering d1/d2 26srdna ( table1 ) . a universal fungal primer set designed by the national institute for occupational safety and health , ff2 and fr1 primers specific for the amplification of 18s rdna , was also used . amplification was performed in a takara pcr thermal cycler dice gradient ( takara bio inc . pcr products were purified by the high pure pcr product purification kit ( roche , indianapolis , in ) . these amplifications were sequenced using its1f , its4r , nl1 , nl4 , ff2 , and fr1 primers by a big dye terminator v3.1 cycle sequencing kit and abi sequence analyzer 3730i ( applied biosystems inc . , carlsbad , ca ) . species identification was determined by a blast search(dna data bank of japan , mishima , japan ) . dnpcs were prepared from nasal polyps of eosinophilic crswnps in five randomly selected patients by enzymatic digestion , as described by okano et al.14 in flat - bottomed 24-well culture plates ( nunc , roskiide , denmark ) , 500 l of 1 10/ml dnpcs were stimulated with serial concentrations ( 2 and 200 g / ml ) of candida parapsilosis , rhodotorula mucilaginosa . the crude extracts of c parapsilosis and r mucilaginosa were provided by teikyo university japanese society for medical mycology research center . after the incubation , freeze - dried fungi were dissolved and sonicated in phosphate - buffered saline ( pbs ) with 0.2% nan3 . the antigen extracts of fungus were dissolved with pbs ( sigma - aldrich inc . , st . the culture supernatant was collected after 72 hours and stored at 80c , after which the levels of il-4 , il-5 , il-6 , il-8 , il-13 , il-17a , il-23 , il-25 , il-33 , eotaxin , rantes ( regulated on activation normal t - cell expressed and secreted ) , tumor necrosis factor ( tnf)- , interferon ( ifn)- , and granulocyte - macrophage colony - stimulating factor ( gm - csf ) were measured by bio - plex suspension array system ( bio - rad laboratories , inc . data were expressed as the fold change relative to that without stimulus of five cell cultures . all analyses were conducted using statmate iv for windows ( atms co. , ltd . , tokyo , japan ) . thirty - five patients with crs with nasal polyps ( 21 males and 14 females , ranging in age from 2377 years , mean age of 49 years ) were consecutively recruited from the department of otorhinolaryngology of juntendo university hospital from april 2011 to march 2012 . crs with nasal polyps was diagnosed based on the criteria of the european position paper.11 none of the patients was treated with antibiotics , systemic or topical corticosteroids , or other immune - modulating drugs for at least 1 month before the surgery . the criteria of afrs of two positive findings , 1 ) specific ige antibodies against fungi , and 2 ) the presence of fungi in the sinus effusion using grocott methanamine cytological silver staining or microbiological examination . serum fungus - specific ige concentrations against alternaria , aspergillus , candida , penicillium , mucor , cladosporium , and pityrosporium were measured . patients with crswnp associated with current signs of purulent nasal discharge , chronic obstructive pulmonary disease , diffuse panbronchiolitis , fungal sinus disease , congenital mucociliary disease , or cystic fibrosis were excluded from this study . the control group consisted of 15 patients with pituitary tumor surgery ( four males and 11 females , age range from 36 to 73 years , mean age of 55 years ) . the study was approved by the ethics committee of the juntendo university faculty of medicine . surgically removed human nasal polyps located in the middle meatus were obtained from the patients with crswnp , and the mucosa of the sphenoid sinus as a control were procured from patients with pituitary tumor . they were treated with 70% ethanol and physiologic saline to eliminate microorganisms outside of the nasal polyps . the samples were placed immediately in a 50% glycerol , then transferred to a 80c freezer for storage until dna extraction could be performed . at the same time , some of these samples were fixed in 10% formalin , embedded in paraffin wax , processed routinely , and stained with hematoxylin - eosin . according to our previous studies,12,13 the eosinophilic and noneosinophilic groups were defined as eosinophil counts of the nasal polyps pf more and less 100/microacopic field ( magnification 400 ) using three fields , respectively . the total number of eosinophils present with a 10 10-mm reticulate present in the eyepiece was determined as the count per high - power field . after the nasal polyps were disrupted with glass beads and 100 l of tris - edta ( 10 mm tris - cl , 1 mm ethylenediaminetetraacetic acid ph 8.0 ) using a homogenizer ( biomasher ) ( takara bio inc . , otsu , japan ) , and 1 l of homogenized polyp samples were spread onto 5% sheep blood agar , chocolate agar , columbia anaerobic blood agar , drigalski agar , and sabouraud agar , and incubated for 2 weeks at 35c . all specimens were stained with grocott methanamine silver staining for confirmation of the existence of fungal organisms . pcr amplification of fungal specific ribosomal dna was performed using its-1f and its-4r primer , the pcr product covering the end of the 18srdna gene to the start of 26srdna gene and nl-1 and nl-4 primer , the pcr product covering d1/d2 26srdna ( table1 ) . a universal fungal primer set designed by the national institute for occupational safety and health , ff2 and fr1 primers specific for the amplification of 18s rdna , was also used . amplification was performed in a takara pcr thermal cycler dice gradient ( takara bio inc . , pcr products were purified by the high pure pcr product purification kit ( roche , indianapolis , in ) . these amplifications were sequenced using its1f , its4r , nl1 , nl4 , ff2 , and fr1 primers by a big dye terminator v3.1 cycle sequencing kit and abi sequence analyzer 3730i ( applied biosystems inc . , carlsbad , ca ) . species identification was determined by a blast search(dna data bank of japan , mishima , japan ) . dnpcs were prepared from nasal polyps of eosinophilic crswnps in five randomly selected patients by enzymatic digestion , as described by okano et al.14 in flat - bottomed 24-well culture plates ( nunc , roskiide , denmark ) , 500 l of 1 10/ml dnpcs were stimulated with serial concentrations ( 2 and 200 g / ml ) of candida parapsilosis , rhodotorula mucilaginosa . the crude extracts of c parapsilosis and r mucilaginosa were provided by teikyo university japanese society for medical mycology research center . after the incubation , freeze - dried fungi were dissolved and sonicated in phosphate - buffered saline ( pbs ) with 0.2% nan3 . the antigen extracts of fungus were dissolved with pbs ( sigma - aldrich inc . , st . the culture supernatant was collected after 72 hours and stored at 80c , after which the levels of il-4 , il-5 , il-6 , il-8 , il-13 , il-17a , il-23 , il-25 , il-33 , eotaxin , rantes ( regulated on activation normal t - cell expressed and secreted ) , tumor necrosis factor ( tnf)- , interferon ( ifn)- , and granulocyte - macrophage colony - stimulating factor ( gm - csf ) were measured by bio - plex suspension array system ( bio - rad laboratories , inc . data were expressed as the fold change relative to that without stimulus of five cell cultures . all analyses were conducted using statmate iv for windows ( atms co. , ltd . , tokyo , japan ) . there was no microbiological growth ( including fungus ) in the culture media of the homogenized nasal polyps . furthermore , grocott methanamine silver staining for the all nasal polyps showed no fungal bodies . sixteen of 35 samples of the nasal polyps showed amplification of fungal dna . using the d1/d2 domain of the large subunit ( 26s ) ribosomal dna primer , nl1 /nl4 , we found that 16 cellular tissues of the homogenized nasal polyps ( 100% ) were positive for fungal dna , whereas using the 18s rdna primer , its1f / its4r amplified sequences four of the 16 ( 25% ) were positive . on the other hand , using the universal fungal primer set , ff2 and fr1 , no fungal dna was detected . incidentally , in none of the mucosa of the sphenoid sinus was fungal dna detected . the amplification products obtained from 16 patients were sequenced to identify the detected species in the tissue of the homogenized nasal polyps , such as c parapsilosis , r mucilaginosa , and aspergillus sp . table2 summarizes the data for all patients . the average number of eosinophils within the nasal polyps in which fungal dna was detected was significantly higher than that in the absence of fungal dna ( p < .01 , fig . f = female ; ige = immunoglobulin e ; m = male ; rast = radioallergosorbent test . comparison of the numbers of tissue eosinophils with nasal polyps between positive samples and those negative for fungal dna . the number of eosinophils in three fields with cell clusters were counted using light microscopy ( 400 magnification ) . the box and whisker plots show the median and the interquartile range of the number . the whiskers extend to the maximum and the minimum data points . to determine the biological and immunological roles of the identified fungi in crswnp in the present study , five samples of dnpcs were stimulated by extracts of c parapsilosis and r mucilaginosa . the expression levels of each cytokine and chemokine are summarized and compared with those of the unstimulated group in figure 2 ) . the secretion levels of the cytokines were set to the maximal level of each standard curve because the levels were above the maximal levels of detection . effect of extracts of candida parapsilosis and rhodotorula mucilaginosa on interleukin ( il)4 , il-5 , il-6 , il-8 , il-13 , il-17a , il-23 , il-25 , il-33 , eotaxin , rantes , interferon ( ifn)- , tumor necrosis factor ( tnf)- , and granulocyte - macrophage colony - stimulating factor ( gm - csf ) production by dispersed nasal polyp cells . five hundred microliters of 1 10/ml dispersed nasal polyp cells were collected after 72 hours , and the levels of the cytokines were determined by multiplex immunoassay . data were expressed as the fold change relative to that without stimulus of five cell cultures ( the means standard errors ) . [ color figure can be viewed in the online issue , which is available at www.laryngoscope.com . ] c parapsilosis stimulation of dnpcs significantly induced the secretion of il-5 , il-6 , il-8 , il-13 , rantes , tnf- , and gm - csf . the most remarkable upregulation was observed in il-6 ( approximately 100-fold ) and il-8 ( approximately 60-fold ) , followed by il-5 , tnf- , rantes , il-13 , and gm - csf . il-4 , il-17a , il-23 , and eotaxin showed no significant increase from stimulation by c parapsilosis extracts . no significant differences were seen in the responses between 2 and 200 g / ml of any cytokines . the stimulation of dnpcs by r mucilaginosa resulted in a significant induction of il-5 , il-8 , il-13 , il-17a , rantes , tnf- , and gm - csf . similar to c parapsilosis , r mucilaginosa most strongly upregulated the production of both il-6 and il-8 , which was followed by il-5 , tnf- , rantes , il-13 , il-17a , and gm - csf . il-4 , il-23 , il-25 , ifn- , and eotaxin showed no significant production with stimulation of r mucilaginosa extract . on the other hand , there was no significant difference in the cytokine releases induced by 2 and 200 g / ml . there was no microbiological growth ( including fungus ) in the culture media of the homogenized nasal polyps . furthermore , grocott methanamine silver staining for the all nasal polyps showed no fungal bodies . sixteen of 35 samples of the nasal polyps showed amplification of fungal dna . using the d1/d2 domain of the large subunit ( 26s ) ribosomal dna primer , nl1 /nl4 , we found that 16 cellular tissues of the homogenized nasal polyps ( 100% ) were positive for fungal dna , whereas using the 18s rdna primer , its1f / its4r amplified sequences four of the 16 ( 25% ) were positive . on the other hand , using the universal fungal primer set , ff2 and fr1 incidentally , in none of the mucosa of the sphenoid sinus was fungal dna detected . the amplification products obtained from 16 patients were sequenced to identify the detected species in the tissue of the homogenized nasal polyps , such as c parapsilosis , r mucilaginosa , and aspergillus sp . table2 summarizes the data for all patients . the average number of eosinophils within the nasal polyps in which fungal dna was detected was significantly higher than that in the absence of fungal dna ( p < .01 , fig . f = female ; ige = immunoglobulin e ; m = male ; rast = radioallergosorbent test . comparison of the numbers of tissue eosinophils with nasal polyps between positive samples and those negative for fungal dna . the number of eosinophils in three fields with cell clusters were counted using light microscopy ( 400 magnification ) . the box and whisker plots show the median and the interquartile range of the number . the whiskers extend to the maximum and the minimum data points . to determine the biological and immunological roles of the identified fungi in crswnp in the present study , five samples of dnpcs were stimulated by extracts of c parapsilosis and r mucilaginosa . the expression levels of each cytokine and chemokine are summarized and compared with those of the unstimulated group in figure 2 ) . the secretion levels of the cytokines were set to the maximal level of each standard curve because the levels were above the maximal levels of detection . effect of extracts of candida parapsilosis and rhodotorula mucilaginosa on interleukin ( il)4 , il-5 , il-6 , il-8 , il-13 , il-17a , il-23 , il-25 , il-33 , eotaxin , rantes , interferon ( ifn)- , tumor necrosis factor ( tnf)- , and granulocyte - macrophage colony - stimulating factor ( gm - csf ) production by dispersed nasal polyp cells . five hundred microliters of 1 10/ml dispersed nasal polyp cells were collected after 72 hours , and the levels of the cytokines were determined by multiplex immunoassay . data were expressed as the fold change relative to that without stimulus of five cell cultures ( the means standard errors ) . [ color figure can be viewed in the online issue , which is available at www.laryngoscope.com . ] c parapsilosis stimulation of dnpcs significantly induced the secretion of il-5 , il-6 , il-8 , il-13 , rantes , tnf- , and gm - csf . the most remarkable upregulation was observed in il-6 ( approximately 100-fold ) and il-8 ( approximately 60-fold ) , followed by il-5 , tnf- , rantes , il-13 , and gm - csf . il-4 , il-17a , il-23 , and eotaxin showed no significant increase from stimulation by c parapsilosis extracts . no significant differences were seen in the responses between 2 and 200 g / ml of any cytokines . the stimulation of dnpcs by r mucilaginosa resulted in a significant induction of il-5 , il-8 , il-13 , il-17a , rantes , tnf- , and gm - csf . similar to c parapsilosis , r mucilaginosa most strongly upregulated the production of both il-6 and il-8 , which was followed by il-5 , tnf- , rantes , il-13 , il-17a , and gm - csf . il-4 , il-23 , il-25 , ifn- , and eotaxin showed no significant production with stimulation of r mucilaginosa extract . on the other hand , there was no significant difference in the cytokine releases induced by 2 and 200 g / ml . in the present study , we found fungal dna in the nasal polyps in spite of the lack of detection of fungus using histology and culture . a variety of studies agree that pcr is superior to both culture and grocott methanamine silver staining for detecting fungal elements.15 because the pcr studies showed the ubiquitous presence of fungi such as aspergillus , penicillium , cladosporium , candida , aureobasidium , and alternaria in the nose and paranasal sinuses of both crs patients and healthy controls,1618 it is unlikely that fungal species and fungal load play a role in disease development.19 gosepath et al.20 reported that fungal dna was detected in all 27 crs specimens with universal pcr primers . 's report and our study may be due to the sampling method , surgical specimens , or environmental factors . on the other hand , rao et al.21 reported that the proper design of pcr primers for fungi and the meticulous harvesting of nasal mucosa resulted in a low but significant incidence of fungi in crs . the primer pairs nl1/nl4 , which encode the d1/d2 domain of large subunit ( 26s ) ribosomal dna , have been widely used for the detection of fungal dna and the identification of fungal species , as compared with two other primers , its1f / its4r and ff2/fr1.22,23 no pcr products could be obtained from normal mucosa as negative controls . the exact reason why the detection rates of three pcr systems showed different values is not known . the large amount of human dna extracted from the nasal polyps might have inhibited several fungus - specific pcr products , because fungi share the same class of ribosomal rna with humans . we detected pcr products derived from four species of fungi including candida , rhodotorula , malasezzia , and aspergillus in the nasal polyps , which were never detected in the normal sinus mucosa . the rhodotorula species are ubiquitous saprophytic yeast that can be recovered from many environmental sources . previously reported as nonpathogenic , rhodotorula species have emerged as opportunistic pathogens with the ability to colonize and infect susceptible patients.24 malasezzia is a monophyletic genus of fungi detected as a ubiquitous component of the human skin microbiome and is associated with a myriad of skin problems.25 although we attempted to observe the localization of the dna of candida and rhodotorula using in situ hybridization in the nasal polyps , the experimental results failed to detect the fungal element , presumably due to the use of inappropriate probes . the fungal dna would be identified within bacterial biofilms26 and/or phagocytic vesicles of antigen - presenting cells such as macrophages and neutrophils in the nasal polyps.27 the detection rate of fugal dna was significantly elevated in the eosinophilic crs as compared with noneosinophilic crs , suggesting that the presence of fungi is closely related with eosinophil accumulation . a concentration - dependent increase in eosinophil migration toward both crs nasal mucin and crs nasal tissue extract was augmented as compared with the mucin of healthy controls.5 exposure of peripheral blood mononuclear cells to fungal antigen in vitro increased il-5 and il-13 production , whereas cells from normal controls did not respond.4 a component of alternaria was shown to degranulate eosinophils from crs patients by acting on pars . moreover , activation of nasal epithelial cells with fungi resulted in the upregulation of par2 and par3 mrna.28 these findings may lead to a hypothesis that fungi on the sinus mucosal surface induce the production of cytokines , which promote eosinophil migration through the epithelial cells and other constitutive cells of the nasal polyps . although we did not employ quantitative assays of pcr products , it was considered that the patients with more fungal dna would have significantly higher eosinophil counts . the final series of experiments aimed to evaluate immunological aspects of the detected fungi in the induction of eosinophilic inflammation . the fungal extracts derived from candida and rhodotorula , which were detected from the nasal polyps , apparently and remarkably upregulated a th1/th2 and th1th2/th17 cytokine profile , respectively , in the ex vivo models of the nasal polyp in the present study . it would be desirable to have stimulated the cytokine - producing cells from healthy sphenoid control tissue . however , it is well known that healthy tissue does not contain eosinophils and very minimal amounts of lymphocytes if any . kaufman et al.29 showed that the interaction of protease present in fungal extracts from the inferior nasal conchae of nonatopic subjects led to morphologic change , cell desquamation , and the induction of proinflammatory th1 cytokines . in addition , par2 stimulation in crs patients did not induce the release of eosinophil attracting cytokines like eotaxins or rantes.30 recent investigations are focusing on the contribution of th17 cells to the pathology of and resistance to fungi . zelante et al.31 found that by affecting fungal clearance and by promoting chronic inflammation and tissue damage , th17 responses at the mucosal surface had a detrimental effect on the course of fungal infections . our previous study revealed that the infiltration of cells positive for both cd4 and il-17a ( th17 cells ) showed a significant correlation with the numbers of eosinophils and mucosal remodeling in japanese crswnp.12 another underlying mechanism that might explain the fungus - mediated secretion of cytokines and chemokines , such as il-6 and il-8 , from dnpcs may involve toll - like receptors ( tlrs ) expressed on the sinonasal mucosa . the recognition of various pathogen - molecular patterns by the tlrs , including mannan , candida and cpg dna induces a cascade of downstream signaling that provokes an inflammatory cytokine profile.32 a quantitative increase in tlr2 mrna was seen in cystic fibrosis polyps as well as in crs,33,34 whereas in some studies35,36 a decrease in mucosal tlr2 and tlr9 mrna in samples from crswnp occurred . despite inconsistencies in previous data , tlr signaling appears to play an important role in mediating host inflammation , with potential derangements contributing to the development of crs.37 moreover , nucleotide - binding oligomerization domain - like receptors ( nlrs ) are newly discovered cytosolic receptors belonging to the pattern - recognition receptor family . nlr mrna was found to be higher in nasal polyps than in normal nasal mucosa.38 further studies are required to elucidate the role of innate immunity in fungus - related crs . the present study offers direct evidence to support the notion that fungal elements modify the inflammatory responses in the nasal polyps of eosinophilic crs , though the underlying mechanisms responsible for the eosinophil - based inflammation require further examination .

objectives / hypothesisthe role of fungi in chronic rhinosinusitis ( crs ) is still controversial . the present study was conducted to detect and identify fungal species from the nasal polyp tissues of eosinophilic and noneosinophilic crs , and to determine the role of fungal antigens in cytokine production.study designprospective study.methodsthirty-five specimens of nasal polyps were collected from patients with crs and examined for fungus using culture , histology , and polymerase chain reaction analysis . the secretion of 14 cytokines stimulated by fungal extracts using dispersed nasal polyp cells ( dnpcs ) was determined by multiplex immunoassay.resultsthere was no microbiological growth ( including fungus ) in the cultures of homogenized nasal polyps . furthermore , grocott methanamine silver staining for all nasal polyps showed no fungal bodies . sixteen of 35 samples of the nasal polyps showed amplification of fungal dna . in none of the mucosa of the sphenoid sinus was fungal dna detected . the number of eosinophils in the nasal polyps in which fungal dna was detected was significantly higher than in the nasal polyps in which fungal dna was not detected ( p < .01 ) . the extract of fungus enhanced the secretion of eosinophil - associated cytokines such as interleukine ( il)-5 , il-13 , il-17a , and rantes ( regulated on activation normal t - cell expressed and secreted ) , and proinflammatory cytokines such as il-6 , il-8 , tumor necrosis factor- , and granulocyte - macrophage colony - stimulating factor from dnpcs.conclusionsthe present study offers direct evidence supporting that fungal elements modify the inflammatory response in the nasal polyps of eosinophilic crs.level of evidence : na

INTRODUCTION MATERIALS AND METHODS Patients Sampling of Tissue and Pretreatment Culture and Histology of Fungi PCR of Fungi in the Nasal Polyps Species Identification Cell Cultures and Multiplex Immunoassay Statistical Analyses RESULTS Culture and Histology of Fungi Detection and Identification of Fungal DNA Cytokine Secretion Stimulated by Fungal Extracts DISCUSSION CONCLUSION

crs has been classified broadly as crs with nasal polyps ( crswnp ) , crs without nasal polyps , and allergic fungal rhinosinusitis ( afrs ) . there is recent evidence suggesting that 1 ) staphylococcus aureus enterotoxins , 2 ) type i hypersensitivity to fungus , and 3 ) non - immunoglobulin e ( ige)-mediated hypersensitivity to fungus may play a role in the pathogenesis of eosinophilic inflammation.1 however , the role of the microorganisms , particularly fungal pathogens , in the etiology of crs remains largely unknown . as further evidence , nasal mucus or tissue from crs patients triggered eosinophil migration,5 and alternaria fungus in particular can directly induce eosinophil degranulation mediated by protease - activated receptor ( par ) activation.6 however , other investigators reported the absence of a universal hyper - responsiveness to fungal antigens in crs patients.7,8 furthermore , a multicenter , randomized clinical trial of topical antifungal agents for crs eventually failed to show any evidence of efficacy,9 and a meta - analysis did not support the routine use of topical antifungals for crs.10 thus , the precise roles of fungi in the etiopathology of crs remain unknown . the present study was conducted to detect and identify fungal species from the nasal polyp tissues of eosinophilic and noneosinophilic crs using grocott methanamine silver staining and polymerase chain reaction ( pcr ) methods . moreover , the effects of fungal extracts identified in the nasal polyps were examined by the ex vivo cellular responses of dispersed nasal polyp cells ( dnpcs ) . thirty - five patients with crs with nasal polyps ( 21 males and 14 females , ranging in age from 2377 years , mean age of 49 years ) were consecutively recruited from the department of otorhinolaryngology of juntendo university hospital from april 2011 to march 2012 . the criteria of afrs of two positive findings , 1 ) specific ige antibodies against fungi , and 2 ) the presence of fungi in the sinus effusion using grocott methanamine cytological silver staining or microbiological examination . surgically removed human nasal polyps located in the middle meatus were obtained from the patients with crswnp , and the mucosa of the sphenoid sinus as a control were procured from patients with pituitary tumor . they were treated with 70% ethanol and physiologic saline to eliminate microorganisms outside of the nasal polyps . according to our previous studies,12,13 the eosinophilic and noneosinophilic groups were defined as eosinophil counts of the nasal polyps pf more and less 100/microacopic field ( magnification 400 ) using three fields , respectively . the total number of eosinophils present with a 10 10-mm reticulate present in the eyepiece was determined as the count per high - power field . after the nasal polyps were disrupted with glass beads and 100 l of tris - edta ( 10 mm tris - cl , 1 mm ethylenediaminetetraacetic acid ph 8.0 ) using a homogenizer ( biomasher ) ( takara bio inc . , otsu , japan ) , and 1 l of homogenized polyp samples were spread onto 5% sheep blood agar , chocolate agar , columbia anaerobic blood agar , drigalski agar , and sabouraud agar , and incubated for 2 weeks at 35c . all specimens were stained with grocott methanamine silver staining for confirmation of the existence of fungal organisms . pcr amplification of fungal specific ribosomal dna was performed using its-1f and its-4r primer , the pcr product covering the end of the 18srdna gene to the start of 26srdna gene and nl-1 and nl-4 primer , the pcr product covering d1/d2 26srdna ( table1 ) . dnpcs were prepared from nasal polyps of eosinophilic crswnps in five randomly selected patients by enzymatic digestion , as described by okano et al.14 in flat - bottomed 24-well culture plates ( nunc , roskiide , denmark ) , 500 l of 1 10/ml dnpcs were stimulated with serial concentrations ( 2 and 200 g / ml ) of candida parapsilosis , rhodotorula mucilaginosa . the culture supernatant was collected after 72 hours and stored at 80c , after which the levels of il-4 , il-5 , il-6 , il-8 , il-13 , il-17a , il-23 , il-25 , il-33 , eotaxin , rantes ( regulated on activation normal t - cell expressed and secreted ) , tumor necrosis factor ( tnf)- , interferon ( ifn)- , and granulocyte - macrophage colony - stimulating factor ( gm - csf ) were measured by bio - plex suspension array system ( bio - rad laboratories , inc . thirty - five patients with crs with nasal polyps ( 21 males and 14 females , ranging in age from 2377 years , mean age of 49 years ) were consecutively recruited from the department of otorhinolaryngology of juntendo university hospital from april 2011 to march 2012 . the criteria of afrs of two positive findings , 1 ) specific ige antibodies against fungi , and 2 ) the presence of fungi in the sinus effusion using grocott methanamine cytological silver staining or microbiological examination . surgically removed human nasal polyps located in the middle meatus were obtained from the patients with crswnp , and the mucosa of the sphenoid sinus as a control were procured from patients with pituitary tumor . according to our previous studies,12,13 the eosinophilic and noneosinophilic groups were defined as eosinophil counts of the nasal polyps pf more and less 100/microacopic field ( magnification 400 ) using three fields , respectively . the total number of eosinophils present with a 10 10-mm reticulate present in the eyepiece was determined as the count per high - power field . all specimens were stained with grocott methanamine silver staining for confirmation of the existence of fungal organisms . pcr amplification of fungal specific ribosomal dna was performed using its-1f and its-4r primer , the pcr product covering the end of the 18srdna gene to the start of 26srdna gene and nl-1 and nl-4 primer , the pcr product covering d1/d2 26srdna ( table1 ) . dnpcs were prepared from nasal polyps of eosinophilic crswnps in five randomly selected patients by enzymatic digestion , as described by okano et al.14 in flat - bottomed 24-well culture plates ( nunc , roskiide , denmark ) , 500 l of 1 10/ml dnpcs were stimulated with serial concentrations ( 2 and 200 g / ml ) of candida parapsilosis , rhodotorula mucilaginosa . the culture supernatant was collected after 72 hours and stored at 80c , after which the levels of il-4 , il-5 , il-6 , il-8 , il-13 , il-17a , il-23 , il-25 , il-33 , eotaxin , rantes ( regulated on activation normal t - cell expressed and secreted ) , tumor necrosis factor ( tnf)- , interferon ( ifn)- , and granulocyte - macrophage colony - stimulating factor ( gm - csf ) were measured by bio - plex suspension array system ( bio - rad laboratories , inc . there was no microbiological growth ( including fungus ) in the culture media of the homogenized nasal polyps . furthermore , grocott methanamine silver staining for the all nasal polyps showed no fungal bodies . sixteen of 35 samples of the nasal polyps showed amplification of fungal dna . using the d1/d2 domain of the large subunit ( 26s ) ribosomal dna primer , nl1 /nl4 , we found that 16 cellular tissues of the homogenized nasal polyps ( 100% ) were positive for fungal dna , whereas using the 18s rdna primer , its1f / its4r amplified sequences four of the 16 ( 25% ) were positive . on the other hand , using the universal fungal primer set , ff2 and fr1 , no fungal dna was detected . incidentally , in none of the mucosa of the sphenoid sinus was fungal dna detected . the amplification products obtained from 16 patients were sequenced to identify the detected species in the tissue of the homogenized nasal polyps , such as c parapsilosis , r mucilaginosa , and aspergillus sp . the average number of eosinophils within the nasal polyps in which fungal dna was detected was significantly higher than that in the absence of fungal dna ( p < .01 , fig . the number of eosinophils in three fields with cell clusters were counted using light microscopy ( 400 magnification ) . to determine the biological and immunological roles of the identified fungi in crswnp in the present study , five samples of dnpcs were stimulated by extracts of c parapsilosis and r mucilaginosa . effect of extracts of candida parapsilosis and rhodotorula mucilaginosa on interleukin ( il)4 , il-5 , il-6 , il-8 , il-13 , il-17a , il-23 , il-25 , il-33 , eotaxin , rantes , interferon ( ifn)- , tumor necrosis factor ( tnf)- , and granulocyte - macrophage colony - stimulating factor ( gm - csf ) production by dispersed nasal polyp cells . five hundred microliters of 1 10/ml dispersed nasal polyp cells were collected after 72 hours , and the levels of the cytokines were determined by multiplex immunoassay . c parapsilosis stimulation of dnpcs significantly induced the secretion of il-5 , il-6 , il-8 , il-13 , rantes , tnf- , and gm - csf . the most remarkable upregulation was observed in il-6 ( approximately 100-fold ) and il-8 ( approximately 60-fold ) , followed by il-5 , tnf- , rantes , il-13 , and gm - csf . il-4 , il-17a , il-23 , and eotaxin showed no significant increase from stimulation by c parapsilosis extracts . the stimulation of dnpcs by r mucilaginosa resulted in a significant induction of il-5 , il-8 , il-13 , il-17a , rantes , tnf- , and gm - csf . similar to c parapsilosis , r mucilaginosa most strongly upregulated the production of both il-6 and il-8 , which was followed by il-5 , tnf- , rantes , il-13 , il-17a , and gm - csf . there was no microbiological growth ( including fungus ) in the culture media of the homogenized nasal polyps . furthermore , grocott methanamine silver staining for the all nasal polyps showed no fungal bodies . sixteen of 35 samples of the nasal polyps showed amplification of fungal dna . using the d1/d2 domain of the large subunit ( 26s ) ribosomal dna primer , nl1 /nl4 , we found that 16 cellular tissues of the homogenized nasal polyps ( 100% ) were positive for fungal dna , whereas using the 18s rdna primer , its1f / its4r amplified sequences four of the 16 ( 25% ) were positive . on the other hand , using the universal fungal primer set , ff2 and fr1 incidentally , in none of the mucosa of the sphenoid sinus was fungal dna detected . the amplification products obtained from 16 patients were sequenced to identify the detected species in the tissue of the homogenized nasal polyps , such as c parapsilosis , r mucilaginosa , and aspergillus sp . the average number of eosinophils within the nasal polyps in which fungal dna was detected was significantly higher than that in the absence of fungal dna ( p < .01 , fig . the number of eosinophils in three fields with cell clusters were counted using light microscopy ( 400 magnification ) . to determine the biological and immunological roles of the identified fungi in crswnp in the present study , five samples of dnpcs were stimulated by extracts of c parapsilosis and r mucilaginosa . effect of extracts of candida parapsilosis and rhodotorula mucilaginosa on interleukin ( il)4 , il-5 , il-6 , il-8 , il-13 , il-17a , il-23 , il-25 , il-33 , eotaxin , rantes , interferon ( ifn)- , tumor necrosis factor ( tnf)- , and granulocyte - macrophage colony - stimulating factor ( gm - csf ) production by dispersed nasal polyp cells . five hundred microliters of 1 10/ml dispersed nasal polyp cells were collected after 72 hours , and the levels of the cytokines were determined by multiplex immunoassay . c parapsilosis stimulation of dnpcs significantly induced the secretion of il-5 , il-6 , il-8 , il-13 , rantes , tnf- , and gm - csf . the most remarkable upregulation was observed in il-6 ( approximately 100-fold ) and il-8 ( approximately 60-fold ) , followed by il-5 , tnf- , rantes , il-13 , and gm - csf . il-4 , il-17a , il-23 , and eotaxin showed no significant increase from stimulation by c parapsilosis extracts . the stimulation of dnpcs by r mucilaginosa resulted in a significant induction of il-5 , il-8 , il-13 , il-17a , rantes , tnf- , and gm - csf . similar to c parapsilosis , r mucilaginosa most strongly upregulated the production of both il-6 and il-8 , which was followed by il-5 , tnf- , rantes , il-13 , il-17a , and gm - csf . in the present study , we found fungal dna in the nasal polyps in spite of the lack of detection of fungus using histology and culture . a variety of studies agree that pcr is superior to both culture and grocott methanamine silver staining for detecting fungal elements.15 because the pcr studies showed the ubiquitous presence of fungi such as aspergillus , penicillium , cladosporium , candida , aureobasidium , and alternaria in the nose and paranasal sinuses of both crs patients and healthy controls,1618 it is unlikely that fungal species and fungal load play a role in disease development.19 gosepath et al.20 reported that fungal dna was detected in all 27 crs specimens with universal pcr primers . we detected pcr products derived from four species of fungi including candida , rhodotorula , malasezzia , and aspergillus in the nasal polyps , which were never detected in the normal sinus mucosa . previously reported as nonpathogenic , rhodotorula species have emerged as opportunistic pathogens with the ability to colonize and infect susceptible patients.24 malasezzia is a monophyletic genus of fungi detected as a ubiquitous component of the human skin microbiome and is associated with a myriad of skin problems.25 although we attempted to observe the localization of the dna of candida and rhodotorula using in situ hybridization in the nasal polyps , the experimental results failed to detect the fungal element , presumably due to the use of inappropriate probes . the fungal dna would be identified within bacterial biofilms26 and/or phagocytic vesicles of antigen - presenting cells such as macrophages and neutrophils in the nasal polyps.27 the detection rate of fugal dna was significantly elevated in the eosinophilic crs as compared with noneosinophilic crs , suggesting that the presence of fungi is closely related with eosinophil accumulation . moreover , activation of nasal epithelial cells with fungi resulted in the upregulation of par2 and par3 mrna.28 these findings may lead to a hypothesis that fungi on the sinus mucosal surface induce the production of cytokines , which promote eosinophil migration through the epithelial cells and other constitutive cells of the nasal polyps . although we did not employ quantitative assays of pcr products , it was considered that the patients with more fungal dna would have significantly higher eosinophil counts . the final series of experiments aimed to evaluate immunological aspects of the detected fungi in the induction of eosinophilic inflammation . the fungal extracts derived from candida and rhodotorula , which were detected from the nasal polyps , apparently and remarkably upregulated a th1/th2 and th1th2/th17 cytokine profile , respectively , in the ex vivo models of the nasal polyp in the present study . kaufman et al.29 showed that the interaction of protease present in fungal extracts from the inferior nasal conchae of nonatopic subjects led to morphologic change , cell desquamation , and the induction of proinflammatory th1 cytokines . our previous study revealed that the infiltration of cells positive for both cd4 and il-17a ( th17 cells ) showed a significant correlation with the numbers of eosinophils and mucosal remodeling in japanese crswnp.12 another underlying mechanism that might explain the fungus - mediated secretion of cytokines and chemokines , such as il-6 and il-8 , from dnpcs may involve toll - like receptors ( tlrs ) expressed on the sinonasal mucosa . nlr mrna was found to be higher in nasal polyps than in normal nasal mucosa.38 further studies are required to elucidate the role of innate immunity in fungus - related crs . the present study offers direct evidence to support the notion that fungal elements modify the inflammatory responses in the nasal polyps of eosinophilic crs , though the underlying mechanisms responsible for the eosinophil - based inflammation require further examination .

renal cell carcinoma ( rcc ) is the most common malignancy arising in the kidney . in the united states alone , each year 39,000 people are diagnosed with rcc and 13,000 people die from the disease [ ] . the classic triad of virchow ( flank pain , hematuria and a palpable abdominal mass ) is only seen in approximately 9% of newly diagnosed patients [ ] . this considerably complicates the diagnosis , since the disease can present with a broad array of ( paraneoplastic ) symptoms [ ] . as a consequence , 30% of patients will present with metastatic disease , whereas of the other 70% treated by nephrectomy , 3040% will eventually relapse [ ] . the 5-year survival rate for small ( less than 7 cm ) tumors limited to the kidney ( pt1 tumor ) is more than 90% [ ] , but prognosis for metastatic disease is bleak , with a median survival of only 10 months [ ] . therefore , therapeutic strategies focus on immunotherapy , neoangiogenesis inhibitors and other targeted approaches . in this review , another approach using antibodies developed for targeting rcc is discussed and particularly their application in the diagnosis and therapy of rcc . since the first description of ehrlich to specifically guide cytotoxic therapy to cancer tissue [ ] , much has been debated on the feasibility of this approach . development of the hybridoma technique [ ] allowed isolation of large quantities of antibodies with predefined specificity . with the identification of the tumor - associated target antigens , real progress has been made on developing treatment and/or diagnostic strategies using mabs . to date , no tumor specific antigen , i.e. an antigen expressed on all tumor cells which is not expressed by normal cells in the body , has been identified . tumor - associated antigens ( taa ) have been identified for a series of human tumor types [ ] . these are either differentiation antigens , ( transiently ) expressed during organogenesis , or aberrantly expressed antigens , ( transiently ) expressed elsewhere in non - related normal tissue(s ) . expression of antigen on the primary tumor or metastases is generally heterogeneous . for tumor targeting with mabs this is a suboptimal feature , since not all cells can be targeted by the mab . heterogeneous expression between different tumor sites , varying degrees of expression in tumor cells of the same tumor and temporal modulation of taa - expression are considered major limitations of effective targeting of tumors with mabs . in addition to intratumoral heterogeneity of antigen expression , other parameters have been defined that may be equally important in hampering tumor targeting with mabs . these are : size of the tumor mass , the antigen density , the fate of antigen / antibody complex , presence of circulating antigen , mab format , mab dose , route of administration and mab circulating half - life [ ] . mab targeting is complicated by large tumor blood vessels as well as impaired blood flow in the tumor by elevated interstitial fluid pressure ( ifp ) [ ] . high vascular density is not equivalent to high perfusion rates in the tumor , which are required for optimal mab delivery . rcc has always been considered a highly vascularized tumor by morphologic standards . however , in comparison with normal kidney tissue rcc is poorly perfused [ ] , thereby impeding adequate mab delivery to the tumor cells . these limitations of delivering the mab to tumor tissue have to be overcome in order to develop a suitable mab - based treatment strategy . several mechanisms to eradicate tumor cells by mabs are available : either via effector cells or complement dependent cytotoxicity or through conjugation of the mab to toxins , drugs or radionuclides . since antigen expression within tumors is heterogeneous , antigen - negative tumor cells may evade tumor cell lysis by effector cell- or complement - mediated cytotoxicity , which may eventually lead to tumor recurrence . the same applies to mabs conjugated to toxins or drugs , since internalization of a mab conjugated to a toxin or drug is required to mediate cell - killing [ ] . radiolabeling of antibodies was developed in 1950 , when eisen observed that proteins could be labeled with i without altering their immunological specificity [ ] . besides i , other radionuclides ( y , lu , re , re and cu ) have since been investigated to induce tumor cell death ( see table 1 ) . the advantage of radiolabeled antibodies is that the mab does not have to bind to every tumor cell to induce cytotoxicity , since the radionuclides emit -particles , which can be effective for up to 50 or more cell diameters . this so - called crossfire effect can thus overcome heterogeneity of antigen expression , as the radiation destroys the antigen - negative cells as well . a disadvantage of this technique is the sensitivity of normal organs to radiation , particularly the bone marrow . the dose limiting toxicity of delivering high - dose radioimmunotherapy ( rit ) , i.e. a radionuclide conjugated to a tumor - associated mab , is therefore generally hematological . table 1radionuclides used in radioimmunotherapy of clear cell renal cell carcinomaradionuclidehalf - life-average ( kev) ( kev)maximum range -particles in tissue ( mm)advantagesdisadvantagesi8.0 days1923623.0easy labeling ; inexpensivehigh radiation burden to personnel / relatives ; hospital admittance requiredre90.7 h3621375.1out - patient treatment possible ; ideal gamma for imaginglaborious labelingy64 h935none12high - energy beta - emission ; prolonged tumor retention ; out - patient treatment possibleno imaging possiblelu6.7 days1492082.5prolonged tumor retention radionuclides used in radioimmunotherapy of clear cell renal cell carcinoma using the previously mentioned hybridoma technique a wide array of mabs against taas has been produced , e.g. mabs against carcino - embryonic antigen ( cea ) ( mainly expressed in colorectal and medullary thyroid carcinomas ) , muc-1 ( mainly ovarian and breast cancer ) , tag72 ( mainly ovarian and colorectal cancer ) , cd-20 ( non - hodgkin 's lymphoma ( nhl ) ) and g250-antigen ( rcc ) . in various clinical trials safety and efficacy of these newly developed mabs labeled with various radionuclides have been investigated [ ] . the effector cell , complement and apoptosis inducing cytotoxicity of the mab give high intrinsic anti - tumor activity as well . extensive research has resulted in the first registered treatment with radiolabeled mabs directed against the surface antigen cd-20 expressed on b - cell nhl ( y - labeled anti - cd20 mab ibritumomab tiuxetan ( zevalin , biogen idec , boston ma , usa and schering , berlin , germany ) and i - labeled anti - cd20 mab tositumomab ( bexxar , gsk , philadelphia , pa , usa ) . in patients with solid tumors , therapeutic strategies with radiolabeled mabs however , as mentioned previously , tumor - related factors also play an important role . the most common types of solid malignancies targeted in clinical trials with rit have been epithelial cancers , e.g. colorectal cancer , ovarian cancer , medullary thyroid cancer , breast cancer , prostate cancer and rcc . results of these trials did not result in registration of radiolabeled mab preparations for regular treatment of these cancer types . however , patients entered in these trials often had bulky metastatic disease and had been heavily pretreated with chemotherapy and/or radiotherapy in most cases . partial responses and stabilization of previously progressive disease have been seen in few patients in most of these trials [ ] . peptides have been used for radionuclide targeting of tumors to overcome the difficulties in tumor targeting with mabs mentioned above . these peptides have a high affinity for specific receptors that are expressed on the tumor cell . tumor targeting peptides have advantages over mabs , as they diffuse rapidly in target tissue and clear rapidly from the blood and from the non - target tissues . peptides are usually non - immunogenic and generally have a low toxicity profile . peptide receptor radionuclide imaging ( prri ) and therapy ( prrt ) are now under investigation . to date , the somatostatin ( sst ) analog in - labeled - octreotide ( octreoscan , mallinckrodt tyco healthcare , petten , the netherlands ) is the most successful radiopeptide for tumor imaging and has been the first to be approved for scintigraphic localization of primary and metastatic neuro - endocrine tumors expressing sst2 and sst5 receptor subtypes [ ] . expression of these receptors was found in 72% of rcc samples analyzed , irrespective of histopathological subtype or grading of the tumor [ ] . the use of [ in]octreotide has been evaluated in patients with metastatic rcc . in this study , 68 rcc metastases in 9 patients , confirmed by diagnostic ct and/or x - ray images were evaluated . forty ( 59% ) of the 68 known sites were visualized [ ] . besides octreotide , a new series of peptides is now being evaluated for targeting of solid tumors . cholecystokinin ( cck ) analogues , vasoactive intestinal peptide ( vip ) , neuropeptide y ( npy ) , bombesin , glucagon - like peptide-1 ( glp-1 ) and rgd peptides have shown promising preclinical tumor - receptor targeting ( for review see reubi [ ] ) . the gastrointestinal peptide gastrin acts as a neurotransmitter in the brain and as a regulator of various functions in the gastrointestinal tract [ ] . these receptors are highly expressed in medullary thyroid carcinoma ( mtc ) , enabling gastrin to visualize metastatic mtc with very high sensitivity [ ] . rcc however , does not express these receptors and gastrin is therefore not suitable for rcc imaging [ ] . vip , a member of the group of secretin - like peptides , is an important neurotransmitter in the gut . its actions are mediated by specific g protein - coupled receptors that can be internalized upon ligand binding [ ] . although expression of the vip receptors has been found on nephroblastomas [ ] , expression on rcc has not been determined . npy is a neurotransmitter that is predominantly found in the central nervous system , where it functions as a stimulator of feeding behavior and inhibition of anxiety [ ] . more recently , expression of npy receptors has been found on rcc and nephroblastomas , suggesting a potential role for rcc targeting using radiolabeled npy [ ] . bombesin has a high and specific affinity for the gastrin releasing peptide receptor ( grp - r ) and this receptor stimulates proliferation of tumor growth in various tumor types [ ] . rcc has been found to have a high expression of grp - r [ ] . targeting of rcc was done using a bombesin analogue labeled with lu . in this study , in vitro autoradiography showed specific uptake of the radioligand in five of the six rcc samples evaluated [ ] . recently , glp-1 receptor expression in solid human tumors has been evaluated extensively and systematically . this study found no glp-1 receptor expression in 20 rcc tissue samples analyzed , excluding glp-1 receptor as a target for in vivo rcc imaging or therapy [ ] . rgd peptides contain the amino acid sequence arg - gly - asp that has high and specific affinity for the v3 integrin [ ] . this integrin is mainly expressed on proliferating endothelial cells , whereas it is not expressed on quiescent endothelial cells [ ] . in growing tumors a continuous formation of new blood vessels is required . the expression of v3 has been found to increase with higher rcc tumor grades . of the rcc metastases examined , 2 of 14 showed high expression of v3 , 8 of 14 showed weak expression and 4 of 14 did not express the v3 integrin [ ] . to date , rgd peptides have not been evaluated for rcc imaging . in rcc , several mabs have been defined that are reactive with rcc - associated antigens [ ] . cross - reactivity with non - kidney tissue was seen in some of these mabs , whereas others were only expressed in kidney / rcc . one of these mabs , which showed relative high tumor - to - blood ratios in mice with rcc xenografts is mab a6h [ ] . this mab recognizes an antigen common to rcc , some lung and colon carcinomas , the proximal renal tubules but no other normal tissues in vivo [ ] . in a clinical study , the imaging and rit potential of this mab was examined [ ] . this low sensitivity was attributed to soluble antigen binding by the mab and the expression of antigen in normal tissue , thereby not allowing the mab to bind to tumor tissue . this clinical finding of antigen expression in normal tissue was not in line with the previous findings . after modification of the dosing regimen , the detection rate of metastatic lesions increased , but the number of detected lesions remained unsatisfactory . as a result , the use of mab a6h for diagnosis and treatment of rcc was discontinued . g250 , a mab against a rcc - associated antigen has been investigated extensively , because the antigen which this mab recognizes showed remarkable tissue distribution and expression . the mab g250 was obtained after fusion of spleen cells from a mouse immunized with fresh rcc homogenates . the antigen that mab g250 targets has been designated in the literature as mn , ca ix and g250 . of the 47 primary rcc specimens initially analyzed , 42 ( 89% ) showed homogeneous g250-antigen expression , whereas four tumors showed heterogeneous expression and one tumor was g250-antigen - negative . of the eight metastases examined , g250-antigen expression was homogeneous in five ( 62% ) , heterogeneous in two , while one did not express the g250-antigen [ ] . expression in normal tissues has been evaluated extensively and has been shown to be restricted to the ( upper ) gastrointestinal mucosa ( stomach , ileum , proximal and middle colon ) and gastrointestinal related structures ( intra- and extrahepatic biliary system , pancreas ) [ ] . later studies showed an almost ubiquitous expression ( > 90% ) of g250-antigen in clear cell rcc ( ccrcc ) , being the most prominent form of rcc ( 80% of cases ) . g250-antigen expression in the different histological subtypes of rcc was determined by rt - pcr and immunohistochemistry . all the clear cell tumors displayed g250-antigen mrna , but expression of g250-antigen by oncocytomas , chromophobe or papillary rcc was low or absent [ ] . an early or a first event in the clear cell rcc tumorigenic pathway is mutations leading to loss of von hippel lindau protein ( pvhl ) in 5075% of sporadic rcc . in normoxic conditions pvhl is responsible for degradation of hypoxia inducible factor-1 ( hif-1 ) , which thereby can not bind to hif-1 to form hif-1 . in hypoxic conditions , however , degradation does not occur and hif-1 can cause the transcription of a number of hypoxia - inducible genes . these include vascular endothelial growth factor ( vegf ) , transforming growth factor- ( tgf ) , erythropoietin , g250-antigen and others . clearly , expression of these proteins is advantageous for tumor growth . with the loss of functional pvhl in rcc this mutational loss of pvhl thus explains why g250-antigen is almost invariably upregulated in ccrcc . g250-antigen is also expressed in various other tumor types ( e.g. cervix , lung ) under hypoxic conditions . various animal and ex vivo experiments have shown the potential of the g250 mab as a targeting modality of rcc [ ] . since g250-antigen is high and homogeneously expressed in rcc tissue , is restricted to a few normal tissues and other tumors , and a low mab dose is needed to obtain antigen saturation , mab g250 seemed a suitable candidate for further investigation in clinical studies . imaging and biodistribution were studied in 16 patients receiving 370 mbq i - labeled mg250 at escalating protein dose levels 1 week prior to nephrectomy . after 34 days clear delineation of tumors was seen in 12 patients , imaged with a gamma camera . ten of these tumors proved to be g250-positive , whereas the other two showed less than 5% g250-antigen expression . after nephrectomy , tumor samples were shown to have high and focal uptake of g250 , up to 0.21 % injected dose / gram ( % id / g ) . tumor targeting was not the result of blood pooling , as the blood volume marker tc - labeled human serum albumin showed significantly lower tumor uptake than [ i]mg250 that had been administered earlier . therefore , this was indicative of true antibody targeting of the tumor by mg250 [ ] . as good targeting of ccrcc by mg250 was seen in this study , a phase i / ii radioimmunotherapy ( rit ) dose escalation study was performed by divgi et al . patients in this study were treated with one high - activity - dose injection of [ i]mg250 . after reaching the maximum tolerated dose ( mtd ) , another 15 patients were enrolled and treated at the mtd to monitor any possible therapeutic effects . in the phase i dose - escalation study , mtd was defined at 3330 mbq / m , due to hematological toxicity . transient hepatic toxicity occurred at dose levels of 1665 mbq / m and higher , but was not dose limiting . fourteen patients had grade 3 hepatic toxicity , that did not last for more than 2 weeks . a total of 33 patients was treated , 18 in the dose - escalating part of the study and another 15 patients at the mtd ( 3330 mbq / m ) , to evaluate therapeutic efficacy . of these 33 patients , 17 stabilized for 3 months , after which patients received other treatments , preventing further follow - up . three patients showed regression of some of their lesions , but no partial or complete responses were noted [ ] . the formation of human anti mouse antibodies ( hama ) in all patients receiving mg250 prohibited retreatment . formation of immune complexes with rapid clearance of the radiolabeled mab to liver and spleen would have occurred in the case of multiple administrations , thereby limiting targeting of the mab to the tumor [ ] . this , in combination with the high potential of g250 as a targeting agent in the treatment of metastasized rcc , led to the development of a chimeric form of g250 ( cg250 ) [ ] . this mab is composed of murine antigen - binding variable domains , that recognize the taa and human constant domains of heavy and light chains derived from the human igg1 isotype [ ] . the rationale behind this construction was the decrease in immunogenicity of the antibody , potentially allowing multiple administrations . unlabeled g250 antibody facilitates antibody - dependent cellular cytotoxicity ( adcc ) of g250-antigen expressing cells , which leads to induction of lysis of these cells [ ] . this finding led to a study where 36 patients with metastatic ccrcc received 50 mg cg250 weekly for 12 weeks . development of human anti chimeric antibody ( haca ) was low and not clinically significant . before treatment , 80% of patients were progressive . after treatment , 11 patients had stable disease and during follow - up one complete and one partial response were seen . the median survival of 15 months suggested that g250 may be able to immunomodulate the natural course of metastatic rcc [ ] . based on these results , an adjuvant phase iii trial has been initiated in high - risk ccrcc patients who are nephrectomized and have no known metastases , using this treatment regimen . since the 1990s , high - dose bolus interleukin-2 ( il-2 ) has been established as a first - line therapy for metastatic rcc . il-2 is a t - cell growth factor that is thought to play a critical role in t - cell dependent immune responses . high - dose bolus il-2 as therapy in metastatic rcc has had varying success , with responses in up to 15% of patients [ ] . it was hypothesized that the immunological specificity of lymphokine - activated killer cells of patients receiving il-2 therapy may be enhanced through the co - administration of cg250 [ ] . vice versa , co - administration of il-2 can enhance the therapeutic efficacy of g250 [ ] . in a phase ii trial 35 patients with progressive ccrcc received weekly i.v . mean survival was 24 months in this trial , compared to 16.3 months median survival with high - dose il-2 therapy [ ] , which also has toxic side effects . the authors considered it unlikely that the increased survival was due to the low - dose il-2 , using a six - fold decrease of normal il-2 dose used to induce clinical efficacy . after cg250 became available , the pharmacokinetics , biodistribution , imaging characteristics and dosimetry of this new radiolabeled targeting vehicle was studied in a protein dose escalation study identical to murine g250 . sixteen presurgical rcc patients received increasing doses of cg250 between 2 and 50 mg labeled with i , given i.v . a week before they underwent nephrectomy . highest tumor uptake was observed in the patients that received 5 and 10 mg [ i]mg250 , with focal tumor uptake as high as 0.52% id / g . at higher protein doses this suggested that antigen - saturation could have occurred at protein doses exceeding 10 mg . excellent images of g250-antigen positive tumors were obtained , with visualization of tumor lesions and metastases , seen earlier on ct or x - ray . dosimetric analysis showed a high radiation - absorbed dose to primary tumors as well as metastases ( up to 1.9 cgy / mbq to primary tumor ) . up to 20 weeks post - injection , human anti chimeric antibody ( haca ) responses were seen in two patients , but titers were considered low and clinically irrelevant [ ] . reducing the immunogenic properties of the antibody opened the possibility of multiple treatments . the mtd of [ i]cg250 in metastatic rcc was determined in a phase i radioactivity dose escalation trial in patients with progressive metastatic rcc at study entry . twelve patients received 5 mg of cg250 labeled with 185 mbq i ( scout dose ) . when accumulation of antibody was seen in any tumor site , patients received escalating radioactivity - doses of [ i]cg250 . in contrast to the trials performed with murine g250 , no hepatic toxicity was seen . this was believed to be the result of saturation of the hepatic compartment by the diagnostic scout dose of [ i]cg250 . besides mild nausea without vomiting and transient fatigue ( both grade 1 ctc ) , no other non - hematological side effects occurred . the mtd was observed to be 2220 mbq / m , with hematological toxicity as the dose - limiting factor . of the 8 patients receiving treatment , 1 showed stable disease and 1 had a partial response [ ] . in subsequent studies two strategies were tested to optimize targeting of metastatic ccrcc with rit . fractionation of the dose was done in a phase i study by divgi et al . , patients received 1110 mbq of [ i]cg250 and whole - body activity was measured after 23 days . this was continued until a whole - body absorbed dose of 0.50 gy was reached . patients without disease progression were retreated after recovery from hematological toxicity . in subsequent cohorts , haca development was measured in two patients , altering pharmacokinetics and excluding them from further treatment . dose - limiting toxicity was again hematopoietic , with the mtd at 0.75 gy as whole - body absorbed dose . this trial therefore provided no evidence of a potential benefit of fractionation of rit doses in treating ccrcc . ( 2220 mbq / m ) , combined with the properties of cg250 allowing multiple administrations , led to a study where two sequential high doses of [ i]cg250 treatment were given . three months later , rapid clearance of the mab by haca development was excluded by imaging of a scout dose of 185 mbq [ i]cg250 . when tumor targeting was seen again , the second high - dose injection [ i]cg250 was given . mtd of the second rit proved to be again due to hematological toxicity and was set at 1665 mbq / m , being 75% of the mtd of the first infusion ) . subsequently , 15 patients were treated at this dose level to evaluate tumor response . in total , 29 patients entered the study , 11 were excluded due to grade 4 hematological toxicity after the first rit ( n=3 ) , palliative treatment ( n=2 ) , rapid progressive disease ( n=2 ) or haca development ( n=4 ) . of the 18 patients evaluated ( 3 not receiving the second rit at mtd ) , 5 patients had stabilization of their disease , lasting 312 months . there proved to be an inverse correlation between the size of metastases and radiation absorbed dose . therapeutic radiation doses ( more than 50 gy ) [ ] were only guided to the lesions smaller than 5 g. the authors concluded that rit in rcc patients could best be given in the setting of small volume disease or as adjuvant therapy [ ] . first , the targeting capabilities of [ in]cg250 have been compared to those of [ i]cg250 . in nude mice - human tumor models , superior targeting of in over i had been shown [ ] . as part of the cg250 antibody - antigen complex is internalized , intracellular [ i]cg250 is metabolized and rapidly excreted by the tumor cell . metallic radionuclides , such as in , y and lu , are trapped in the lysosomes and residualize after internalization of the mab - antigen complex by the target cells [ ] . to investigate whether this phenomenon may also occur in humans , five patients with metastatic rcc were i.v . injected with 185 mbq of [ in]dtpa - cg250 on day 0 and 185 mbq of [ i]cg250 on day 4 . gamma images were made directly and on day 4 after both injections and compared ( fig . [ in]dtpa - cg250 images revealed more lesions than [ i]cg250 ( 47 vs. 30 ) and quantitative analysis showed higher accumulation of [ in]dtpa - cg250 in 20 of 25 lesions measured in terms of % id / g [ ] . figure 1(a ) [ in]cg 250 immunoscintigram of a patient with metastatic ccrcc , acquired 6 days after injection of 185 mbq of [ in]cg250 . green arrows mark a lesion not seen on the fluorodeoxyglucose ( fdg)-positron emission tomography ( pet)-computed tomography(ct ) images shown in ( b ) . red arrows indicate the injection standard . the anterior image is shown in the left panel , ( b ) pet - ct scan of the same patient acquired after injection of 250 mbq of [ f]fdg . ( a ) [ in]cg 250 immunoscintigram of a patient with metastatic ccrcc , acquired 6 days after injection of 185 mbq of [ in]cg250 . green arrows mark a lesion not seen on the fluorodeoxyglucose ( fdg)-positron emission tomography ( pet)-computed tomography(ct ) images shown in ( b ) . red arrows indicate the injection standard . the anterior image is shown in the left panel , ( b ) pet - ct scan of the same patient acquired after injection of 250 mbq of [ f]fdg . the therapeutic properties of cg250 labeled with four radionuclides have been tested in nude mice with human rcc xenografts . the four radionuclides under investigation were y and lu ( both residualizing ) , and i and re ( both non - residualizing ) . after determining the mtd for each radionuclide conjugated to cg250 in mice , an rit experiment was done comparing tumor growth and survival after treatment with each radiolabeled cg250 preparation . tumor growth was delayed most effectively by lu , followed by y and re and least by i ( 185 , 125 , 90 and 25 days , respectively ) . the best median survival was observed for lu ( 300 days ) , with the control group having a median survival of less than 150 days . these radionuclides should be considered better candidates for rit with cg250 than i [ ] . based on these preclinical and clinical data ( table 2 ) , an ongoing phase i / ii [ lu]dota - cg250 dose escalation rit study in progressive , metastatic rcc patients was designed . patients in whom cg250 targeting of rcc metastases is observed are treated with up to three cycles of [ lu]dota - cg250 to determine mtd . so far , minor responses have been noted at the lower dose levels and dose escalation is ongoing . table 2phase i / ii radioimmunotherapy trials in clear cell renal cell carcinomareferenceradiophar - maceuticaltarget antigenmab typeno . of patientsresponsesspecial featuresdivgi et al.[][i]g250g250murine g2503317 sdsteffens et al.[][i]g250g250chimeric g250121 pr ; 1 sddivgi et al.[][i]cg250g250chimeric g250157 sdfractionated ritbrouwers et al.[][i]cg250g250chimeric g250275 sdtwo high - dose treatment phase i / ii radioimmunotherapy trials in clear cell renal cell carcinoma future strategies to improve clinical efficacy of cg250 could be : ( 1 ) tumor pretargeting ; ( 2 ) high dose rit with bone marrow support or transplant or ( 3 ) use of high linear energy transfer ( let ) particles emitting radionuclides . in pretarging rit , administration of the mab is separated from the injection of the radionuclide . this allows the unlabeled mab to bind to the tumor as well as to clear from circulation and normal organs . the radionuclide is administered in a second injection as a rapidly clearing agent with high affinity to the previously administered mab . affinity of the radionuclide binding to the mab may be achieved through , e.g. an avidin - biotin complex or with bispecific mabs ( bsmabs ) . biotinylated mab and radionuclides can be coupled through an extremely avid interaction with avidin ( for review see boerman et al . a bsmab ( cg250 x dtin-1 ) was produced and pretargeting experiments in nude mice with rcc xenografts targeted with in - labeled bivalent peptide showed excellent tumor targeting [ ] . to date , however , this approach has not been tested clinically for cg250 . autologous marrow transplantation or peripheral blood stem cell reinfusion has been investigated as a means to overcome bone marrow toxicity in rit , thereby allowing administration of activity doses more than twice as high as without any bone marrow support [ ] . the use of let ( alpha ) particle emitting radionuclides has the advantage of high cytotoxic potency , combined with a low range and thus not reaching most normal tissue surrounding the tumor . however , most alpha - emitters have a half - life of less than 1 h , which is hardly compatible with mabs targeting tumors . nevertheless , to date clinical impact of rit for treatment of ccrcc has been minimal . it remains to be established whether the use of more powerful radionuclides can lead to alteration of the clinical course of metastatic ccrcc . second , g250 may be more valuable in an adjuvant setting and/or as a diagnostic means . finally , with the advent of new treatment possibilities for rcc such as angiogenesis inhibitors , combination treatment with g250 rit and these new substances may play a role in more effective management of ccrcc .

abstractrenal cell carcinoma ( rcc ) is a radio- and chemotherapy resistant tumor , which has a very high morbidity and mortality when metastasized . the current treatment options demonstrate limited efficacy and severe side - effects . therefore , there is a need for new therapeutic strategies for rcc . as for other malignancies , monoclonal antibodies ( mabs ) targeting tumor - associated antigens have been developed for rcc . one of these , mab g250 , targets the mn / caix / g250 antigen , which is ubiquitously expressed in clear cell rcc ( ccrcc ) . ccrcc is the most common form of rcc with a prevalence of 80% . expression of g250 in normal tissue is restricted to the gastrointestinal mucosa and related structures , thereby making it a suitable candidate for targeting ccrcc . in several clinical studies the efficient accumulation of mab g250 in ccrcc has been demonstrated , resulting in high contrast images . g250-imaging could prove to be a valuable tool in diagnosing metastases in patients with a g250-antigen positive primary tumor and/or in the differential diagnosis of suspect kidney lesions . furthermore , the therapeutic efficacy of radiolabeled g250 has been investigated in a series of studies . thus far , most efforts have been devoted to g250 labeled with high doses of 131i . other radionuclides which may enhance the therapeutic index of this radiolabeled mab are currently under investigation . in our institution , an activity dose escalation study is currently ongoing to investigate the therapeutic potential of 177lu - labeled g250 in metastatic ccrcc patients . in this review , the current status of the diagnostic and therapeutic properties of radiolabeled antibodies in rcc is described .

Introduction Monoclonal antibodies Radiolabeled peptides in renal cell carcinoma Monoclonal antibodies in renal cell carcinoma Discovery and use of G250: from mG250 to cG250 Use of unlabeled antibody cG250 Studies with cG250 labeled with indium, iodine and lutetium

renal cell carcinoma ( rcc ) is the most common malignancy arising in the kidney . the classic triad of virchow ( flank pain , hematuria and a palpable abdominal mass ) is only seen in approximately 9% of newly diagnosed patients [ ] . as a consequence , 30% of patients will present with metastatic disease , whereas of the other 70% treated by nephrectomy , 3040% will eventually relapse [ ] . the 5-year survival rate for small ( less than 7 cm ) tumors limited to the kidney ( pt1 tumor ) is more than 90% [ ] , but prognosis for metastatic disease is bleak , with a median survival of only 10 months [ ] . therefore , therapeutic strategies focus on immunotherapy , neoangiogenesis inhibitors and other targeted approaches . in this review , another approach using antibodies developed for targeting rcc is discussed and particularly their application in the diagnosis and therapy of rcc . since the first description of ehrlich to specifically guide cytotoxic therapy to cancer tissue [ ] , much has been debated on the feasibility of this approach . with the identification of the tumor - associated target antigens , real progress has been made on developing treatment and/or diagnostic strategies using mabs . to date , no tumor specific antigen , i.e. an antigen expressed on all tumor cells which is not expressed by normal cells in the body , has been identified . tumor - associated antigens ( taa ) have been identified for a series of human tumor types [ ] . expression of antigen on the primary tumor or metastases is generally heterogeneous . these are : size of the tumor mass , the antigen density , the fate of antigen / antibody complex , presence of circulating antigen , mab format , mab dose , route of administration and mab circulating half - life [ ] . mab targeting is complicated by large tumor blood vessels as well as impaired blood flow in the tumor by elevated interstitial fluid pressure ( ifp ) [ ] . high vascular density is not equivalent to high perfusion rates in the tumor , which are required for optimal mab delivery . however , in comparison with normal kidney tissue rcc is poorly perfused [ ] , thereby impeding adequate mab delivery to the tumor cells . these limitations of delivering the mab to tumor tissue have to be overcome in order to develop a suitable mab - based treatment strategy . since antigen expression within tumors is heterogeneous , antigen - negative tumor cells may evade tumor cell lysis by effector cell- or complement - mediated cytotoxicity , which may eventually lead to tumor recurrence . besides i , other radionuclides ( y , lu , re , re and cu ) have since been investigated to induce tumor cell death ( see table 1 ) . the advantage of radiolabeled antibodies is that the mab does not have to bind to every tumor cell to induce cytotoxicity , since the radionuclides emit -particles , which can be effective for up to 50 or more cell diameters . a disadvantage of this technique is the sensitivity of normal organs to radiation , particularly the bone marrow . a radionuclide conjugated to a tumor - associated mab , is therefore generally hematological . table 1radionuclides used in radioimmunotherapy of clear cell renal cell carcinomaradionuclidehalf - life-average ( kev) ( kev)maximum range -particles in tissue ( mm)advantagesdisadvantagesi8.0 days1923623.0easy labeling ; inexpensivehigh radiation burden to personnel / relatives ; hospital admittance requiredre90.7 h3621375.1out - patient treatment possible ; ideal gamma for imaginglaborious labelingy64 h935none12high - energy beta - emission ; prolonged tumor retention ; out - patient treatment possibleno imaging possiblelu6.7 days1492082.5prolonged tumor retention radionuclides used in radioimmunotherapy of clear cell renal cell carcinoma using the previously mentioned hybridoma technique a wide array of mabs against taas has been produced , e.g. mabs against carcino - embryonic antigen ( cea ) ( mainly expressed in colorectal and medullary thyroid carcinomas ) , muc-1 ( mainly ovarian and breast cancer ) , tag72 ( mainly ovarian and colorectal cancer ) , cd-20 ( non - hodgkin 's lymphoma ( nhl ) ) and g250-antigen ( rcc ) . in various clinical trials safety and efficacy of these newly developed mabs labeled with various radionuclides have been investigated [ ] . extensive research has resulted in the first registered treatment with radiolabeled mabs directed against the surface antigen cd-20 expressed on b - cell nhl ( y - labeled anti - cd20 mab ibritumomab tiuxetan ( zevalin , biogen idec , boston ma , usa and schering , berlin , germany ) and i - labeled anti - cd20 mab tositumomab ( bexxar , gsk , philadelphia , pa , usa ) . in patients with solid tumors , therapeutic strategies with radiolabeled mabs however , as mentioned previously , tumor - related factors also play an important role . the most common types of solid malignancies targeted in clinical trials with rit have been epithelial cancers , e.g. results of these trials did not result in registration of radiolabeled mab preparations for regular treatment of these cancer types . partial responses and stabilization of previously progressive disease have been seen in few patients in most of these trials [ ] . to date , the somatostatin ( sst ) analog in - labeled - octreotide ( octreoscan , mallinckrodt tyco healthcare , petten , the netherlands ) is the most successful radiopeptide for tumor imaging and has been the first to be approved for scintigraphic localization of primary and metastatic neuro - endocrine tumors expressing sst2 and sst5 receptor subtypes [ ] . expression of these receptors was found in 72% of rcc samples analyzed , irrespective of histopathological subtype or grading of the tumor [ ] . the use of [ in]octreotide has been evaluated in patients with metastatic rcc . in this study , 68 rcc metastases in 9 patients , confirmed by diagnostic ct and/or x - ray images were evaluated . besides octreotide , a new series of peptides is now being evaluated for targeting of solid tumors . the gastrointestinal peptide gastrin acts as a neurotransmitter in the brain and as a regulator of various functions in the gastrointestinal tract [ ] . these receptors are highly expressed in medullary thyroid carcinoma ( mtc ) , enabling gastrin to visualize metastatic mtc with very high sensitivity [ ] . vip , a member of the group of secretin - like peptides , is an important neurotransmitter in the gut . although expression of the vip receptors has been found on nephroblastomas [ ] , expression on rcc has not been determined . npy is a neurotransmitter that is predominantly found in the central nervous system , where it functions as a stimulator of feeding behavior and inhibition of anxiety [ ] . more recently , expression of npy receptors has been found on rcc and nephroblastomas , suggesting a potential role for rcc targeting using radiolabeled npy [ ] . bombesin has a high and specific affinity for the gastrin releasing peptide receptor ( grp - r ) and this receptor stimulates proliferation of tumor growth in various tumor types [ ] . rcc has been found to have a high expression of grp - r [ ] . targeting of rcc was done using a bombesin analogue labeled with lu . in this study , in vitro autoradiography showed specific uptake of the radioligand in five of the six rcc samples evaluated [ ] . the expression of v3 has been found to increase with higher rcc tumor grades . of the rcc metastases examined , 2 of 14 showed high expression of v3 , 8 of 14 showed weak expression and 4 of 14 did not express the v3 integrin [ ] . in rcc , several mabs have been defined that are reactive with rcc - associated antigens [ ] . cross - reactivity with non - kidney tissue was seen in some of these mabs , whereas others were only expressed in kidney / rcc . one of these mabs , which showed relative high tumor - to - blood ratios in mice with rcc xenografts is mab a6h [ ] . in a clinical study , the imaging and rit potential of this mab was examined [ ] . this low sensitivity was attributed to soluble antigen binding by the mab and the expression of antigen in normal tissue , thereby not allowing the mab to bind to tumor tissue . this clinical finding of antigen expression in normal tissue was not in line with the previous findings . after modification of the dosing regimen , the detection rate of metastatic lesions increased , but the number of detected lesions remained unsatisfactory . as a result , the use of mab a6h for diagnosis and treatment of rcc was discontinued . g250 , a mab against a rcc - associated antigen has been investigated extensively , because the antigen which this mab recognizes showed remarkable tissue distribution and expression . the antigen that mab g250 targets has been designated in the literature as mn , ca ix and g250 . of the eight metastases examined , g250-antigen expression was homogeneous in five ( 62% ) , heterogeneous in two , while one did not express the g250-antigen [ ] . expression in normal tissues has been evaluated extensively and has been shown to be restricted to the ( upper ) gastrointestinal mucosa ( stomach , ileum , proximal and middle colon ) and gastrointestinal related structures ( intra- and extrahepatic biliary system , pancreas ) [ ] . later studies showed an almost ubiquitous expression ( > 90% ) of g250-antigen in clear cell rcc ( ccrcc ) , being the most prominent form of rcc ( 80% of cases ) . g250-antigen expression in the different histological subtypes of rcc was determined by rt - pcr and immunohistochemistry . all the clear cell tumors displayed g250-antigen mrna , but expression of g250-antigen by oncocytomas , chromophobe or papillary rcc was low or absent [ ] . an early or a first event in the clear cell rcc tumorigenic pathway is mutations leading to loss of von hippel lindau protein ( pvhl ) in 5075% of sporadic rcc . in normoxic conditions pvhl is responsible for degradation of hypoxia inducible factor-1 ( hif-1 ) , which thereby can not bind to hif-1 to form hif-1 . clearly , expression of these proteins is advantageous for tumor growth . with the loss of functional pvhl in rcc this mutational loss of pvhl thus explains why g250-antigen is almost invariably upregulated in ccrcc . g250-antigen is also expressed in various other tumor types ( e.g. various animal and ex vivo experiments have shown the potential of the g250 mab as a targeting modality of rcc [ ] . since g250-antigen is high and homogeneously expressed in rcc tissue , is restricted to a few normal tissues and other tumors , and a low mab dose is needed to obtain antigen saturation , mab g250 seemed a suitable candidate for further investigation in clinical studies . ten of these tumors proved to be g250-positive , whereas the other two showed less than 5% g250-antigen expression . after nephrectomy , tumor samples were shown to have high and focal uptake of g250 , up to 0.21 % injected dose / gram ( % id / g ) . therefore , this was indicative of true antibody targeting of the tumor by mg250 [ ] . as good targeting of ccrcc by mg250 was seen in this study , a phase i / ii radioimmunotherapy ( rit ) dose escalation study was performed by divgi et al . patients in this study were treated with one high - activity - dose injection of [ i]mg250 . in the phase i dose - escalation study , mtd was defined at 3330 mbq / m , due to hematological toxicity . a total of 33 patients was treated , 18 in the dose - escalating part of the study and another 15 patients at the mtd ( 3330 mbq / m ) , to evaluate therapeutic efficacy . the formation of human anti mouse antibodies ( hama ) in all patients receiving mg250 prohibited retreatment . formation of immune complexes with rapid clearance of the radiolabeled mab to liver and spleen would have occurred in the case of multiple administrations , thereby limiting targeting of the mab to the tumor [ ] . this , in combination with the high potential of g250 as a targeting agent in the treatment of metastasized rcc , led to the development of a chimeric form of g250 ( cg250 ) [ ] . unlabeled g250 antibody facilitates antibody - dependent cellular cytotoxicity ( adcc ) of g250-antigen expressing cells , which leads to induction of lysis of these cells [ ] . this finding led to a study where 36 patients with metastatic ccrcc received 50 mg cg250 weekly for 12 weeks . based on these results , an adjuvant phase iii trial has been initiated in high - risk ccrcc patients who are nephrectomized and have no known metastases , using this treatment regimen . high - dose bolus il-2 as therapy in metastatic rcc has had varying success , with responses in up to 15% of patients [ ] . vice versa , co - administration of il-2 can enhance the therapeutic efficacy of g250 [ ] . in a phase ii trial 35 patients with progressive ccrcc received weekly i.v . mean survival was 24 months in this trial , compared to 16.3 months median survival with high - dose il-2 therapy [ ] , which also has toxic side effects . after cg250 became available , the pharmacokinetics , biodistribution , imaging characteristics and dosimetry of this new radiolabeled targeting vehicle was studied in a protein dose escalation study identical to murine g250 . sixteen presurgical rcc patients received increasing doses of cg250 between 2 and 50 mg labeled with i , given i.v . reducing the immunogenic properties of the antibody opened the possibility of multiple treatments . the mtd of [ i]cg250 in metastatic rcc was determined in a phase i radioactivity dose escalation trial in patients with progressive metastatic rcc at study entry . when accumulation of antibody was seen in any tumor site , patients received escalating radioactivity - doses of [ i]cg250 . in contrast to the trials performed with murine g250 , no hepatic toxicity was seen . this was believed to be the result of saturation of the hepatic compartment by the diagnostic scout dose of [ i]cg250 . fractionation of the dose was done in a phase i study by divgi et al . ( 2220 mbq / m ) , combined with the properties of cg250 allowing multiple administrations , led to a study where two sequential high doses of [ i]cg250 treatment were given . when tumor targeting was seen again , the second high - dose injection [ i]cg250 was given . mtd of the second rit proved to be again due to hematological toxicity and was set at 1665 mbq / m , being 75% of the mtd of the first infusion ) . therapeutic radiation doses ( more than 50 gy ) [ ] were only guided to the lesions smaller than 5 g. the authors concluded that rit in rcc patients could best be given in the setting of small volume disease or as adjuvant therapy [ ] . first , the targeting capabilities of [ in]cg250 have been compared to those of [ i]cg250 . metallic radionuclides , such as in , y and lu , are trapped in the lysosomes and residualize after internalization of the mab - antigen complex by the target cells [ ] . to investigate whether this phenomenon may also occur in humans , five patients with metastatic rcc were i.v . figure 1(a ) [ in]cg 250 immunoscintigram of a patient with metastatic ccrcc , acquired 6 days after injection of 185 mbq of [ in]cg250 . the anterior image is shown in the left panel , ( b ) pet - ct scan of the same patient acquired after injection of 250 mbq of [ f]fdg . the anterior image is shown in the left panel , ( b ) pet - ct scan of the same patient acquired after injection of 250 mbq of [ f]fdg . the therapeutic properties of cg250 labeled with four radionuclides have been tested in nude mice with human rcc xenografts . the four radionuclides under investigation were y and lu ( both residualizing ) , and i and re ( both non - residualizing ) . based on these preclinical and clinical data ( table 2 ) , an ongoing phase i / ii [ lu]dota - cg250 dose escalation rit study in progressive , metastatic rcc patients was designed . patients in whom cg250 targeting of rcc metastases is observed are treated with up to three cycles of [ lu]dota - cg250 to determine mtd . so far , minor responses have been noted at the lower dose levels and dose escalation is ongoing . table 2phase i / ii radioimmunotherapy trials in clear cell renal cell carcinomareferenceradiophar - maceuticaltarget antigenmab typeno . [][i]cg250g250chimeric g250275 sdtwo high - dose treatment phase i / ii radioimmunotherapy trials in clear cell renal cell carcinoma future strategies to improve clinical efficacy of cg250 could be : ( 1 ) tumor pretargeting ; ( 2 ) high dose rit with bone marrow support or transplant or ( 3 ) use of high linear energy transfer ( let ) particles emitting radionuclides . the radionuclide is administered in a second injection as a rapidly clearing agent with high affinity to the previously administered mab . affinity of the radionuclide binding to the mab may be achieved through , e.g. autologous marrow transplantation or peripheral blood stem cell reinfusion has been investigated as a means to overcome bone marrow toxicity in rit , thereby allowing administration of activity doses more than twice as high as without any bone marrow support [ ] . the use of let ( alpha ) particle emitting radionuclides has the advantage of high cytotoxic potency , combined with a low range and thus not reaching most normal tissue surrounding the tumor . however , most alpha - emitters have a half - life of less than 1 h , which is hardly compatible with mabs targeting tumors . nevertheless , to date clinical impact of rit for treatment of ccrcc has been minimal . it remains to be established whether the use of more powerful radionuclides can lead to alteration of the clinical course of metastatic ccrcc . finally , with the advent of new treatment possibilities for rcc such as angiogenesis inhibitors , combination treatment with g250 rit and these new substances may play a role in more effective management of ccrcc .

in this article , we present a view of health care spending in the u.s . that focuses on the sectors that finance or sponsor health care . this view allows us to examine each of the sponsor 's ability to pay for their health care obligations . the basis for these estimates is the national health expenditure accounts , the official federal government estimates of total u.s . the nhea structure is a matrix comprised of expenditures for health care goods and services and of funding sources that pay for these goods and services . these sources of funds are classified into private health insurance ( phi ) , out - of - pocket spending , other private revenues , and specific government programs such as medicare and medicaid . for national accounting , this structure is useful in measuring changes in spending trends associated with policy initiatives in the government and private sectors , along with the amounts paid by each source . the analysis in this article is based on a subset of the national health expenditures . this subset , health services and supplies ( hss ) , represents spending for health care provided during the year , including personal health care , government public health and program administration . in 2003 , hss was about 96 percent of national health expenditures , which also include investment , research , and construction expenditures . to determine where the responsibility for financing health care falls , we reorganize spending into business , household , and government sectors . this reallocation to the sponsors of health care is as follows : phi allocated to businesses , federal , and state and local government employers and employees ( or households ) who pay for employer - sponsored health insurance premiums , and to individuals ( or households ) who purchase health insurance directly . medicare distributed between employers ( businesses , federal , and state and local governments ) , and employees ( households ) are the payroll taxes through the federal insurance contributions act ( fica ) and the self - employment contributions act ( seca ) for the hospital insurance ( hi ) fund . supplementary medical insurance ( smi ) premiums paid by beneficiaries are allocated to households and the corresponding smi general revenues are allocated to the federal government . buy - ins ( payments by state medicaid programs and matched by the federal government of hi and smi premiums for individuals who are eligible for both medicaid and medicare)are classified with the medicaid program . medicaid distributed to federal and state and local governments . workers ' compensation spending , temporary disability insurance , and industrial inplant health services allocated to employers who sponsor these benefits . a small portion of the health spending is estimated for other private revenues philanthropic giving and revenues received by some health care providers from non - health services ( for example , cafeteria , and gift shop revenues ) . after the nhea sources of funds are allocated to these sponsor categories , we construct ways to compare sponsor 's health care financing amounts with measures of their overall income or revenues . these relative measures help track the changes in the sponsors ' ability to finance health care . in the private business sector , we compare health care spending to total employee compensation and to aggregate wages and salaries . the burden measure for households federal , state , and local government burden is measured by comparing spending on health to tax receipts . although we categorize sponsors into businesses , households , and governments direct financers of health insurance individuals ultimately bear the full responsibility of paying for increasing health care costs through higher taxes , reduced wages , and higher product costs ( pauly , 1995 ) . more information regarding the methodology and definitions is available at the cms web site : http://www.cms.hhs.gov/statistics/burden-of-health-care-costs/ businesses , households , and governments are sponsors of health care , and therefore pay the costs of consuming medical care . the changing obligations placed on each of these sponsors can result in changes to the types of health insurance that is offered or selected , scope of benefits and cost - sharing arrangements . in this article , we have constructed measures to track the changes in the ability to finance health care faced by these sponsors . in 2003 , spending growth for health services and supplies decelerated for the first time in 7 years ( smith et al . , 2005 ) . even with this slowdown , the burden placed on each of the sponsors continued to grow . the portion of health spending as a share of total compensation continued to grow even as businesses passed on more of the growth in health care costs to employees by increasing their portion of phi premiums and raising copays and deductibles . household income did not keep pace with the increased premiums and out - of - pocket health care spending . for federal programs , while health care costs slowed due to legislative changes , federal revenues declined in 2003 . states are also struggling with ways to pay for health costs despite seeing this cost growth slow in 2003 . in the near future states have had a slight increase in the growth of revenues in 2004 ( national governors association and national association of state budget officers , 2004 ) . the medicare prescription drug , improvement , and modernization act of 2003 expands the medicare program to include prescription drug coverage that lessens the burden that households face in paying for health costs . this legislation also provides subsidies to employers to help them offset the costs of providing health insurance coverage for retirees and is intended to reduce states ' contributions to prescription drug spending for dually eligible beneficiaries . however , a few of these changes will shift the health care financing to governments , particularly the federal government , which raises long term sustainability questions for the federal government programs as highlighted in the 2005 annual report of the board of trustees of the federal hospital insurance and federal supplementary medical insurance trust fund ( 2005 ) . other private revenues , such as philanthropy , have maintained a generally steady share of hss , going from 5 percent in 1987 to 4 percent in 2003 . the share of hss financed by businesses , households , and other private sources decreased from 69 percent in 1987 to 61 percent in 2003 as the public share grew from 31 to 39 percent narrowing the gap between public and private financing . although government - financed health cost growth moderated somewhat in 2003 , it was still the third consecutive year that government expenditures grew faster than overall businesses , households , and other private expenditures . during the early 1990s this was especially true for the federal government in financing these expenditures , prompting changes to the medicare program , such as those that occurred with the balanced budget act ( bba ) of 1997 . ( subsequent legislation , such as the balanced budget refinement act of 1999 , the benefits improvement and protection act of 2000 , and the consolidated appropriations resolution of 2003 offset some of the bba reductions . ) government expenditures include spending by federal , and state and local governments for programs such as medicaid and medicare ( 81 percent ) plus phi premiums and medicare hi trust funds payroll taxes paid on behalf of government employees ( 19 percent ) . most of the shift in share to the government sector from 1987 to 1997 was offset by a decrease in the share of household spending , which fell from 39 to 32 percent . this was particularly true during the managed care era ( 1993 - 1997 ) when strong competition among health plans for the employer market , together with effective price negotiation with health care providers and other cost management techniques , helped slow health costs increases and reduce the proportion of costs that were paid out of pocket . the share of spending by business has been virtually unchanged at 26 - 27 percent over the past 16 years . private businesses expenditures reached $ 423.0 billion in 2003 , 7.1 percent higher than in 2002 . the employer portion of employer - sponsored health insurance premiums is the largest share of private businesses health expenditures , 76 percent , followed by employer payroll taxes for the medicare hi trust fund , 15 percent . despite the recent slowing growth in phi premiums , businesses still faced an average annual growth in phi premiums of 8.3 percent from 1999 - 2003 , compared to 5.9 percent during the 1991 - 1998 period , when enrollment in health maintenance organizations ( hmos ) was at an all - time high . such plans were reasonably successful at controlling health care costs , but became unpopular with consumers , resulting in declining hmo enrollment , from 31 percent of covered workers in 1996 to 25 percent in 2004 ( claxton et al . , 2004 ) . during that time , workers increasingly have shown preference for preferred provider organization and point - of - service plans , trading broader access to providers for higher costs ( levit et al . , 2003 ) . during 2001 and 2002 , the mild recession marked by falling employment , very low inflation , and stagnant wages and salaries resulted in a deceleration , then decline , in private employer paid payroll taxes collected for the medicare hi trust fund . in fact , from 2000 to 2002 , employer - paid medicare payroll taxes experienced the slowest period of growth in the history of the fund . in general , since these taxes are based on wages and salaries and do not directly reflect increases in health care costs , growth in payroll taxes has not kept up with other private business - paid health care costs . as a result , the payroll tax share of total private business expenditure for health has steadily declined ( from 20 percent in 1987 to 15 percent in 2003 ) . temporary disability insurance provides workers with partial compensation for loss of wages caused by temporary nonoccupational disability . industrial inplant health services are the employer costs associated with directly operating facilities or providing supplies for the health care needs of employees , either on- or off - site . these three programs were 9 percent of spending by private business on health care in 2003 . rising medical costs in the late 1980s prompted employers to adopt managed care workers ' compensation plans , a step that is , in part , credited with slowing workers ' compensation cost growth from 1992 - 1997 . additionally , lower injury rates , benefit changes , safety and return - to - work programs , antifraud measures , and tightening of eligibility standards likely contributed to slowing growth during that same period ( mont et al . since 1998 , businesses health spending ( bhs ) , as a share of total compensation , has been on the rise . in 2003 , health spending for private businesses was 8.3 percent of total compensation , up from 7.0 percent in 1998 . breaking from prior and subsequent trends , private sector employees saw their wages and salaries grow faster than private businesses spending on health care from 19951998 . over the last 4 years , however , private bhs outpaced wage and salary growth by an average annual rate of nearly 5 percent ( u.s . bureau of economic analysis , 2005 ) . for almost a decade between 1991 and 2000 , health care spending by private business as a share of compensation remained fairly stable . between 2000 and 2003 , however , this share rose sharply from 7.2 to 8.3 percent , driven by escalating employer - paid health insurance premiums and workers ' compensation medical costs . most economists believe that employers trade wages for rising benefit costs , resulting in slower wage or non - medical benefit growth for workers ( monaco and phelps , 1995 ; pauly , 1995 ) . as a percent of total compensation total phi premiums reached $ 600.6 billion in 2003 and per enrollee estimates were $ 3,088 . over the last decade , total phi premiums more than doubled , while the number of enrollees has grown less than 6 percent . this means that almost all of the growth in phi premiums is a result of increasing costs and utilization per enrollee , rather than increases in the number of persons covered by phi . overall , in 2003 , the share of premiums paid by private and government employers declined to 75.8 percent , a level last seen in the late 1990s . while 2001 and 2002 was a period of double - digit phi premium growth , some private employers were able to shift expenses to employees through higher premiums ( levit et al . this resulted in the percent of phi paid by private employers declining from a high in 2000 of 76.0 to 74.6 in 2003 . the federal government share of employer - sponsored health insurance has remained relatively stable over the past 3 years . however , federal employee and employer contributions to phi have experienced 3 years of double - digit growth , higher than most past years since 1987 . the number of enrollees with phi continued to decline , from a peak of 197.6 million in 2000 to 194.5 million in 2003 , a level last seen in 1999 . the recent decline of manufacturing jobs and increase in service sector employment has impacted worker benefits because service sector jobs typically are less likely to provide health insurance . this continued structural change , intensified by the recent recession , may have partly contributed to the decline in the enrollment in employment - based health insurance plans ( fronstin , 2004 ) . in addition , for the manufacturing jobs that remain , the likelihood of coverage by employer - sponsored health insurance diminished , also contributing to the decline in employment - based health insurance ( fronstin , 2004 ) . however , other research has attributed a majority of the decline in the number of insured to premium increases for employees , not employment changes . a study estimates that since 1987 , workforce changes have had little effect on the rates of coverage . this study suggests that declines in coverage have resulted almost entirely from increases in premiums in relation to personal income ( gilmer and kronic , 2005 ) . households ' spending reached $ 512.6 billion in 2003 , about one - third of total spending on hss . spending for phi premiums , including the employee share of employer - sponsored health insurance and individually purchased health insurance , was $ 174 billion while out - of - pocket spending for copays , deductibles , and for goods and services not covered by insurance was $ 230.5 billion . since 1987 , the share of households ' health spending going to phi premiums increased from 23 to 34 percent while the share for out - of - pocket spending dropped from 58 to 45 percent . spending by households grew 7.8 percent in 2003 , the second consecutive year of over 7 percent growth . other periods with growth in this range include 1988 to 1990 and 1997 to 1998 . the earlier period of higher growth occurred before the expansion of enrollment in the more tightly managed health plans , while the later period reflected stabilization in enrollment in these plans ( claxton et al . the latest period of slow growth , 1999 to 2001 , occurred as the overall economy grew rapidly and labor markets were tight , providing employers with incentives not to pass on rising health care costs to employees . in 2002 , this changed as employers began passing more costs to individuals , primarily through higher copays and deductibles . by looking at the share of household personal income that goes to health care , we can assess the burden that health care costs place on households . between 2001 and 2003 , the share of personal income consumed by health care grew rapidly , increasing 0.6 percentage points from 5.3 to 5.9 percent . this is the fastest increase in share since the 1987 - 1988 period . for 2002 and 2003 , personal income growth of 3.1 percent in 2001 , 1.3 percent in 2002 , and 3.0 percent in 2003 were slow by historical standards ( u.s . bureau of economic analysis , 2004 ) . during this period , household spending for health care grew at rates that at 5.8 percent in 2001 , 7.5 percent in 2002 , and 7.8 percent in 2003were two or more times as fast as income growth . in 2003 , spending of $ 278.1 billion by state and local governments for health care represented a marked deceleration in spending growth . the 7.7 percent increase in spending was over 3 percentage points slower than in 2002 . the primary driver of this lower growth rate was slowing medicaid expenditures , whose growth slowed by 4.1 percentage points in 2003from 11.4 percent to 7.3 percent . recently , states have been experiencing fiscal pressures , and by mid-2003 , when states were beginning their fiscal year , nearly all states had implemented some kind of cost containment on medicaid spending ( smith et al . , 2004 ) . at the same time , states ' ability to utilize various creative financing schemes to increase federal medicaid funding were limited by federal regulation ( smith et al . , 2005 ) . since state and local governments are also employers , contributions to phi premiums for active and retired workers accounted for almost one - third of state and local health expenditures in 2003 . though growth in the states ' payments for phi premiums decelerated in 2003 , it still marked the fourth consecutive year of double - digit growth . other state and local government programs such as general assistance , maternal and child health , and public health activities accounted for 26 percent of state and local government health spending in 2003 . spending by the federal government for health care reached $ 344.0 billion in 2003 , or 21 percent of hss . federal health care spending growth decelerated sharply in 2003 , at roughly one - half the 2001 and 2002 rates . growth for all federal health programs , except the department of veterans affairs and indian health service , decelerated between 2002 and 2003 ( cms , 2005 ) . federal government medicare expenditures are calculated as nhea medicare expenditures for benefits and administration less the sum of hi payroll taxes paid by employers , employees , and the self - employed , hi and smi premiums , and hi income from taxation of social security benefits . this difference is roughly equal to trust fund interest income and federal general revenue contributions to medicare . medicaid spending , which was 47 percent of federal health care costs in 2003 , also decelerated sharply , slowing from 12.6 percent growth in 2002 to 6.9 percent in 2003 . while nearly all states implemented cost containment efforts in 2003 , many states specifically controlled growth in medicaid spending and enrollment by tightening eligibility and restricting benefits ( national governors association and national association of state budget officers , 2004 ) . growth in these programs decelerated from 10.3 percent in 2002 to 6.7 percent in 2003 . they account for about one - fifth of federal government spending on health care . from 1998 to 2000 , growth in federal general revenue and interest income financing for medicare steadily decelerated . during this period , this coincided with more revenue collected through payroll taxes levied on rapidly rising wages in a growing economy . in 2001 and 2002 as economic growth slowed , the amount of medicare spending financed through general revenues increased substantially , due in part to a rapid growth in overall medicare spending coupled with a slowdown in income received from payroll taxes . in 2003 , as overall growth in medicare spending slowed and growth in income from payroll taxes accelerated slightly , the growth in medicare expenditures financed by general revenues slowed . however , the share of medicare financed through general revenues and premiums increased because total medicare expenditures continued to grow faster than income from payroll taxes . the burden on governments to pay for health costs is increasing . federal government 's burden , measured by comparing its employer and general revenue spending on health against its nonpayroll - tax revenues , increased significantly in 2003 to about one - third of federal revenues . while federal health care spending growth slowed in 2003 , the federal government 's revenues declined as income tax cuts were implemented ( u.s . state and local government burden , using a similar measure as that used for the federal government comparison , also increased in 2003 . about one - quarter of state and local governments ' revenues go to health care . several states have reached a financial crisis as they struggle to find ways to finance increasing health care costs , especially for medicaid . unlike the federal government that can support deficit spending by borrowing , almost all state governments must balance their budgets each year , making the pressure they face from rising state health care costs particularly acute . higher than expected state revenues from taxes in states fiscal year 2004 , which includes part of calendar year 2003 , partially offset increased growth in state health spending , resulting in only a modest increase in burden in 2003 ( national governors association and national association of state budget officers , 2004 ) . overall , health - related tax expenditures accounted for 16 percent , or $ 116.2 billion , of all tax expenditures in 2003 , ranking third behind commerce and housing , at 32 percent , and income security , at 21 percent . the federal government receives less income tax revenue then would otherwise occur because of certain allowed tax deductions and income exclusions . tax exclusions for employer contributions for medical insurance premiums and medical care were the largest health - related tax expenditure in 2003 , at $ 101.9 billion . the second largest health - related tax expenditure was deductible medical expenses at $ 6.2 billion . current national income accounting principles recognized by the bureau of economic analysis national income and product accounts ( nipa ) , the united nation 's system of national accounts , and the organization of economic cooperation and development ( oecd ) health accounts , do not include tax expenditures in their estimates . cms , the bureau of economic analysis , and oecd recognize that tax expenditures provide important economic incentives that influence the level and distribution of costs throughout the entire health care market . however , because no explicit taxes are collected or spending incurred , the nhea , like the nipa and the oecd , do not include tax expenditures in their official national accounting practices . , so does the debate about how to show estimates of health - related tax expenditures and compare them with the nhea . the office of management and budget ( omb ) offers some insight on the difficulty of directly integrating tax expenditures with the nhea stating that individual tax expenditures will not necessarily equal the increase in federal revenues by repealing these special provisions . tax expenditures alter economic behavior through various incentives and the estimates provided are interdependent , meaning they do not reflect any interactions between other programs and individual and corporate income tax receipts ( executive office of the president , 2004 ) . currently , cms , along with omb and the congressional budget office ( cbo ) use sidebars , such as this discussion , to show tax expenditure estimates .

this article provides estimates of health care expenditures by businesses , households , and governments for 1987 - 2003 . sponsors that finance public and private health insurance programs and other payers face increasing challenges as health care cost rise . their capacity to support rising costs was particularly strained during the recent economic recession , with the federal government 's burden measured against revenue available for this purpose growing faster than for other sponsors .

Introduction Summary

in this article , we present a view of health care spending in the u.s . that focuses on the sectors that finance or sponsor health care . this view allows us to examine each of the sponsor 's ability to pay for their health care obligations . the basis for these estimates is the national health expenditure accounts , the official federal government estimates of total u.s . the nhea structure is a matrix comprised of expenditures for health care goods and services and of funding sources that pay for these goods and services . these sources of funds are classified into private health insurance ( phi ) , out - of - pocket spending , other private revenues , and specific government programs such as medicare and medicaid . for national accounting , this structure is useful in measuring changes in spending trends associated with policy initiatives in the government and private sectors , along with the amounts paid by each source . the analysis in this article is based on a subset of the national health expenditures . this subset , health services and supplies ( hss ) , represents spending for health care provided during the year , including personal health care , government public health and program administration . in 2003 , hss was about 96 percent of national health expenditures , which also include investment , research , and construction expenditures . to determine where the responsibility for financing health care falls , we reorganize spending into business , household , and government sectors . this reallocation to the sponsors of health care is as follows : phi allocated to businesses , federal , and state and local government employers and employees ( or households ) who pay for employer - sponsored health insurance premiums , and to individuals ( or households ) who purchase health insurance directly . medicare distributed between employers ( businesses , federal , and state and local governments ) , and employees ( households ) are the payroll taxes through the federal insurance contributions act ( fica ) and the self - employment contributions act ( seca ) for the hospital insurance ( hi ) fund . supplementary medical insurance ( smi ) premiums paid by beneficiaries are allocated to households and the corresponding smi general revenues are allocated to the federal government . buy - ins ( payments by state medicaid programs and matched by the federal government of hi and smi premiums for individuals who are eligible for both medicaid and medicare)are classified with the medicaid program . medicaid distributed to federal and state and local governments . workers ' compensation spending , temporary disability insurance , and industrial inplant health services allocated to employers who sponsor these benefits . a small portion of the health spending is estimated for other private revenues philanthropic giving and revenues received by some health care providers from non - health services ( for example , cafeteria , and gift shop revenues ) . after the nhea sources of funds are allocated to these sponsor categories , we construct ways to compare sponsor 's health care financing amounts with measures of their overall income or revenues . these relative measures help track the changes in the sponsors ' ability to finance health care . in the private business sector , we compare health care spending to total employee compensation and to aggregate wages and salaries . the burden measure for households federal , state , and local government burden is measured by comparing spending on health to tax receipts . although we categorize sponsors into businesses , households , and governments direct financers of health insurance individuals ultimately bear the full responsibility of paying for increasing health care costs through higher taxes , reduced wages , and higher product costs ( pauly , 1995 ) . more information regarding the methodology and definitions is available at the cms web site : http://www.cms.hhs.gov/statistics/burden-of-health-care-costs/ businesses , households , and governments are sponsors of health care , and therefore pay the costs of consuming medical care . the changing obligations placed on each of these sponsors can result in changes to the types of health insurance that is offered or selected , scope of benefits and cost - sharing arrangements . in this article , we have constructed measures to track the changes in the ability to finance health care faced by these sponsors . in 2003 , spending growth for health services and supplies decelerated for the first time in 7 years ( smith et al . , 2005 ) . even with this slowdown , the burden placed on each of the sponsors continued to grow . the portion of health spending as a share of total compensation continued to grow even as businesses passed on more of the growth in health care costs to employees by increasing their portion of phi premiums and raising copays and deductibles . household income did not keep pace with the increased premiums and out - of - pocket health care spending . for federal programs , while health care costs slowed due to legislative changes , federal revenues declined in 2003 . in the near future states have had a slight increase in the growth of revenues in 2004 ( national governors association and national association of state budget officers , 2004 ) . the medicare prescription drug , improvement , and modernization act of 2003 expands the medicare program to include prescription drug coverage that lessens the burden that households face in paying for health costs . this legislation also provides subsidies to employers to help them offset the costs of providing health insurance coverage for retirees and is intended to reduce states ' contributions to prescription drug spending for dually eligible beneficiaries . however , a few of these changes will shift the health care financing to governments , particularly the federal government , which raises long term sustainability questions for the federal government programs as highlighted in the 2005 annual report of the board of trustees of the federal hospital insurance and federal supplementary medical insurance trust fund ( 2005 ) . other private revenues , such as philanthropy , have maintained a generally steady share of hss , going from 5 percent in 1987 to 4 percent in 2003 . the share of hss financed by businesses , households , and other private sources decreased from 69 percent in 1987 to 61 percent in 2003 as the public share grew from 31 to 39 percent narrowing the gap between public and private financing . although government - financed health cost growth moderated somewhat in 2003 , it was still the third consecutive year that government expenditures grew faster than overall businesses , households , and other private expenditures . during the early 1990s this was especially true for the federal government in financing these expenditures , prompting changes to the medicare program , such as those that occurred with the balanced budget act ( bba ) of 1997 . ( subsequent legislation , such as the balanced budget refinement act of 1999 , the benefits improvement and protection act of 2000 , and the consolidated appropriations resolution of 2003 offset some of the bba reductions . ) government expenditures include spending by federal , and state and local governments for programs such as medicaid and medicare ( 81 percent ) plus phi premiums and medicare hi trust funds payroll taxes paid on behalf of government employees ( 19 percent ) . most of the shift in share to the government sector from 1987 to 1997 was offset by a decrease in the share of household spending , which fell from 39 to 32 percent . this was particularly true during the managed care era ( 1993 - 1997 ) when strong competition among health plans for the employer market , together with effective price negotiation with health care providers and other cost management techniques , helped slow health costs increases and reduce the proportion of costs that were paid out of pocket . the share of spending by business has been virtually unchanged at 26 - 27 percent over the past 16 years . private businesses expenditures reached $ 423.0 billion in 2003 , 7.1 percent higher than in 2002 . the employer portion of employer - sponsored health insurance premiums is the largest share of private businesses health expenditures , 76 percent , followed by employer payroll taxes for the medicare hi trust fund , 15 percent . despite the recent slowing growth in phi premiums , businesses still faced an average annual growth in phi premiums of 8.3 percent from 1999 - 2003 , compared to 5.9 percent during the 1991 - 1998 period , when enrollment in health maintenance organizations ( hmos ) was at an all - time high . such plans were reasonably successful at controlling health care costs , but became unpopular with consumers , resulting in declining hmo enrollment , from 31 percent of covered workers in 1996 to 25 percent in 2004 ( claxton et al . , 2004 ) . during that time , workers increasingly have shown preference for preferred provider organization and point - of - service plans , trading broader access to providers for higher costs ( levit et al . , 2003 ) . during 2001 and 2002 , the mild recession marked by falling employment , very low inflation , and stagnant wages and salaries resulted in a deceleration , then decline , in private employer paid payroll taxes collected for the medicare hi trust fund . in general , since these taxes are based on wages and salaries and do not directly reflect increases in health care costs , growth in payroll taxes has not kept up with other private business - paid health care costs . as a result , the payroll tax share of total private business expenditure for health has steadily declined ( from 20 percent in 1987 to 15 percent in 2003 ) . temporary disability insurance provides workers with partial compensation for loss of wages caused by temporary nonoccupational disability . industrial inplant health services are the employer costs associated with directly operating facilities or providing supplies for the health care needs of employees , either on- or off - site . these three programs were 9 percent of spending by private business on health care in 2003 . rising medical costs in the late 1980s prompted employers to adopt managed care workers ' compensation plans , a step that is , in part , credited with slowing workers ' compensation cost growth from 1992 - 1997 . additionally , lower injury rates , benefit changes , safety and return - to - work programs , antifraud measures , and tightening of eligibility standards likely contributed to slowing growth during that same period ( mont et al . since 1998 , businesses health spending ( bhs ) , as a share of total compensation , has been on the rise . in 2003 , health spending for private businesses was 8.3 percent of total compensation , up from 7.0 percent in 1998 . breaking from prior and subsequent trends , private sector employees saw their wages and salaries grow faster than private businesses spending on health care from 19951998 . over the last 4 years , however , private bhs outpaced wage and salary growth by an average annual rate of nearly 5 percent ( u.s . bureau of economic analysis , 2005 ) . for almost a decade between 1991 and 2000 , health care spending by private business as a share of compensation remained fairly stable . between 2000 and 2003 , however , this share rose sharply from 7.2 to 8.3 percent , driven by escalating employer - paid health insurance premiums and workers ' compensation medical costs . most economists believe that employers trade wages for rising benefit costs , resulting in slower wage or non - medical benefit growth for workers ( monaco and phelps , 1995 ; pauly , 1995 ) . as a percent of total compensation total phi premiums reached $ 600.6 billion in 2003 and per enrollee estimates were $ 3,088 . this means that almost all of the growth in phi premiums is a result of increasing costs and utilization per enrollee , rather than increases in the number of persons covered by phi . overall , in 2003 , the share of premiums paid by private and government employers declined to 75.8 percent , a level last seen in the late 1990s . while 2001 and 2002 was a period of double - digit phi premium growth , some private employers were able to shift expenses to employees through higher premiums ( levit et al . this resulted in the percent of phi paid by private employers declining from a high in 2000 of 76.0 to 74.6 in 2003 . the federal government share of employer - sponsored health insurance has remained relatively stable over the past 3 years . the number of enrollees with phi continued to decline , from a peak of 197.6 million in 2000 to 194.5 million in 2003 , a level last seen in 1999 . the recent decline of manufacturing jobs and increase in service sector employment has impacted worker benefits because service sector jobs typically are less likely to provide health insurance . this continued structural change , intensified by the recent recession , may have partly contributed to the decline in the enrollment in employment - based health insurance plans ( fronstin , 2004 ) . in addition , for the manufacturing jobs that remain , the likelihood of coverage by employer - sponsored health insurance diminished , also contributing to the decline in employment - based health insurance ( fronstin , 2004 ) . this study suggests that declines in coverage have resulted almost entirely from increases in premiums in relation to personal income ( gilmer and kronic , 2005 ) . households ' spending reached $ 512.6 billion in 2003 , about one - third of total spending on hss . spending for phi premiums , including the employee share of employer - sponsored health insurance and individually purchased health insurance , was $ 174 billion while out - of - pocket spending for copays , deductibles , and for goods and services not covered by insurance was $ 230.5 billion . since 1987 , the share of households ' health spending going to phi premiums increased from 23 to 34 percent while the share for out - of - pocket spending dropped from 58 to 45 percent . spending by households grew 7.8 percent in 2003 , the second consecutive year of over 7 percent growth . other periods with growth in this range include 1988 to 1990 and 1997 to 1998 . the earlier period of higher growth occurred before the expansion of enrollment in the more tightly managed health plans , while the later period reflected stabilization in enrollment in these plans ( claxton et al . the latest period of slow growth , 1999 to 2001 , occurred as the overall economy grew rapidly and labor markets were tight , providing employers with incentives not to pass on rising health care costs to employees . in 2002 , this changed as employers began passing more costs to individuals , primarily through higher copays and deductibles . by looking at the share of household personal income that goes to health care , we can assess the burden that health care costs place on households . between 2001 and 2003 , the share of personal income consumed by health care grew rapidly , increasing 0.6 percentage points from 5.3 to 5.9 percent . this is the fastest increase in share since the 1987 - 1988 period . for 2002 and 2003 , personal income growth of 3.1 percent in 2001 , 1.3 percent in 2002 , and 3.0 percent in 2003 were slow by historical standards ( u.s . during this period , household spending for health care grew at rates that at 5.8 percent in 2001 , 7.5 percent in 2002 , and 7.8 percent in 2003were two or more times as fast as income growth . in 2003 , spending of $ 278.1 billion by state and local governments for health care represented a marked deceleration in spending growth . the 7.7 percent increase in spending was over 3 percentage points slower than in 2002 . the primary driver of this lower growth rate was slowing medicaid expenditures , whose growth slowed by 4.1 percentage points in 2003from 11.4 percent to 7.3 percent . recently , states have been experiencing fiscal pressures , and by mid-2003 , when states were beginning their fiscal year , nearly all states had implemented some kind of cost containment on medicaid spending ( smith et al . , 2004 ) . at the same time , states ' ability to utilize various creative financing schemes to increase federal medicaid funding were limited by federal regulation ( smith et al . , 2005 ) . since state and local governments are also employers , contributions to phi premiums for active and retired workers accounted for almost one - third of state and local health expenditures in 2003 . though growth in the states ' payments for phi premiums decelerated in 2003 , it still marked the fourth consecutive year of double - digit growth . other state and local government programs such as general assistance , maternal and child health , and public health activities accounted for 26 percent of state and local government health spending in 2003 . spending by the federal government for health care reached $ 344.0 billion in 2003 , or 21 percent of hss . federal health care spending growth decelerated sharply in 2003 , at roughly one - half the 2001 and 2002 rates . growth for all federal health programs , except the department of veterans affairs and indian health service , decelerated between 2002 and 2003 ( cms , 2005 ) . federal government medicare expenditures are calculated as nhea medicare expenditures for benefits and administration less the sum of hi payroll taxes paid by employers , employees , and the self - employed , hi and smi premiums , and hi income from taxation of social security benefits . this difference is roughly equal to trust fund interest income and federal general revenue contributions to medicare . medicaid spending , which was 47 percent of federal health care costs in 2003 , also decelerated sharply , slowing from 12.6 percent growth in 2002 to 6.9 percent in 2003 . while nearly all states implemented cost containment efforts in 2003 , many states specifically controlled growth in medicaid spending and enrollment by tightening eligibility and restricting benefits ( national governors association and national association of state budget officers , 2004 ) . they account for about one - fifth of federal government spending on health care . from 1998 to 2000 , growth in federal general revenue and interest income financing for medicare steadily decelerated . in 2003 , as overall growth in medicare spending slowed and growth in income from payroll taxes accelerated slightly , the growth in medicare expenditures financed by general revenues slowed . however , the share of medicare financed through general revenues and premiums increased because total medicare expenditures continued to grow faster than income from payroll taxes . federal government 's burden , measured by comparing its employer and general revenue spending on health against its nonpayroll - tax revenues , increased significantly in 2003 to about one - third of federal revenues . while federal health care spending growth slowed in 2003 , the federal government 's revenues declined as income tax cuts were implemented ( u.s . state and local government burden , using a similar measure as that used for the federal government comparison , also increased in 2003 . about one - quarter of state and local governments ' revenues go to health care . several states have reached a financial crisis as they struggle to find ways to finance increasing health care costs , especially for medicaid . unlike the federal government that can support deficit spending by borrowing , almost all state governments must balance their budgets each year , making the pressure they face from rising state health care costs particularly acute . overall , health - related tax expenditures accounted for 16 percent , or $ 116.2 billion , of all tax expenditures in 2003 , ranking third behind commerce and housing , at 32 percent , and income security , at 21 percent . the federal government receives less income tax revenue then would otherwise occur because of certain allowed tax deductions and income exclusions . the second largest health - related tax expenditure was deductible medical expenses at $ 6.2 billion . current national income accounting principles recognized by the bureau of economic analysis national income and product accounts ( nipa ) , the united nation 's system of national accounts , and the organization of economic cooperation and development ( oecd ) health accounts , do not include tax expenditures in their estimates . cms , the bureau of economic analysis , and oecd recognize that tax expenditures provide important economic incentives that influence the level and distribution of costs throughout the entire health care market . however , because no explicit taxes are collected or spending incurred , the nhea , like the nipa and the oecd , do not include tax expenditures in their official national accounting practices . , so does the debate about how to show estimates of health - related tax expenditures and compare them with the nhea . the office of management and budget ( omb ) offers some insight on the difficulty of directly integrating tax expenditures with the nhea stating that individual tax expenditures will not necessarily equal the increase in federal revenues by repealing these special provisions . tax expenditures alter economic behavior through various incentives and the estimates provided are interdependent , meaning they do not reflect any interactions between other programs and individual and corporate income tax receipts ( executive office of the president , 2004 ) . currently , cms , along with omb and the congressional budget office ( cbo ) use sidebars , such as this discussion , to show tax expenditure estimates .

free energy is probably the most important quantity in thermodynamics and one of the central topics in biophysics . nevertheless , for many relevant systems with local minimum energy configurations separated by energy barriers , efficient and accurate calculation of this property is still a big challenge in computational chemistry . free energy differences between different states can be calculated through free energy perturbation ( fep ) and thermodynamic integration ( ti ) methods . since the first application of the methodology to the calculation of the relative free energies of ligand binding and solvation of the organic molecules methanol and ethane , fep and ti have been widely used to study a wide range of processes such as solvation , phase transitions , ligand binding , and proteinprotein interactions , just to name a few . these methods , which are firmly rooted in statistical mechanics , are usually combined with molecular dynamics ( md ) or monte carlo ( mc ) simulations . the data obtained from these simulations allows us to quantitatively evaluate free energy changes and understand , at molecular level , the structural and energetic factors governing the process . however , to obtain accurate free energy values , major issues like free energy convergence and conformational sampling still need to be addressed . although these topics have been mainly discussed as independent issues , convergence and the amount of sampling are strictly connected.(17 ) for instance , for processes that involve large conformational changes and reorganization of solvent , poor sampling can trap the system in local minima and , as a consequence , lead to apparent but false convergence . in other words , in these cases the calculated free energy might correspond to pseudoconverged values obtained from trapped local conformations . as we will show in this paper , even for a very simple system , like propane - to - propane transformation , independent free energy calculations carried out with conventional md simulation may not be able to reproduce accurately the correct free energy difference , though the simulations may show apparently converged values . quantitative prediction of free energy change is only obtained when configuration sampling is efficiently improved . a large number of techniques have been introduced to enhance sampling over configuration space . a straightforward way of modifying the potential energy surface to enhance sampling has been proposed by hamelberg et al.(33 ) this approach , which is based on earlier work of voter , has proved to be efficient in accelerating not only conformational transitions but also millisecond time scale motions of a protein in explicit water.(40 ) in this work we propose a simple and efficient approach to improve accuracy and convergence of free energy simulations in condensed - phase systems . although the formulation and the results presented here were obtained by coupling ti with the accelerated md method ( amd ) , the procedure can be easily extended to the fep approach . to check convergence and accuracy of the ti simulations , this system was chosen because i ) the correct free energy result is rigorously equal to zero and ii ) similar zero - free energy change systems have been used before as model systems to compare the efficiency and convergence of different approaches to free energy calculations . in order to enhance sampling by increasing the escape rate from potential energy wells , the accelerated md approach modifies the energy landscape by adding a boost potential , v(r ) , to the original potential surface every time v(r ) is below a predefined energy level e ( figure 1 ) . in other words , implementation , v(r ) is given by where modulates the depth and the local roughness of the energy basins in the modified potential . since the torsional potential governs the rate of sampling of biomolecular rotameric states , the boost potential has been largely applied to the torsional term of the potential energy function . this approach , which will be referred to as amdt , has been successfully applied to study several biological systems and processes . schematic representation of a hypothetical true ( solid line ) and modified ( dashed line ) potential energy function with different values of . the modified potential ( generated with eq 1 ) converges to the true potential at large values of . the dotted line corresponds to the boost energy e. more recently , hamelberg et al . introduced a dual boost approach in order to efficiently sample both the torsional degrees of freedom and the diffusive motions.(36 ) in this implementation , two boost potentials are applied separately to the potential energy . while the first one is applied only to the torsional terms , the second one is added to the total potential energy ( amdtt ) . the modified potential is given by where vt(r ) and vt(r ) are the boost potentials applied to the torsional terms vt(r ) and the total potential vt(r ) . vt(r ) is defined as vt(r ) = v0(r ) + vt(r ) + vt(r ) . the correct canonical averages of an observable , calculated from configurations sampled on the modified potential energy surface , is then fully recovered from the accelerated md simulations by reweighting each point in the configuration space by exp{[v(r)]}. in the dual boost approach , the boost factor is given by exp{[vt(r)+vt(r)]}. in this work , a third approach is introduced in which molecular conformational transitions are accelerated by lowering the energy barriers , while the potential surfaces near the minima are left unchanged . the idea behind this approach has been used before by darve et al . to calculate free energies by applying a scale - force molecular dynamics algorithm.(27 ) owing to the symmetry of eq 1 in relation to e and v(r ) , this approach can be easily implemented by simply redefining eq 1 as in this implementation , the boost potential , v(r ) , is subtracted from the true potential v(r ) whenever the potential v(r ) is greater than the boost energy e ; in this case , the simulation is performed on the modified potential v*(r ) = v(r ) v(r ) . on the other hand , when v(r ) is below the energy level e , the simulation is performed on the true potential v*(r ) = v(r ) . hereafter , this approach will be referred to as amd and amdt when applied to the torsional terms of the potential energy . figures 1 and 2 illustrate a schematic representation of a hypothetical one - dimensional potential modified using eqs 1 and 3 , respectively . in both cases , as decreases , the modified potential becomes flatter , and as increases , the modified landscape asymptotically approaches the unmodified potential . schematic representation of a hypothetical true ( solid line ) and modified ( dashed line ) potential energy function with different values of . the modified potential ( generated with eq 3 ) converges to the true potential at large values of . the dotted line corresponds to the boost energy e. in this approach , analogously to hamelberg et al.s implementation , the correct canonical ensemble averages of an observable are fully recovered by reweighting each configuration by the boltzmann factor of the negative of the boost potential energy , exp{[v(r)]}. the application of this schema into the dual boost approach is straightforward . in this case , eq 2 is simply redefined as v*(r ) = { v0(r)+[vt(r)vt(r)]}vt(r ) , and the boost factor as exp{[vt(r)+vt(r)]}. this implementation will be referred to as amdtt . thermodynamic integration is a commonly used technique to compute the difference in free energy between two thermodynamic states , which differ from each other according to their intermolecular or intramolecular interaction potentials . in this case , the interaction potential can be expressed as a function of a coupling parameter , , that determines the state of the system.(10 ) thus , by defining the free energy , f , as a continuous function of , the difference in free energy between two states is given by where = 0 and 1 correspond to the initial and final states , respectively . since f( ) can be written as f can be rewritten as(45 ) where q is the partition function of the system , = 1/kbt , kb is boltzmann s constant , and t is the temperature . here , we use the partition function for canonical ensemble , which is defined as(46 ) where n is the number of particles , h is planck s constant , p and r are the momenta and positions of the particles , and h is hamiltonian of the system . substituting eq 7 into eq 6 and deriving in respect to ,(45 ) we obtain assuming that the kinetic energy term is separable and not dependent on , eq 8 can be rewritten in terms of the potential energy v(r ) of the system and , finally where the integrand is the ensemble average of v/ calculated on the original potential v(r ) at a specific value of , and f is the free energy difference between the initial ( = 0 ) and final ( = 1 ) states obtained on the unmodified potential surface , v(r ) . similarly , for the modified potential we have now , the ensemble average of true v(r)/ is performed over the modified potential v*(r ) . since both approaches can be coupled with ti simulations , we will first express v*(r ) as in this case , eq 11 can be rewritten as the boltzmann distribution can be extracted from the non - boltzmann distribution using the method introduced by torrie et al.(19 ) the corrected canonical distribution can then be recovered by reweighting the phase space of the modified potential by multiplying the integrand by the strength of the bias at each position , which in this case corresponds to exp[v ] . thus , the corrected ensemble average of f can be obtained by dividing both the numerator and the denominator of eq 11 by dr exp[v(r ) ] exp[v ] = dr exp[v*(r ) ] and integrating over . analogously , if the modified potential is defined as v*(r ) = v(r ) v , eq 16 can be redefined as therefore , independent of the approach applied , the accelerated molecular dynamics simulation method converges to the canonical distribution , and the corrected canonical ensemble average of the system is obtained by simply reweighting each point in the configuration phase space on the modified potential by the strength of the boltzmann factor of the bias energy , exp[v ] or exp[v ] , at that particular point . the first question to be answered about the amd approach is if this method is able to improve conformational transitions . to address this question , we performed md simulations of a butane molecule in explicit water and monitored the dihedral angle shown in figure 3 . it is worth noting that even for a simple system like this , the number of conformational transitions is still very limited . figure 4-middle displays the results obtained from the amd approach . in this simulation , only the torsional term of the potential energy was applied in the boost potential ( amdt ) . parameters e and were set to 0.5 and 0.2 kcal / mol , respectively . for comparison , figure 4-bottom shows the dihedral transitions calculated with the amdt approach . in this case , parameters e and were set to 5.0 and 0.5 kcal / mol , respectively . as expected , more conformational transitions are observed with amdt and amdt than with normal md simulations . figure 4 also reveals that , even though both methods improved conformational sampling , amdt still produces a much larger number of transitions than amdt . plots of dihedral angle of the butane molecule , as defined in figure 3 , sampled with normal md , amdt , and amdt approaches . the propane - to - propane system ( figure 5 ) was used to test convergence and accuracy of the accelerated ti simulations . a similar system , ethane - to - ethane transformation , has been used before by other groups to test the performance of different approaches to calculate free energy changes . this transformation is particularly interesting because independent of the force field , water model , or simulation method used , the free energy change should be equal to zero ( g = 0 ) . the free energy changes obtained with normal md simulations are compared to the ones obtained with amdt , amdt , and amdtt . figures 6 , 7 , 8 , 9 , and 10 show the free energy change , the average free energy , and the error associated with the propane - to - propane transformation calculated from five independent ti simulations . in all simulations , the same amount of sampling was performed at each window , and an equal amount of time was spent in equilibration and data collection . the error was estimated by calculating the standard deviation of the five independent simulations as a function of time . all ti calculations with normal md fail to reproduce the expected free energy value ( figure 6 ) , converging to free energy values of 0.4 kcal / mol . the change with time of the average free energy toward the expected free energy value is rather slow , and it is clear that this normal md requires much longer simulations to reproduce accurate results . although the calculated average free energy change is closer to the corrected free energy value , like normal md , longer simulations are still required to reproduce the correct average free energy change . nevertheless , both approaches perform better than normal ti simulations , and this improvement can be mainly attributed to the increasing in conformational sampling . free energy change in kcal / mol , calculated for the propane - to - propane simulations as a function of time from five independent simulations ( top ) . the inset plot shows in detail the standard deviation as a function of time for the points with at least 80 10 of data collection steps per . the same number of equilibration and data collection steps were used for each . average free energy change and the error associated with each point were calculated from the five independent simulations ( bottom ) . free energy change in kcal / mol , calculated for the propane - to - propane simulations as a function of time from five independent simulations ( top ) . the inset plot shows in detail the standard deviation as a function of time for the points with at least 80 10(3 ) of data collection steps per . the same number of equilibration and data collection steps were used for each . average free energy change and the error associated with each point were calculated from the five independent simulations ( bottom ) . free energy change in kcal / mol , calculated for the propane - to - propane simulations as a function of time from five independent simulations ( top ) . the inset plot shows in detail the standard deviation as a function of time for the points with at least 80 10(3 ) of data collection steps per . the same number of equilibration and data collection steps were used for each . average free energy change and the error associated with each point were calculated from the five independent simulations ( bottom ) . free energy change in kcal / mol , calculated for the propane - to - propane simulations as a function of time from five independent simulations ( top ) . the inset plot shows in detail the standard deviation as a function of time for the points with at least 80 10(3 ) of data collection steps per . the same number of equilibration and data collection steps were used for each . average free energy change and the error associated with each point were calculated from the five independent simulations ( bottom ) . units are in kcal / mol . accumulated free energy change for the propane - to - propane simulations calculated with 250 10 of data collection steps per . the same number of equilibration and data collection steps were used for each . as mentioned before , amdt and amdt approaches modify the energy landscape by adding a boost potential to the potential surface , and , in these cases , the boost potential is based on the torsional terms of the potential energy . even though the conformational sampling is clearly enhanced in both approaches , the reason for that might be the absence of energy terms in the boost potential describing solutesolvent and solventsolvent interactions . therefore , in order to also accelerate the solvent response along the propane - to - propane transformation , the dual boost approach was also tested with ti calculations ( vt was applied with parameters e and set to 3.0 and 30.0 kcal / mol per atom , respectively ) . owing to instabilities introduced by the application of amdtt approach in ti simulations , only results obtained with amdtt are displayed in figure 9 . by comparing amdtt and amdt , we see clearly that inclusion of the potential energy terms describing solutesolvent and solventsolvent interactions in eq 3 dramatically improves the accuracy and convergence of the ti simulations . it is worth mentioning that all calculated free energy values using amdtt , with at least 100 ps of data collection , converged to the correct value and are within the estimated error . for the system studied in this work , amdtt was the only approach to achieve converged and accurate results from ti simulation ( g = 0.027 0.04 kcal / mol ) . figure 10 shows the accumulated free energy for each value of . in this plot , the accumulated free energy was calculated by using equilibration = data collection time = 250 ps for each window . except for the amdtt , which performed remarkably well , all approaches failed to reproduce the corrected free energy change for the propane - to - propane transformation . the main difference between the two approaches ( eqs 1 and 3 ) consists of how the modified potential surface is generated . for methods based on the amd approach , although this method proved to be excellent to enhance conformational sampling of biomolecules , some issues concerning the calculation of thermodynamics properties still need to be addressed . for instance , to fully recover ensemble average properties , each point in the phase space should be multiplied by its respective boltzmann factor of the boost energy , exp(v ) . in some cases , when this procedure is applied , relatively few configurations in the entire trajectory have significant contributions to the ensemble average . as a consequence this is the main issue to be addressed when the amd method is coupled with ti calculations . it is worth mentioning that in this implementation v is a non - negative number , and its respective boltzmann factor produces numbers in the interval [ 1 ) . thus , large values of exp(v ) correspond to configurations near energy minima of the potential surface , while small values correspond to relatively high - energy regions . figure 11 , on the right , displays the distribution of the boost factor along the amdt simulations at three different values of . it is clear from those plots that , even for this rather low acceleration condition , the system spends almost the entire simulation in regions of relatively high energy . only very few configurations ( for instance configurations with exp(v)>50 ) distribution of boltzmann factor of the boost potential calculated from propane - to - propane simulations using the amdtt ( left ) and amdt ( right ) approaches . in order to address this issue , here , we introduce the amd approach aiming to improve sampling without compromising the statistics in the ti calculations . as mentioned before , in this approach , regions near the minima in the potential surface are left unchanged , and the boltzmann factor of the boost energy is now defined as exp(v ) . in this implementation , exp(v ) assumes values in the interval ( 01 ] . thus , unlike the amd approach , all configurations near the low - energy regions of the potential surface ( v = 0 ) , which are the ones that most contribute to the ensemble average , have the same weight of exp(v ) 1 . besides that , configurations sampled in high - energy regions of the conformational space have rather small weights , exp(v ) 1 , and , as a consequence , have a fairly small contribution to the ensemble average . figure 11 , on the left , shows the boost factor distribution along the amdtt simulations at three different values of . it is clear that the amd approach is not only able to sample both low- and high - energy regions of the potential surface but also to keep regions near the minima well populated . as a consequence , the statistics are not compromised in the ti calculations , and the ensemble average is recovered ( figure 9 ) . all calculations were performed using the sander module in the amber8(47 ) package that was modified to carry out the accelerated md simulations . the gaff force field was used to describe the solute in all simulations . the butane molecule was solvated in a periodic box of explicit tip3p waters,(48 ) which extends on each side 10 from the closest atom of the solute , by using the leap module in amber . to bring the system to its correct density , we carried out an md simulation for 1 ns in which the npt ensemble ( t = 300 k , p = 1 atm ) was applied . all data collection was carried out over md simulations of 1 ns , during which the nvt ensemble ( t = 300 k , density= 0.984 g / ml ) was applied . the final configuration was then used as the starting point for the propane propane simulations . in both systems , butane and propane propane simulations , the free energy change was calculated by varying form 0 ( initial state ) to 1 ( final state ) . all ti simulations were carried out using seven discrete points of , which were determined by gaussian quadrature formulas . normal and accelerated md simulations of 500 ps were carried out for each point . temperature and pressure were controlled via a weak coupling to external temperature and pressure baths(49 ) with coupling constants of 0.5 and 1.0 ps , respectively . apart from all ti simulations where the time step was set to 1 fs , the equations of motion were integrated with a step length of 2.0 fs using the verlet leapfrog algorithm.(50 ) for further analysis , the trajectory was saved every 1.0 ps . the pme summation method was used to treat the long - range electrostatic interactions in the minimization and simulation steps . the short - range nonbonded interactions were truncated using a 8 cutoff , and the nonbonded pair list was updated every 20 steps . in this work , we showed a straightforward way of coupling the thermodynamic integration approach with the accelerated md method . we also introduced a new approach , amd , aiming to improve convergence and efficiency of free energy calculations in condensed - phase systems . our results showed that both accelerated md approaches improve conformation sampling when compared to normal md simulations . when applied to just torsion terms of potential energy , both approaches , amdt and amdt , increased substantially the number of conformation transitions of the butane molecule in explicit water when compared to normal md simulations . in addition , the accuracy of free energy simulations was significantly improved when sampling of internal degrees of freedom of solute was enhanced . however , accurate and converged results were only achieved when the solvent interactions were taken into account in the accelerated md approaches . when combined with amd , the application of dual - boost approach improved markedly the convergence and accuracy of ti calculations . by analyzing the distribution of the boost potential along the free energy simulations , we observed that the amd approach efficiently samples both low- and high - energy regions of the potential surface . since this approach also maintains well populated regions near the minima , the statistics are not compromised in the ti calculations , and , as a result , the ensemble average can be recovered .

in this work we propose a straightforward and efficient approach to improve accuracy and convergence of free energy simulations in condensed - phase systems . we also introduce a new accelerated molecular dynamics ( md ) approach in which molecular conformational transitions are accelerated by lowering the energy barriers while the potential surfaces near the minima are left unchanged . all free energy calculations were performed on the propane - to - propane model system . the accuracy of free energy simulations was significantly improved when sampling of internal degrees of freedom of solute was enhanced . however , accurate and converged results were only achieved when the solvent interactions were taken into account in the accelerated md approaches . the analysis of the distribution of boost potential along the free energy simulations showed that the new accelerated md approach samples efficiently both low- and high - energy regions of the potential surface . since this approach also maintains substantial populations in regions near the minima , the statistics are not compromised in the thermodynamic integration calculations , and , as a result , the ensemble average can be recovered .

Introduction Theory Results Methods Conclusions

free energy is probably the most important quantity in thermodynamics and one of the central topics in biophysics . free energy differences between different states can be calculated through free energy perturbation ( fep ) and thermodynamic integration ( ti ) methods . since the first application of the methodology to the calculation of the relative free energies of ligand binding and solvation of the organic molecules methanol and ethane , fep and ti have been widely used to study a wide range of processes such as solvation , phase transitions , ligand binding , and proteinprotein interactions , just to name a few . these methods , which are firmly rooted in statistical mechanics , are usually combined with molecular dynamics ( md ) or monte carlo ( mc ) simulations . the data obtained from these simulations allows us to quantitatively evaluate free energy changes and understand , at molecular level , the structural and energetic factors governing the process . however , to obtain accurate free energy values , major issues like free energy convergence and conformational sampling still need to be addressed . (17 ) for instance , for processes that involve large conformational changes and reorganization of solvent , poor sampling can trap the system in local minima and , as a consequence , lead to apparent but false convergence . in other words , in these cases the calculated free energy might correspond to pseudoconverged values obtained from trapped local conformations . as we will show in this paper , even for a very simple system , like propane - to - propane transformation , independent free energy calculations carried out with conventional md simulation may not be able to reproduce accurately the correct free energy difference , though the simulations may show apparently converged values . quantitative prediction of free energy change is only obtained when configuration sampling is efficiently improved . a straightforward way of modifying the potential energy surface to enhance sampling has been proposed by hamelberg et al. (33 ) this approach , which is based on earlier work of voter , has proved to be efficient in accelerating not only conformational transitions but also millisecond time scale motions of a protein in explicit water. (40 ) in this work we propose a simple and efficient approach to improve accuracy and convergence of free energy simulations in condensed - phase systems . although the formulation and the results presented here were obtained by coupling ti with the accelerated md method ( amd ) , the procedure can be easily extended to the fep approach . to check convergence and accuracy of the ti simulations , this system was chosen because i ) the correct free energy result is rigorously equal to zero and ii ) similar zero - free energy change systems have been used before as model systems to compare the efficiency and convergence of different approaches to free energy calculations . in order to enhance sampling by increasing the escape rate from potential energy wells , the accelerated md approach modifies the energy landscape by adding a boost potential , v(r ) , to the original potential surface every time v(r ) is below a predefined energy level e ( figure 1 ) . in other words , implementation , v(r ) is given by where modulates the depth and the local roughness of the energy basins in the modified potential . since the torsional potential governs the rate of sampling of biomolecular rotameric states , the boost potential has been largely applied to the torsional term of the potential energy function . this approach , which will be referred to as amdt , has been successfully applied to study several biological systems and processes . introduced a dual boost approach in order to efficiently sample both the torsional degrees of freedom and the diffusive motions. (36 ) in this implementation , two boost potentials are applied separately to the potential energy . while the first one is applied only to the torsional terms , the second one is added to the total potential energy ( amdtt ) . the correct canonical averages of an observable , calculated from configurations sampled on the modified potential energy surface , is then fully recovered from the accelerated md simulations by reweighting each point in the configuration space by exp{[v(r)]}. in the dual boost approach , the boost factor is given by exp{[vt(r)+vt(r)]}. in this work , a third approach is introduced in which molecular conformational transitions are accelerated by lowering the energy barriers , while the potential surfaces near the minima are left unchanged . the idea behind this approach has been used before by darve et al . to calculate free energies by applying a scale - force molecular dynamics algorithm. (27 ) owing to the symmetry of eq 1 in relation to e and v(r ) , this approach can be easily implemented by simply redefining eq 1 as in this implementation , the boost potential , v(r ) , is subtracted from the true potential v(r ) whenever the potential v(r ) is greater than the boost energy e ; in this case , the simulation is performed on the modified potential v*(r ) = v(r ) v(r ) . on the other hand , when v(r ) is below the energy level e , the simulation is performed on the true potential v*(r ) = v(r ) . hereafter , this approach will be referred to as amd and amdt when applied to the torsional terms of the potential energy . in both cases , as decreases , the modified potential becomes flatter , and as increases , the modified landscape asymptotically approaches the unmodified potential . the dotted line corresponds to the boost energy e. in this approach , analogously to hamelberg et al.s implementation , the correct canonical ensemble averages of an observable are fully recovered by reweighting each configuration by the boltzmann factor of the negative of the boost potential energy , exp{[v(r)]}. in this case , eq 2 is simply redefined as v*(r ) = { v0(r)+[vt(r)vt(r)]}vt(r ) , and the boost factor as exp{[vt(r)+vt(r)]}. thermodynamic integration is a commonly used technique to compute the difference in free energy between two thermodynamic states , which differ from each other according to their intermolecular or intramolecular interaction potentials . in this case , the interaction potential can be expressed as a function of a coupling parameter , , that determines the state of the system. (10 ) thus , by defining the free energy , f , as a continuous function of , the difference in free energy between two states is given by where = 0 and 1 correspond to the initial and final states , respectively . since f( ) can be written as f can be rewritten as(45 ) where q is the partition function of the system , = 1/kbt , kb is boltzmann s constant , and t is the temperature . here , we use the partition function for canonical ensemble , which is defined as(46 ) where n is the number of particles , h is planck s constant , p and r are the momenta and positions of the particles , and h is hamiltonian of the system . substituting eq 7 into eq 6 and deriving in respect to ,(45 ) we obtain assuming that the kinetic energy term is separable and not dependent on , eq 8 can be rewritten in terms of the potential energy v(r ) of the system and , finally where the integrand is the ensemble average of v/ calculated on the original potential v(r ) at a specific value of , and f is the free energy difference between the initial ( = 0 ) and final ( = 1 ) states obtained on the unmodified potential surface , v(r ) . similarly , for the modified potential we have now , the ensemble average of true v(r)/ is performed over the modified potential v*(r ) . since both approaches can be coupled with ti simulations , we will first express v*(r ) as in this case , eq 11 can be rewritten as the boltzmann distribution can be extracted from the non - boltzmann distribution using the method introduced by torrie et al. (19 ) the corrected canonical distribution can then be recovered by reweighting the phase space of the modified potential by multiplying the integrand by the strength of the bias at each position , which in this case corresponds to exp[v ] . thus , the corrected ensemble average of f can be obtained by dividing both the numerator and the denominator of eq 11 by dr exp[v(r ) ] exp[v ] = dr exp[v*(r ) ] and integrating over . analogously , if the modified potential is defined as v*(r ) = v(r ) v , eq 16 can be redefined as therefore , independent of the approach applied , the accelerated molecular dynamics simulation method converges to the canonical distribution , and the corrected canonical ensemble average of the system is obtained by simply reweighting each point in the configuration phase space on the modified potential by the strength of the boltzmann factor of the bias energy , exp[v ] or exp[v ] , at that particular point . the first question to be answered about the amd approach is if this method is able to improve conformational transitions . it is worth noting that even for a simple system like this , the number of conformational transitions is still very limited . in this simulation , only the torsional term of the potential energy was applied in the boost potential ( amdt ) . in this case , parameters e and were set to 5.0 and 0.5 kcal / mol , respectively . as expected , more conformational transitions are observed with amdt and amdt than with normal md simulations . plots of dihedral angle of the butane molecule , as defined in figure 3 , sampled with normal md , amdt , and amdt approaches . the propane - to - propane system ( figure 5 ) was used to test convergence and accuracy of the accelerated ti simulations . a similar system , ethane - to - ethane transformation , has been used before by other groups to test the performance of different approaches to calculate free energy changes . this transformation is particularly interesting because independent of the force field , water model , or simulation method used , the free energy change should be equal to zero ( g = 0 ) . the free energy changes obtained with normal md simulations are compared to the ones obtained with amdt , amdt , and amdtt . figures 6 , 7 , 8 , 9 , and 10 show the free energy change , the average free energy , and the error associated with the propane - to - propane transformation calculated from five independent ti simulations . in all simulations , the same amount of sampling was performed at each window , and an equal amount of time was spent in equilibration and data collection . the error was estimated by calculating the standard deviation of the five independent simulations as a function of time . the change with time of the average free energy toward the expected free energy value is rather slow , and it is clear that this normal md requires much longer simulations to reproduce accurate results . although the calculated average free energy change is closer to the corrected free energy value , like normal md , longer simulations are still required to reproduce the correct average free energy change . nevertheless , both approaches perform better than normal ti simulations , and this improvement can be mainly attributed to the increasing in conformational sampling . free energy change in kcal / mol , calculated for the propane - to - propane simulations as a function of time from five independent simulations ( top ) . the inset plot shows in detail the standard deviation as a function of time for the points with at least 80 10 of data collection steps per . average free energy change and the error associated with each point were calculated from the five independent simulations ( bottom ) . free energy change in kcal / mol , calculated for the propane - to - propane simulations as a function of time from five independent simulations ( top ) . the inset plot shows in detail the standard deviation as a function of time for the points with at least 80 10(3 ) of data collection steps per . average free energy change and the error associated with each point were calculated from the five independent simulations ( bottom ) . free energy change in kcal / mol , calculated for the propane - to - propane simulations as a function of time from five independent simulations ( top ) . average free energy change and the error associated with each point were calculated from the five independent simulations ( bottom ) . free energy change in kcal / mol , calculated for the propane - to - propane simulations as a function of time from five independent simulations ( top ) . the inset plot shows in detail the standard deviation as a function of time for the points with at least 80 10(3 ) of data collection steps per . average free energy change and the error associated with each point were calculated from the five independent simulations ( bottom ) . accumulated free energy change for the propane - to - propane simulations calculated with 250 10 of data collection steps per . as mentioned before , amdt and amdt approaches modify the energy landscape by adding a boost potential to the potential surface , and , in these cases , the boost potential is based on the torsional terms of the potential energy . even though the conformational sampling is clearly enhanced in both approaches , the reason for that might be the absence of energy terms in the boost potential describing solutesolvent and solventsolvent interactions . therefore , in order to also accelerate the solvent response along the propane - to - propane transformation , the dual boost approach was also tested with ti calculations ( vt was applied with parameters e and set to 3.0 and 30.0 kcal / mol per atom , respectively ) . owing to instabilities introduced by the application of amdtt approach in ti simulations , only results obtained with amdtt are displayed in figure 9 . by comparing amdtt and amdt , we see clearly that inclusion of the potential energy terms describing solutesolvent and solventsolvent interactions in eq 3 dramatically improves the accuracy and convergence of the ti simulations . it is worth mentioning that all calculated free energy values using amdtt , with at least 100 ps of data collection , converged to the correct value and are within the estimated error . for the system studied in this work , amdtt was the only approach to achieve converged and accurate results from ti simulation ( g = 0.027 0.04 kcal / mol ) . figure 10 shows the accumulated free energy for each value of . in this plot , the accumulated free energy was calculated by using equilibration = data collection time = 250 ps for each window . except for the amdtt , which performed remarkably well , all approaches failed to reproduce the corrected free energy change for the propane - to - propane transformation . the main difference between the two approaches ( eqs 1 and 3 ) consists of how the modified potential surface is generated . for methods based on the amd approach , although this method proved to be excellent to enhance conformational sampling of biomolecules , some issues concerning the calculation of thermodynamics properties still need to be addressed . for instance , to fully recover ensemble average properties , each point in the phase space should be multiplied by its respective boltzmann factor of the boost energy , exp(v ) . in some cases , when this procedure is applied , relatively few configurations in the entire trajectory have significant contributions to the ensemble average . as a consequence this is the main issue to be addressed when the amd method is coupled with ti calculations . it is worth mentioning that in this implementation v is a non - negative number , and its respective boltzmann factor produces numbers in the interval [ 1 ) . thus , large values of exp(v ) correspond to configurations near energy minima of the potential surface , while small values correspond to relatively high - energy regions . figure 11 , on the right , displays the distribution of the boost factor along the amdt simulations at three different values of . it is clear from those plots that , even for this rather low acceleration condition , the system spends almost the entire simulation in regions of relatively high energy . only very few configurations ( for instance configurations with exp(v)>50 ) distribution of boltzmann factor of the boost potential calculated from propane - to - propane simulations using the amdtt ( left ) and amdt ( right ) approaches . in order to address this issue , here , we introduce the amd approach aiming to improve sampling without compromising the statistics in the ti calculations . as mentioned before , in this approach , regions near the minima in the potential surface are left unchanged , and the boltzmann factor of the boost energy is now defined as exp(v ) . in this implementation , exp(v ) assumes values in the interval ( 01 ] . thus , unlike the amd approach , all configurations near the low - energy regions of the potential surface ( v = 0 ) , which are the ones that most contribute to the ensemble average , have the same weight of exp(v ) 1 . besides that , configurations sampled in high - energy regions of the conformational space have rather small weights , exp(v ) 1 , and , as a consequence , have a fairly small contribution to the ensemble average . figure 11 , on the left , shows the boost factor distribution along the amdtt simulations at three different values of . it is clear that the amd approach is not only able to sample both low- and high - energy regions of the potential surface but also to keep regions near the minima well populated . as a consequence , the statistics are not compromised in the ti calculations , and the ensemble average is recovered ( figure 9 ) . all calculations were performed using the sander module in the amber8(47 ) package that was modified to carry out the accelerated md simulations . the butane molecule was solvated in a periodic box of explicit tip3p waters,(48 ) which extends on each side 10 from the closest atom of the solute , by using the leap module in amber . to bring the system to its correct density , we carried out an md simulation for 1 ns in which the npt ensemble ( t = 300 k , p = 1 atm ) was applied . in both systems , butane and propane propane simulations , the free energy change was calculated by varying form 0 ( initial state ) to 1 ( final state ) . normal and accelerated md simulations of 500 ps were carried out for each point . (50 ) for further analysis , the trajectory was saved every 1.0 ps . the pme summation method was used to treat the long - range electrostatic interactions in the minimization and simulation steps . the short - range nonbonded interactions were truncated using a 8 cutoff , and the nonbonded pair list was updated every 20 steps . in this work , we showed a straightforward way of coupling the thermodynamic integration approach with the accelerated md method . we also introduced a new approach , amd , aiming to improve convergence and efficiency of free energy calculations in condensed - phase systems . our results showed that both accelerated md approaches improve conformation sampling when compared to normal md simulations . in addition , the accuracy of free energy simulations was significantly improved when sampling of internal degrees of freedom of solute was enhanced . however , accurate and converged results were only achieved when the solvent interactions were taken into account in the accelerated md approaches . when combined with amd , the application of dual - boost approach improved markedly the convergence and accuracy of ti calculations . by analyzing the distribution of the boost potential along the free energy simulations , we observed that the amd approach efficiently samples both low- and high - energy regions of the potential surface . since this approach also maintains well populated regions near the minima , the statistics are not compromised in the ti calculations , and , as a result , the ensemble average can be recovered .

the management of severely agitated elderly patients in the acute hospital setting is not easy.14 urgent safety issues for both patient and staff coexist with diagnostic priorities in a setting not geared to first - line nonpharmacological strategies of meeting psychosocial and emotional needs of patients.5 the adverse outcomes of cognitively impaired older people in hospital are well documented and include prolonged length of stay , increased mortality , polypharmacy , and psychotropic use.68 however , agitation among elderly patients in the hospital setting is common , and needs to be managed given its association with some of these adverse outcomes.9 the most common causes of agitation in the elderly are delirium or behavioral and psychological symptoms of dementia ( bpsd ) , and less commonly , a primary psychosis.1,2 delirium affects an estimated 14%56% of all hospitalized elderly patients,10 while an estimated 60% of people with dementia will experience symptoms of agitation or aggression at some stage during the course of their disease.11 there is broad consensus that the first - line management of both delirium and bpsd involves thorough assessment and treatment of the underlying cause of the delirium or trigger for the bpsd in conjunction with nonpharmacological strategies.10,12 acute hospital settings , particularly emergency departments ( eds ) , are not geared to this , but rather to pharmacological strategies , ideally used as a last resort due to high risk - to - benefit ratios in the elderly . however , despite the existence of various guidelines on the management of both delirium and dementia , including within australia,1315 they often lack detailed advice on the pharmacological and nonpharmacological management of patients with severe agitation.16 this is probably a reflection of the lack of high - quality randomized control trials in this domain.1721 while there are guidelines for the management of the agitated patients,22 these are not age - specific , an important issue given the distinctive etiologies of behavioral disturbance in the elderly compared with younger adult groups . as much as posing a risk to themselves , despite their frailty , severely agitated elderly patients can pose a physical risk to others . as part of a strategy to prevent and manage violence in the workplace , many hospitals in australia have access to a duress response team,23 whose remit includes older persons . however , there is limited literature addressing the management of severely agitated elderly patients by duress response teams with the exception of 1 study of agitated adult patients ( mean aged 32 years ) in an ed.24 a prospective study of parenteral sedation for agitated elderly patients ( 65 years and older ) in an ed focused on the use of parenteral sedation only rather than the reasons for sedation and the use of alternative nonpharmacological strategies.25 the aim of this study was to describe the management of severely agitated elderly patients in an acute hospital setting , by analyzing a cohort of patients who required a response from the hospital s duress response team , known as the aggression response team ( art ) . we aimed to describe the older patient population referred for art calls , the reasons for and interventions during art calls ( including the use and nature of pharmacological sedation ) , complications arising from these interventions , and the follow - up of art calls . elderly patients are predominantly admitted under either general medical or surgical teams , with the availability of both geriatric and psychiatry consult - liaison services . the art can be alerted via the pager system when there is a severely agitated patient and a staff member has concerns for the safety of either the patient or others . the team comprises a medical and psychiatry registrar ( each with 2 or more years of post - graduate experience ) , a senior nurse acting as team leader , and security guards . the team leaders and security guards have mandatory annual face - to - face training in de - escalation strategies and restraint techniques . it is hospital policy that an incident report is recorded in the incident information management system ( iims ) for all art calls . patients 65 years and older who were referred for art calls during 2014 in the ed or wards ( excluding the psychiatry ward ) were identified using the iims database26 and their medical records were reviewed . ethical approval for the study was obtained from the northern sydney local health district human research ethics committee . patient consent was deemed not necessary by the northern sydney local health district human research ethics committee as the research was based on a de - identified file audit . data was collected from the medical notes regarding demographics , medical history , regular medications , presenting symptom , and progress prior to the art call . art call details included time of activation ( taken from the pager system ) , reasons for the art call , working diagnosis documented in medical progress notes prior to the art call , and interventions . details of pharmacological sedation were taken from the medication chart , including dose , route , and time of administration . details regarding post - sedation monitoring were taken from the standard observation chart , or progress notes . adverse events relating to observations were defined in alignment with the standard adult general observation chart calling criteria for clinical reviews,25 that is respiratory depression ( respiratory rate [ rr ] < 12 ) , hypotension ( systolic blood pressure [ sbp ] < 100 ) , and over - sedation ( avpu [ alert , voice , pain , unresponsive ] score = p or u ) ( nsw ministry of health).27 other adverse events identified from the notes included extrapyramidal side effects , missed or delayed usual medication , and falls over the 24 hours following sedation . whether a patient had settled following sedation simple statistical calculations such as mean , median , and standard deviation were performed using microsoft excel . elderly patients are predominantly admitted under either general medical or surgical teams , with the availability of both geriatric and psychiatry consult - liaison services . the art can be alerted via the pager system when there is a severely agitated patient and a staff member has concerns for the safety of either the patient or others . the team comprises a medical and psychiatry registrar ( each with 2 or more years of post - graduate experience ) , a senior nurse acting as team leader , and security guards . the team leaders and security guards have mandatory annual face - to - face training in de - escalation strategies and restraint techniques . it is hospital policy that an incident report is recorded in the incident information management system ( iims ) for all art calls . patients 65 years and older who were referred for art calls during 2014 in the ed or wards ( excluding the psychiatry ward ) were identified using the iims database26 and their medical records were reviewed . ethical approval for the study was obtained from the northern sydney local health district human research ethics committee . patient consent was deemed not necessary by the northern sydney local health district human research ethics committee as the research was based on a de - identified file audit . data was collected from the medical notes regarding demographics , medical history , regular medications , presenting symptom , and progress prior to the art call . art call details included time of activation ( taken from the pager system ) , reasons for the art call , working diagnosis documented in medical progress notes prior to the art call , and interventions . details of pharmacological sedation were taken from the medication chart , including dose , route , and time of administration . details regarding post - sedation monitoring were taken from the standard observation chart , or progress notes . adverse events relating to observations were defined in alignment with the standard adult general observation chart calling criteria for clinical reviews,25 that is respiratory depression ( respiratory rate [ rr ] < 12 ) , hypotension ( systolic blood pressure [ sbp ] < 100 ) , and over - sedation ( avpu [ alert , voice , pain , unresponsive ] score = p or u ) ( nsw ministry of health).27 other adverse events identified from the notes included extrapyramidal side effects , missed or delayed usual medication , and falls over the 24 hours following sedation . whether a patient had settled following sedation simple statistical calculations such as mean , median , and standard deviation were performed using microsoft excel . of 73 art calls identified from the iims database , there were 43 unique patients , including 26 with only 1 art call , 12 with 2 calls , and 5 with 3 or more calls ( maximum 6 art calls , 1 patient ) . the mean age was 81 years ( range 6694 years , standard deviation 7.2 years ) , and 29 ( 67% ) patients were male . thirteen ( 18% ) art calls occurred between 8 am and 4 pm , 35 ( 48% ) occurred between 4 pm and midnight , and 22 ( 30% ) occurred between midnight and 8 am ( art call timing was unavailable for 3 cases ) . five ( 7% ) of art calls occurred in the ed , while 68 ( 93% ) occurred on the medical and surgical wards . the median time from triage to art call was 1.9 days ( range 21 minutes16 days ) . when looking at the pager system database ( which did not include patient - specific identifiers ) , of the 326 art calls regardless of age , there were 209 art calls in the ed and 117 on the wards . iims reports were only filed for 50% of these art calls ; however , the rate was much better on the wards ( 71% ) compared to the ed ( 38% ) . for those patients who did have iims reports , those on the wards ( 83% ) were 65 years or older , while in the ed only 10% were 65 years or older . reasons for art calls were documented by both medical and nursing staff in 56 ( 77% ) of all the art calls , by nursing staff alone in 15 art calls and medical staff alone in 2 art calls . multiple reasons for art calls were given in 53 ( 73% ) cases , and a single reason was given in 20 ( 27% ) . the most common reasons were wandering ( 78% ) , physical aggression ( 66% ) , and verbal aggression ( 47% ) . documentation limited the rating of severity of physical aggression , although examples included staff being physically struck , patients throwing files or equipment at staff , and 1 patient using a fire extinguisher as a weapon . for patients who had art calls for wandering , often there was concern that the patient would abscond , and in some cases they had already left the building . examples of documentation included , combative , resisting care . verbally aggressive and threatening , climbing out of bed . hitting and kicking staff , wanting to leave . entered the lifts . other reasons for art calls included pulling out indwelling catheters or intravenous access . in regards to diagnoses made by medical teams at the time of the art call , 11 ( 26% ) had a working diagnosis of bpsd , 10 ( 23% ) had delirium , 9 ( 21% ) had bpsd and delirium , 2 ( 4% ) had primary psychosis , and 1 ( 2% ) had alcohol withdrawal , while 10 ( 23% ) did not have a documented working diagnosis . there was documentation that verbal de - escalation had been attempted in the period , leading up to 31 ( 42% ) of the art calls . in 14 ( 19% ) cases , the family was called in or spoke to the patient over the phone . in 16 ( 22% ) cases , a 1:1 special nurse was requested by the medical team , although it is unclear how often it was provided . mechanical restraints were used in 10 ( 14% ) cases . in terms of pharmacological agents used , table 2 presents the first agents used . the specific routes used were intramuscular in 32 ( 50% ) cases , intravenous in 12 ( 19% ) cases , subcutaneous in 4 ( 6% ) cases , and oral in 16 ( 25% ) cases . of 48 cases where parenteral sedation was used , in only 16 ( 33% ) was it documented that oral sedation had been refused . the median time from the art call to administration of the first agent was 13 minutes ( range 045 minutes ) . there were 5 cases in which dual agents were used simultaneously , and a further 9 cases in which the time from administration of the first agent to the second agent was less than 15 minutes . overall , patients received further benzodiazepines or antipsychotics in 53 ( 73% ) of all art calls with a mean time between first and second doses of 202 minutes ( range 01,201 minutes ) . for those patients who received parenteral pharmacological sedation , the median time from sedation to the sedation given in each of these cases is listed in table 3 . regarding follow - up of art calls , there was review within 48 hours by psychiatry in 41% of cases , by geriatrics in 22% , and by dementia clinical nurse consultant in 36% . there was review by at least one of the aforementioned departments in 62% of the cases . of 73 art calls identified from the iims database , there were 43 unique patients , including 26 with only 1 art call , 12 with 2 calls , and 5 with 3 or more calls ( maximum 6 art calls , 1 patient ) . the mean age was 81 years ( range 6694 years , standard deviation 7.2 years ) , and 29 ( 67% ) patients were male . thirteen ( 18% ) art calls occurred between 8 am and 4 pm , 35 ( 48% ) occurred between 4 pm and midnight , and 22 ( 30% ) occurred between midnight and 8 am ( art call timing was unavailable for 3 cases ) . five ( 7% ) of art calls occurred in the ed , while 68 ( 93% ) occurred on the medical and surgical wards . the median time from triage to art call was 1.9 days ( range 21 minutes16 days ) . when looking at the pager system database ( which did not include patient - specific identifiers ) , of the 326 art calls regardless of age , there were 209 art calls in the ed and 117 on the wards . iims reports were only filed for 50% of these art calls ; however , the rate was much better on the wards ( 71% ) compared to the ed ( 38% ) . for those patients who did have iims reports , those on the wards ( 83% ) were 65 years or older , while in the ed only 10% were 65 years or older . reasons for art calls were documented by both medical and nursing staff in 56 ( 77% ) of all the art calls , by nursing staff alone in 15 art calls and medical staff alone in 2 art calls . multiple reasons for art calls were given in 53 ( 73% ) cases , and a single reason was given in 20 ( 27% ) . the most common reasons were wandering ( 78% ) , physical aggression ( 66% ) , and verbal aggression ( 47% ) . documentation limited the rating of severity of physical aggression , although examples included staff being physically struck , patients throwing files or equipment at staff , and 1 patient using a fire extinguisher as a weapon . for patients who had art calls for wandering , often there was concern that the patient would abscond , and in some cases they had already left the building . examples of documentation included , combative , resisting care . verbally aggressive and threatening , climbing out of bed . hitting and kicking staff , wanting to leave . other reasons for art calls included pulling out indwelling catheters or intravenous access . in regards to diagnoses made by medical teams at the time of the art call , 11 ( 26% ) had a working diagnosis of bpsd , 10 ( 23% ) had delirium , 9 ( 21% ) had bpsd and delirium , 2 ( 4% ) had primary psychosis , and 1 ( 2% ) had alcohol withdrawal , while 10 ( 23% ) did not have a documented working diagnosis . there was documentation that verbal de - escalation had been attempted in the period , leading up to 31 ( 42% ) of the art calls . in 14 ( 19% ) cases , the family was called in or spoke to the patient over the phone . in 16 ( 22% ) cases , a 1:1 special nurse was requested by the medical team , although it is unclear how often it was provided . mechanical restraints were used in 10 ( 14% ) cases . in terms of pharmacological agents used , table 2 presents the first agents used . the specific routes used were intramuscular in 32 ( 50% ) cases , intravenous in 12 ( 19% ) cases , subcutaneous in 4 ( 6% ) cases , and oral in 16 ( 25% ) cases . of 48 cases where parenteral sedation was used , in only 16 ( 33% ) was it documented that oral sedation had been refused . the median time from the art call to administration of the first agent was 13 minutes ( range 045 minutes ) . there were 5 cases in which dual agents were used simultaneously , and a further 9 cases in which the time from administration of the first agent to the second agent was less than 15 minutes . overall , patients received further benzodiazepines or antipsychotics in 53 ( 73% ) of all art calls with a mean time between first and second doses of 202 minutes ( range 01,201 minutes ) . for those patients who received parenteral pharmacological sedation , the median time from sedation to their next set of observations was 120 minutes ( range 51,530 minutes ) . the sedation given in each of these cases is listed in table 3 . regarding follow - up of art calls , there was review within 48 hours by psychiatry in 41% of cases , by geriatrics in 22% , and by dementia clinical nurse consultant in 36% . there was review by at least one of the aforementioned departments in 62% of the cases . to our knowledge , this is the first study to document the management of behavioral emergencies in the elderly in an acute hospital setting . notwithstanding the significant limitations outlined in the limitations section , our descriptive study provides an insight into the challenges of providing safe and effective management for the extremely agitated older persons in the acute hospital setting . not surprisingly , delirium and dementia were found to be the most common diagnoses in elderly referred to the art . this is in contrast to studies of severe agitation in hospitalized younger adult patients,24 which have shown that functional psychiatric illness and substance abuse were the most common diagnoses , which only featured in 4% of our patients . first , it reinforces the need for age - specific management guidelines for the behavioral emergency . clearly , the management of agitation due to amphetamine - induced psychosis in a 22-year - old male must differ from agitation secondary to a urinary tract infection in an 82-year - old male . this finding reinforces what is already known in the literature on delirium , namely that we need to be attentive to high - risk groups for delirium and bpsd and target modifiable risk factors as part of a prevention strategy.10 if a patient comes in to ed with a history of bpsd , we must be alert for the potential for agitation . a study of older patients admitted to a tertiary care teaching hospital through ed showed that patients with dementia and/or delirium who become symptomatic after admission first show mental signs and symptoms , then show behavioral disturbances , which appear to be the proximate causes of greater length of stay.9 interestingly , our clinical experience of the typical nursing home patient referred to hospital with bpsd was not reflected in this data , which although based on a small sample size showed that the majority of patients referred for art calls were from home or low - level care , not high - level residential care . are patients with cognitive impairment from home even more at risk of becoming frightened and agitated in the acute hospital ? are they underestimated in terms of their propensity for escalation compared with nursing home patients ? given the known risk of functional decline and institutionalization following severe or prolonged delirium or hospitalization per se,68 this provides further impetus for the need for prevention strategies . consistent with previous literature,28 and presumably reflective of both staffing levels and the inherent nature of behavior disturbance associated with delirium and dementia , referrals to the art team frequently occur outside of normal working hours . if recourse to the use of 1:1 nursing specials is an option , could this be geared to more vulnerable hours ? could there be negotiation with families to support patients during some of these hours , while recognizing the burden to families ? notably , as our study demonstrated , adverse effects from pharmacological sedation are not uncommon , and these are also potentially more dangerous in a less resourced after - hours setting . surprisingly , the majority of art calls occurred on the wards rather than in the ed . however , it does not follow that there are fewer incidents of severe agitation in the ed compared to the wards , or that these patients are better managed in the ed given their lower reliance on art calls . rather , we believe this reflects less submission of iims reports due to time constraints , and also the different staffing in the ed , where there is less reliance on the art ( with its security guards ) in order to manage these patients . indeed , many patients received parenteral pharmacological sedation in the ed prior to having an art call . there is clearly an equal obligation on eds to be cognizant of the common reasons for agitation in elderly patients and to apply appropriate management strategies . the reasons for art calls were usually multifactorial ; however , a common theme was a concern for the safety of the patient , staff , or others . there was a high prevalence of wandering among patients having art calls , and we believe that this reflects the absence of both locked wards and a dedicated delirium unit at the study hospital . notwithstanding the contextual basis for this , it must be emphasized that there is consensus in the literature that neither wandering nor verbal aggression ( also commonly precipitating an art call ) is an indication for psychotropic use.29 as hypothesized , there was an extremely high reliance on pharmacological sedation . based on the limited documentation , it is not clear that nonpharmacological interventions were exhausted in all cases . there were also some cases where the level of aggression did not appear to represent imminent harm to the patient or others , although this is difficult to conclude with any certainty without better documentation or the use of objective sedation and agitation scales . midazolam was commonly used , which is of concern given the lack of evidence for benzodiazepines in managing acute agitation in the elderly , their potential to worsen delirium,10 and the limited circumstances in which they are appropriate in the management of bpsd.30 doses of haloperidol up to maximum 5 mg are of significant concern given its propensity for extrapyramidal side effects , dose - related mortality , and significant risk in dementia with lewy bodies . there was a high reliance on parenteral sedation , which may reflect patient noncompliance with oral sedation ; however , this was not formally documented in the majority of cases . the lack of regular post - sedation monitoring is of great concern , given the vulnerability of elderly patients to adverse effects . there may be several factors contributing to this , including : a lack of specific guidelines on the frequency of observations required post - sedation , a fear from nursing and medical staff that regular examination of the patient may further agitate the patient , and perhaps a lack of awareness of side effects of sedation . however , our study demonstrated a relatively high rate of adverse events following the use of pharmacological sedation , and hence should be a cautionary reminder of the need to monitor these patients carefully . first , and foremost , we recognize that these are site - specific findings , acknowledging the primary role of staffing and environment in the management of behavioral symptoms.5 it is a small cohort from a single center over a 1 year time period , the resources and staffing of which may have influenced the findings , limiting their generalizability . specifically , the lack of a secured ward or specialized delirium unit is likely to have influenced the management strategies utilized . second , the data captured only those registered on the incident monitoring system , and therefore represented only a proportion of actual incidents of aggression . this may have led to the inclusion of more severely agitated patients . notwithstanding this , we need to know how to manage inpatients on the very severe end of the behavior spectrum , within tier 7 of an australian hierarchical model of bpsd service delivery.31 third , as a retrospective chart - based study , the quality of the data was determined by the quality of naturalistic clinical documentation . this probably had impact on several variables such as diagnosis , such that subjects may have been misclassified as dementia or delirium , or , as commonly seen in acute settings , delirium may have been missed.10 additionally , the bare minimum approach to documentation , particularly of behavioral or nonpharmacological strategies may have led to underestimation of the utilization of these . in this retrospective chart audit of patients referred to a duress response team , we observed a high frequency of recourse to pharmacological sedation of severely agitated elderly patients , with significant variance in the choice and dose of sedation and a high rate of complications arising from sedation . albeit that our findings may be site specific , in the absence of any other such data in the literature , and indeed guidelines for such clinical scenarios , and in the face of management approaches of older patients being subsumed within general adult guidelines , this is a start . it is a start toward the development of some consensual guidelines for the management of the behavioral emergency in the older person , not for emergency sedation of the elderly which is not our goal , but our default option . we recommend the development of guidelines and education on the management of behavioral emergency in the elderly patients , including nonpharmacological de - escalation strategies tailored toward patients with dementia and/or delirium , and standardized psychotropic guidelines .

aimthe management of severely agitated elderly patients is not easy , and limited guidelines are available to assist practitioners . at a sydney hospital , an aggression response team ( art ) comprising clinical and security staff can be alerted when a staff member has safety concerns . our aims were to describe the patient population referred for art calls , reasons for and interventions during art calls , and complications following them.methodspatients 65 years and older referred for art calls in the emergency department or wards during 2014 were identified using the incident information management system database and medical records were reviewed . demographic and clinical data were collected.resultsof 43 elderly patients with art calls , 30 had repeat art calls . thirty - one patients ( 72% ) had underlying dementia , and 22 ( 51% ) were agitated at the time of admission . the main reasons for art calls were wandering and physical aggression . pharmacological sedation was used in 88% of the art calls , with a range of psychotropics , doses , and routes of administration , including intravenous ( 19% ) and , most commonly , midazolam ( 53% ) . complications were documented in 14% of cases where sedation was used.conclusionwe observed a high frequency of pharmacological sedation among the severely agitated elderly , with significant variance in the choice and dose of sedation and a high rate of complications arising from sedation , which may be an underestimate given the lack of post - sedation monitoring . we recommend the development of guidelines on the management of behavioral emergency in the elderly patients , including de - escalation strategies and standardized psychotropic guidelines .

Introduction Methods Setting Study design Data collection and analysis Results Patient demographics Timing and location of ART calls Reasons for ART calls Interventions during ART calls Post-sedation monitoring, complications, and follow-up of ART calls Discussion Limitations Conclusion and recommendations

the management of severely agitated elderly patients in the acute hospital setting is not easy.14 urgent safety issues for both patient and staff coexist with diagnostic priorities in a setting not geared to first - line nonpharmacological strategies of meeting psychosocial and emotional needs of patients.5 the adverse outcomes of cognitively impaired older people in hospital are well documented and include prolonged length of stay , increased mortality , polypharmacy , and psychotropic use.68 however , agitation among elderly patients in the hospital setting is common , and needs to be managed given its association with some of these adverse outcomes.9 the most common causes of agitation in the elderly are delirium or behavioral and psychological symptoms of dementia ( bpsd ) , and less commonly , a primary psychosis.1,2 delirium affects an estimated 14%56% of all hospitalized elderly patients,10 while an estimated 60% of people with dementia will experience symptoms of agitation or aggression at some stage during the course of their disease.11 there is broad consensus that the first - line management of both delirium and bpsd involves thorough assessment and treatment of the underlying cause of the delirium or trigger for the bpsd in conjunction with nonpharmacological strategies.10,12 acute hospital settings , particularly emergency departments ( eds ) , are not geared to this , but rather to pharmacological strategies , ideally used as a last resort due to high risk - to - benefit ratios in the elderly . however , despite the existence of various guidelines on the management of both delirium and dementia , including within australia,1315 they often lack detailed advice on the pharmacological and nonpharmacological management of patients with severe agitation.16 this is probably a reflection of the lack of high - quality randomized control trials in this domain.1721 while there are guidelines for the management of the agitated patients,22 these are not age - specific , an important issue given the distinctive etiologies of behavioral disturbance in the elderly compared with younger adult groups . as much as posing a risk to themselves , despite their frailty , severely agitated elderly patients can pose a physical risk to others . however , there is limited literature addressing the management of severely agitated elderly patients by duress response teams with the exception of 1 study of agitated adult patients ( mean aged 32 years ) in an ed.24 a prospective study of parenteral sedation for agitated elderly patients ( 65 years and older ) in an ed focused on the use of parenteral sedation only rather than the reasons for sedation and the use of alternative nonpharmacological strategies.25 the aim of this study was to describe the management of severely agitated elderly patients in an acute hospital setting , by analyzing a cohort of patients who required a response from the hospital s duress response team , known as the aggression response team ( art ) . we aimed to describe the older patient population referred for art calls , the reasons for and interventions during art calls ( including the use and nature of pharmacological sedation ) , complications arising from these interventions , and the follow - up of art calls . the art can be alerted via the pager system when there is a severely agitated patient and a staff member has concerns for the safety of either the patient or others . the team comprises a medical and psychiatry registrar ( each with 2 or more years of post - graduate experience ) , a senior nurse acting as team leader , and security guards . the team leaders and security guards have mandatory annual face - to - face training in de - escalation strategies and restraint techniques . it is hospital policy that an incident report is recorded in the incident information management system ( iims ) for all art calls . patients 65 years and older who were referred for art calls during 2014 in the ed or wards ( excluding the psychiatry ward ) were identified using the iims database26 and their medical records were reviewed . data was collected from the medical notes regarding demographics , medical history , regular medications , presenting symptom , and progress prior to the art call . art call details included time of activation ( taken from the pager system ) , reasons for the art call , working diagnosis documented in medical progress notes prior to the art call , and interventions . details of pharmacological sedation were taken from the medication chart , including dose , route , and time of administration . details regarding post - sedation monitoring were taken from the standard observation chart , or progress notes . elderly patients are predominantly admitted under either general medical or surgical teams , with the availability of both geriatric and psychiatry consult - liaison services . the art can be alerted via the pager system when there is a severely agitated patient and a staff member has concerns for the safety of either the patient or others . the team comprises a medical and psychiatry registrar ( each with 2 or more years of post - graduate experience ) , a senior nurse acting as team leader , and security guards . the team leaders and security guards have mandatory annual face - to - face training in de - escalation strategies and restraint techniques . it is hospital policy that an incident report is recorded in the incident information management system ( iims ) for all art calls . patients 65 years and older who were referred for art calls during 2014 in the ed or wards ( excluding the psychiatry ward ) were identified using the iims database26 and their medical records were reviewed . data was collected from the medical notes regarding demographics , medical history , regular medications , presenting symptom , and progress prior to the art call . art call details included time of activation ( taken from the pager system ) , reasons for the art call , working diagnosis documented in medical progress notes prior to the art call , and interventions . details of pharmacological sedation were taken from the medication chart , including dose , route , and time of administration . details regarding post - sedation monitoring were taken from the standard observation chart , or progress notes . adverse events relating to observations were defined in alignment with the standard adult general observation chart calling criteria for clinical reviews,25 that is respiratory depression ( respiratory rate [ rr ] < 12 ) , hypotension ( systolic blood pressure [ sbp ] < 100 ) , and over - sedation ( avpu [ alert , voice , pain , unresponsive ] score = p or u ) ( nsw ministry of health).27 other adverse events identified from the notes included extrapyramidal side effects , missed or delayed usual medication , and falls over the 24 hours following sedation . of 73 art calls identified from the iims database , there were 43 unique patients , including 26 with only 1 art call , 12 with 2 calls , and 5 with 3 or more calls ( maximum 6 art calls , 1 patient ) . thirteen ( 18% ) art calls occurred between 8 am and 4 pm , 35 ( 48% ) occurred between 4 pm and midnight , and 22 ( 30% ) occurred between midnight and 8 am ( art call timing was unavailable for 3 cases ) . five ( 7% ) of art calls occurred in the ed , while 68 ( 93% ) occurred on the medical and surgical wards . when looking at the pager system database ( which did not include patient - specific identifiers ) , of the 326 art calls regardless of age , there were 209 art calls in the ed and 117 on the wards . iims reports were only filed for 50% of these art calls ; however , the rate was much better on the wards ( 71% ) compared to the ed ( 38% ) . for those patients who did have iims reports , those on the wards ( 83% ) were 65 years or older , while in the ed only 10% were 65 years or older . reasons for art calls were documented by both medical and nursing staff in 56 ( 77% ) of all the art calls , by nursing staff alone in 15 art calls and medical staff alone in 2 art calls . multiple reasons for art calls were given in 53 ( 73% ) cases , and a single reason was given in 20 ( 27% ) . the most common reasons were wandering ( 78% ) , physical aggression ( 66% ) , and verbal aggression ( 47% ) . documentation limited the rating of severity of physical aggression , although examples included staff being physically struck , patients throwing files or equipment at staff , and 1 patient using a fire extinguisher as a weapon . for patients who had art calls for wandering , often there was concern that the patient would abscond , and in some cases they had already left the building . other reasons for art calls included pulling out indwelling catheters or intravenous access . in regards to diagnoses made by medical teams at the time of the art call , 11 ( 26% ) had a working diagnosis of bpsd , 10 ( 23% ) had delirium , 9 ( 21% ) had bpsd and delirium , 2 ( 4% ) had primary psychosis , and 1 ( 2% ) had alcohol withdrawal , while 10 ( 23% ) did not have a documented working diagnosis . there was documentation that verbal de - escalation had been attempted in the period , leading up to 31 ( 42% ) of the art calls . in 14 ( 19% ) cases , the family was called in or spoke to the patient over the phone . the specific routes used were intramuscular in 32 ( 50% ) cases , intravenous in 12 ( 19% ) cases , subcutaneous in 4 ( 6% ) cases , and oral in 16 ( 25% ) cases . of 48 cases where parenteral sedation was used , in only 16 ( 33% ) was it documented that oral sedation had been refused . the median time from the art call to administration of the first agent was 13 minutes ( range 045 minutes ) . there were 5 cases in which dual agents were used simultaneously , and a further 9 cases in which the time from administration of the first agent to the second agent was less than 15 minutes . overall , patients received further benzodiazepines or antipsychotics in 53 ( 73% ) of all art calls with a mean time between first and second doses of 202 minutes ( range 01,201 minutes ) . for those patients who received parenteral pharmacological sedation , the median time from sedation to the sedation given in each of these cases is listed in table 3 . regarding follow - up of art calls , there was review within 48 hours by psychiatry in 41% of cases , by geriatrics in 22% , and by dementia clinical nurse consultant in 36% . of 73 art calls identified from the iims database , there were 43 unique patients , including 26 with only 1 art call , 12 with 2 calls , and 5 with 3 or more calls ( maximum 6 art calls , 1 patient ) . thirteen ( 18% ) art calls occurred between 8 am and 4 pm , 35 ( 48% ) occurred between 4 pm and midnight , and 22 ( 30% ) occurred between midnight and 8 am ( art call timing was unavailable for 3 cases ) . five ( 7% ) of art calls occurred in the ed , while 68 ( 93% ) occurred on the medical and surgical wards . when looking at the pager system database ( which did not include patient - specific identifiers ) , of the 326 art calls regardless of age , there were 209 art calls in the ed and 117 on the wards . for those patients who did have iims reports , those on the wards ( 83% ) were 65 years or older , while in the ed only 10% were 65 years or older . reasons for art calls were documented by both medical and nursing staff in 56 ( 77% ) of all the art calls , by nursing staff alone in 15 art calls and medical staff alone in 2 art calls . multiple reasons for art calls were given in 53 ( 73% ) cases , and a single reason was given in 20 ( 27% ) . the most common reasons were wandering ( 78% ) , physical aggression ( 66% ) , and verbal aggression ( 47% ) . documentation limited the rating of severity of physical aggression , although examples included staff being physically struck , patients throwing files or equipment at staff , and 1 patient using a fire extinguisher as a weapon . for patients who had art calls for wandering , often there was concern that the patient would abscond , and in some cases they had already left the building . other reasons for art calls included pulling out indwelling catheters or intravenous access . in regards to diagnoses made by medical teams at the time of the art call , 11 ( 26% ) had a working diagnosis of bpsd , 10 ( 23% ) had delirium , 9 ( 21% ) had bpsd and delirium , 2 ( 4% ) had primary psychosis , and 1 ( 2% ) had alcohol withdrawal , while 10 ( 23% ) did not have a documented working diagnosis . there was documentation that verbal de - escalation had been attempted in the period , leading up to 31 ( 42% ) of the art calls . in 14 ( 19% ) cases , the family was called in or spoke to the patient over the phone . the specific routes used were intramuscular in 32 ( 50% ) cases , intravenous in 12 ( 19% ) cases , subcutaneous in 4 ( 6% ) cases , and oral in 16 ( 25% ) cases . of 48 cases where parenteral sedation was used , in only 16 ( 33% ) was it documented that oral sedation had been refused . there were 5 cases in which dual agents were used simultaneously , and a further 9 cases in which the time from administration of the first agent to the second agent was less than 15 minutes . overall , patients received further benzodiazepines or antipsychotics in 53 ( 73% ) of all art calls with a mean time between first and second doses of 202 minutes ( range 01,201 minutes ) . for those patients who received parenteral pharmacological sedation , the median time from sedation to their next set of observations was 120 minutes ( range 51,530 minutes ) . regarding follow - up of art calls , there was review within 48 hours by psychiatry in 41% of cases , by geriatrics in 22% , and by dementia clinical nurse consultant in 36% . to our knowledge , this is the first study to document the management of behavioral emergencies in the elderly in an acute hospital setting . clearly , the management of agitation due to amphetamine - induced psychosis in a 22-year - old male must differ from agitation secondary to a urinary tract infection in an 82-year - old male . a study of older patients admitted to a tertiary care teaching hospital through ed showed that patients with dementia and/or delirium who become symptomatic after admission first show mental signs and symptoms , then show behavioral disturbances , which appear to be the proximate causes of greater length of stay.9 interestingly , our clinical experience of the typical nursing home patient referred to hospital with bpsd was not reflected in this data , which although based on a small sample size showed that the majority of patients referred for art calls were from home or low - level care , not high - level residential care . are patients with cognitive impairment from home even more at risk of becoming frightened and agitated in the acute hospital ? consistent with previous literature,28 and presumably reflective of both staffing levels and the inherent nature of behavior disturbance associated with delirium and dementia , referrals to the art team frequently occur outside of normal working hours . surprisingly , the majority of art calls occurred on the wards rather than in the ed . however , it does not follow that there are fewer incidents of severe agitation in the ed compared to the wards , or that these patients are better managed in the ed given their lower reliance on art calls . rather , we believe this reflects less submission of iims reports due to time constraints , and also the different staffing in the ed , where there is less reliance on the art ( with its security guards ) in order to manage these patients . there is clearly an equal obligation on eds to be cognizant of the common reasons for agitation in elderly patients and to apply appropriate management strategies . the reasons for art calls were usually multifactorial ; however , a common theme was a concern for the safety of the patient , staff , or others . there was a high prevalence of wandering among patients having art calls , and we believe that this reflects the absence of both locked wards and a dedicated delirium unit at the study hospital . notwithstanding the contextual basis for this , it must be emphasized that there is consensus in the literature that neither wandering nor verbal aggression ( also commonly precipitating an art call ) is an indication for psychotropic use.29 as hypothesized , there was an extremely high reliance on pharmacological sedation . based on the limited documentation , it is not clear that nonpharmacological interventions were exhausted in all cases . there were also some cases where the level of aggression did not appear to represent imminent harm to the patient or others , although this is difficult to conclude with any certainty without better documentation or the use of objective sedation and agitation scales . midazolam was commonly used , which is of concern given the lack of evidence for benzodiazepines in managing acute agitation in the elderly , their potential to worsen delirium,10 and the limited circumstances in which they are appropriate in the management of bpsd.30 doses of haloperidol up to maximum 5 mg are of significant concern given its propensity for extrapyramidal side effects , dose - related mortality , and significant risk in dementia with lewy bodies . there was a high reliance on parenteral sedation , which may reflect patient noncompliance with oral sedation ; however , this was not formally documented in the majority of cases . the lack of regular post - sedation monitoring is of great concern , given the vulnerability of elderly patients to adverse effects . there may be several factors contributing to this , including : a lack of specific guidelines on the frequency of observations required post - sedation , a fear from nursing and medical staff that regular examination of the patient may further agitate the patient , and perhaps a lack of awareness of side effects of sedation . however , our study demonstrated a relatively high rate of adverse events following the use of pharmacological sedation , and hence should be a cautionary reminder of the need to monitor these patients carefully . first , and foremost , we recognize that these are site - specific findings , acknowledging the primary role of staffing and environment in the management of behavioral symptoms.5 it is a small cohort from a single center over a 1 year time period , the resources and staffing of which may have influenced the findings , limiting their generalizability . specifically , the lack of a secured ward or specialized delirium unit is likely to have influenced the management strategies utilized . second , the data captured only those registered on the incident monitoring system , and therefore represented only a proportion of actual incidents of aggression . notwithstanding this , we need to know how to manage inpatients on the very severe end of the behavior spectrum , within tier 7 of an australian hierarchical model of bpsd service delivery.31 third , as a retrospective chart - based study , the quality of the data was determined by the quality of naturalistic clinical documentation . in this retrospective chart audit of patients referred to a duress response team , we observed a high frequency of recourse to pharmacological sedation of severely agitated elderly patients , with significant variance in the choice and dose of sedation and a high rate of complications arising from sedation . albeit that our findings may be site specific , in the absence of any other such data in the literature , and indeed guidelines for such clinical scenarios , and in the face of management approaches of older patients being subsumed within general adult guidelines , this is a start . it is a start toward the development of some consensual guidelines for the management of the behavioral emergency in the older person , not for emergency sedation of the elderly which is not our goal , but our default option . we recommend the development of guidelines and education on the management of behavioral emergency in the elderly patients , including nonpharmacological de - escalation strategies tailored toward patients with dementia and/or delirium , and standardized psychotropic guidelines .

essentially , slrh is a form of dilution haplotyping similar to previous approaches based on fosmid clones or long fragment reads ( lfr ) . it involves placing a small number of large ~710 kbp dna fragments into separate pools . each pool has a unique barcode that identifies its fragments , which are then recovered from short - read sequences and assembled into long haplotype blocks using a phasing algorithm ( fig . the preparation of each phasing library starts by shearing dna into fragments of approximately 10 kbp in size ; these fragments are gel - purified and ligated with amplification adaptors at both ends . fragments are then diluted into a 384-well plate containing 3,0006,000 molecules per well and pcr - amplified using adapter - specific primers ( supplementary fig . the number of fragments amplified ( 3,0006,000 ) is chosen to minimize the probability of two fragments overlapping , which later simplifies fragment calling in the informatics pipeline . each resulting pool of amplified molecules is prepared into a sequencing library using the nextera dna transposase , and sequencing adapters with barcodes unique to each well are incorporated through limited - cycle pcr . the resulting sub - libraries are pooled and sequenced on the illumina platform ( fig . a single phasing library typically corresponds to about 30 gbp of 101-base , paired - end illumina reads . the sequenced reads are then aligned to the reference genome and mapped back to their original wells as specified by the barcode adapters . mapped reads within each variants in each fragment are called based on the subject 's whole - genome genotyping ; in our experiments , the subject 's genotypes were determined from a 50x read coverage on the illumina platform ( online methods ) . fragments called at this stage have n50 lengths of about 79 kbp ( i.e. at least half of all sequenced bases are within fragments of at least 7 - 9kbp ) and cover the genome to a depth of about 48x ( supplementary tables 3 and 4 ) . the relatively short length of these reads compared to older technologies required the development of a haplotyping algorithm , prism ( fig . 1b and supplementary data ) , which augments dilution haplotyping with statistical techniques . in brief , first , in a local_stage , it assembles fragments into haplotype blocks by connecting them together at their overlapping heterozygous snvs . then , in a global stage , prism exploits linkage disequilibrium patterns to assemble local blocks into long and accurate global haplotype contigs . such contigs can phase up to 99% of heterozygous snvs and up to 95% of heterozygous variants . between each local block , prism produces confidence scores that indicate the likelihood of introducing a switch error owing to statistical phasing . in applications where a high level of accuracy is desired , the user may introduce breaks in statistically assembled haplotype contigs whenever a confidence score falls below a certain threshold . the ability to trade - off between accuracy and completeness is a feature of prism that to our knowledge is not provided by other phasing algorithms that we expect to be useful in applications that demand a high a level of precision . the global phasing stage is an essential component of the slrh pipeline . simply connecting long dna fragments at heterozygous snvs results in local blocks of about 60 kbp in length ( supplementary the global phasing performed by prism increases the lengths of these blocks by more than ten - fold ; it also increases snv phasing rates from 94% to more than 99% . the key algorithmic tool used by prism is a hidden markov model ( hmm ; supplementary material ) . at a high level , the hmm tries to represent the partially phased haplotypes of the sample as an imperfect mosaic of haplotypes from a pre - phased reference panel from the 1000 genomes project . confidence scores between adjacent blocks are derived from the forwards - backwards variables of the hidden markov model . the prism hmm extends the work of li and stephens , on which modern statistical phasing packages such as impute2 ( ref . ) or shape - it are based . unlike existing packages , our method is able to use partially phased information obtained from long reads , which allows us to achieve greater accuracy than traditional statistical methods . the only statistical package able to leverage partial phase information is the recently introduced shapeit2 ; its underlying algorithm follows the same basic approach as prism . as a demonstration of the ability of slrh to accurately phase human genomes , we prepared libraries for three hapmap samples : na12878 , na12891 and na12892 ( supplementary table 2 ) . we evaluated the performance of slrh at different accuracy thresholds by introducing breaks in the global haplotype contigs whenever the confidence score of a local block was below a threshold . depending on the threshold , the n50 lengths of haplotype blocks varied between 130 and 750 kbp ; the percentage of snvs phased varied between 96.0% and 99.4% ( fig . 2 and supplementary tables 5 , 6 ) . in particular , at the 0.9 confidence score threshold , about 99% of all snvs were phased in blocks with n50 lengths of 560650 kbp ( supplementary tables 7 , 8) . about 89% of 23,645 genes derived from the ucsc dataset were fully contained in a single haplotype block ( table 2 and supplementary table 9 ) . the haplotype blocks also contained more than 73% of novel snvs and indels ( supplementary table 9 ) , although the accuracy over these variants was lower than average , partly because of the increased difficulty in genotyping such rare variants . the majority of phased genes contained compound heterozygous snvs ( about 75% of all genes with snvs ) ; hla - c , a gene whose haplotypes are used to predict immune response during organ transplantation is an example of a compound heterozygous gene ( supplementary fig . 2 ) . phased genes also contained about 2,500 snvs that were found to be damaging by the sift software package . about 1,500 genes were affected by these mutations , and about 500 were found to have at least one damaging snv on both the maternal and the paternal copy ( table 2 ) . to assess the accuracy of slrh , we compared the above haplotypes to ones derived from applying mendelian inheritance rules to our three samples . even the long statistically assembled haplotypes obtained from a 0.5 probability threshold were highly accurate : for sample na12878 , slrh produced long blocks with n50 lengths of 1.1 mbp that contained long switching events at an average rate of 0.85 per mbp ( supplementary tables 5 , 6 ) . breaking these haplotypes at low - confidence positions further improved their precision : in every sample , long switch errors occurred at an average rate between 0.2 and 0.9 per mbp , depending on the chosen accuracy threshold ( fig . 2 and supplementary tables 5 , 6 ) . at the 0.9 threshold , long switch events occurred at a rate of 0.47 per mbp for sample na12878 ( supplementary tables 7 , 8) . at a small number of positions , we observed short ( one - base ) discordances with parental haplotyping . this affected about 0.15% of heterozygous positions ( supplementary tables 5 , 6 ) , which were often associated with centromeres and copy number variations . finally , the absolute accuracy of the haplotype blocks ( i.e. without correcting for haplotype switching ) varied between 93% and 96% , depending on the sample . next , to demonstrate the low sequencing requirements of slrh , we ran the prism algorithm separately on each individual phasing library . we observed only a small loss of haplotyping performance , highlighting the robustness of our statistically aided approach . overall , we obtained haplotype blocks that were almost as accurate and only 100 kbp shorter than ones derived from 60 gbp of sequencing ( fig . 9899% of all snvs were phased in blocks with n50 lengths of 400500 kbp , depending on the sample ( supplementary fig . 3 and supplementary tables 10 , 11 ) . long switching events always occurred at rates below one switch per mbp , and at the 0.9 threshold , we measured 0.50.8 switches per mbp . this corresponds to accuracies of 99.8799.90% , a drop of only about 0.01% with respect to two phasing libraries per sample . finally , results for the two replicate libraries of the hapmap sample na12878 were highly concordant ( fig . 3 and supplementary table 12 ) . the two libraries were prepared with exactly the same input parameters ( e.g. fragment length , number of fragments per well ) ; their performance metrics differed by less than 1% and the two replicates assigned the same phase to most snvs ( online methods ) . next , as a demonstration of scientific applications based on the phased genome made possible by slrh , we performed an analysis of differential dna methylation across the genome of the hapmap sample na12878 ( lymphoblastoid cell line gm12878 ) based on its haplotype information . we thus obtained a detailed map of allele - specific methylation patterns within a human genome ; such maps are useful for understanding biological mechanisms such as genomic imprinting . in brief , we performed methylc - seq experiment on gm12878 cell line and assigned methylated short reads to their closest haplotype . with the reads coverage and bisulfite conversion rates on both alleles , we then quantified allele - specific dna methylation ( asm ) using fisher 's exact test ( online methods ) . we found 216,034 statistically significant asm events that clustered in 992 differentially methylated regions ( dmrs ) ranging in size from 6 to 3,181 bp ( median size 190 bp , online methods ) . ten of the dmrs were located at previously studied areas of the genome , such as in the upstream region of the h19 gene ( fig . the full list of dmrs and their associated genes is available in the supplementary material . to gather more insight into how differential methylation may affect gene expression , we determined the overlap between the dmrs and transcription start sites ( tsss ) , transcription end sites ( tess ) , exons and intergenic regions defined by genecode v14 . consistently with previous findings , the dmrs were significantly enriched at gene promoters ( p < 2.2e-16 , binomial test ) . about 20% of the dmrs were located at gene tsss , and an additional 42% were located within annotated genes ( which include tess , introns and exons ) ; the remaining 38% were found at distal intergenic regions ( supplementary fig . we further explored the regulatory role of the majority of dmrs that are not in gene promoters by assessing the overlap between the dmrs and dnase i hypersensitive sites and tf binding sites identified by encode . we found that about 55% of the dmrs overlapped with tf binding sites and 82% overlapped with dnasei hypersensitive sites ( supplementary figs . 4 and 5 ) . overall , the above findings support the fact that differential methylation plays a role in gene regulation , particularly in the differential expression of genes . we compared the asm events we found with a previous study that studied methylation patterns within the hapmap sample na12878 using reduced - representation bisulfite sequencing ( rrbs ) . we discovered substantially more asm events ( 216,034 , compared to 2,998 ) than were previously found using rrbs , a method that targets only gc - enriched regions . since methylc - seq can detect dna methylation in the whole genome while rrbs only detect dna methylation in gc- enriched regions , our results suggest the prevalence of asm events outside of cpg islands captured by rrbs technology . to our surprise , although 326 cytosines that were identified as asm in the rrbs study also passed the criteria for testing in our study , only 96 were significantly ( p<0.05 , fisher 's exact test ) differentially methylated between the two alleles . we suspect the rrbs technology may introduce high bias from the amplification that leads to high false positive rates . both pcr and the nextera transposase introduce errors in the haplotyping process ; we assessed the significance of these errors by running prism on a high - quality synthetic data set obtained by sampling 7 kbp reads uniformly at random from the trio - phased genome of na12878 ( online methods ) . analysis of the synthetic data resulted in more complete haplotypes with a 0.4% higher snv phasing rate . a further analysis of pcr amplification bias ( online methods ) suggested that some areas of the genome exhibit a systematically lower amplification rate , and are covered by fewer long fragments . the long - range switch accuracy on both datasets was similar , but the short switch accuracy was much higher on the synthetic dataset . this suggests that pcr and nextera mainly introduce gaps in the phased haplotypes as well as point errors at individual variants ; however , their impact on long - rage phase information appears to be small . essentially , slrh is a form of dilution haplotyping similar to previous approaches based on fosmid clones or long fragment reads ( lfr ) . it involves placing a small number of large ~710 kbp dna fragments into separate pools . each pool has a unique barcode that identifies its fragments , which are then recovered from short - read sequences and assembled into long haplotype blocks using a phasing algorithm ( fig . the preparation of each phasing library starts by shearing dna into fragments of approximately 10 kbp in size ; these fragments are gel - purified and ligated with amplification adaptors at both ends . fragments are then diluted into a 384-well plate containing 3,0006,000 molecules per well and pcr - amplified using adapter - specific primers ( supplementary fig . the number of fragments amplified ( 3,0006,000 ) is chosen to minimize the probability of two fragments overlapping , which later simplifies fragment calling in the informatics pipeline . each resulting pool of amplified molecules is prepared into a sequencing library using the nextera dna transposase , and sequencing adapters with barcodes unique to each well are incorporated through limited - cycle pcr . the resulting sub - libraries are pooled and sequenced on the illumina platform ( fig . a single phasing library typically corresponds to about 30 gbp of 101-base , paired - end illumina reads . the sequenced reads are then aligned to the reference genome and mapped back to their original wells as specified by the barcode adapters . mapped reads within each variants in each fragment are called based on the subject 's whole - genome genotyping ; in our experiments , the subject 's genotypes were determined from a 50x read coverage on the illumina platform ( online methods ) . fragments called at this stage have n50 lengths of about 79 kbp ( i.e. at least half of all sequenced bases are within fragments of at least 7 - 9kbp ) and cover the genome to a depth of about 48x ( supplementary tables 3 and 4 ) . the relatively short length of these reads compared to older technologies required the development of a haplotyping algorithm , prism ( fig . 1b and supplementary data ) , which augments dilution haplotyping with statistical techniques . in brief , first , in a local_stage , it assembles fragments into haplotype blocks by connecting them together at their overlapping heterozygous snvs . then , in a global stage , prism exploits linkage disequilibrium patterns to assemble local blocks into long and accurate global haplotype contigs . such contigs can phase up to 99% of heterozygous snvs and up to 95% of heterozygous variants . between each local block , prism produces confidence scores that indicate the likelihood of introducing a switch error owing to statistical phasing . in applications where a high level of accuracy is desired , the user may introduce breaks in statistically assembled haplotype contigs whenever a confidence score falls below a certain threshold . the ability to trade - off between accuracy and completeness is a feature of prism that to our knowledge is not provided by other phasing algorithms that we expect to be useful in applications that demand a high a level of precision . the global phasing stage is an essential component of the slrh pipeline . simply connecting long dna fragments at heterozygous snvs results in local blocks of about 60 kbp in length ( supplementary the global phasing performed by prism increases the lengths of these blocks by more than ten - fold ; it also increases snv phasing rates from 94% to more than 99% . the key algorithmic tool used by prism is a hidden markov model ( hmm ; supplementary material ) . at a high level , the hmm tries to represent the partially phased haplotypes of the sample as an imperfect mosaic of haplotypes from a pre - phased reference panel from the 1000 genomes project . confidence scores between adjacent blocks are derived from the forwards - backwards variables of the hidden markov model . the prism hmm extends the work of li and stephens , on which modern statistical phasing packages such as impute2 ( ref . ) or shape - it are based . unlike existing packages , our method is able to use partially phased information obtained from long reads , which allows us to achieve greater accuracy than traditional statistical methods . the only statistical package able to leverage partial phase information is the recently introduced shapeit2 ; its underlying algorithm follows the same basic approach as prism . as a demonstration of the ability of slrh to accurately phase human genomes , we prepared libraries for three hapmap samples : na12878 , na12891 and na12892 ( supplementary table 2 ) . we evaluated the performance of slrh at different accuracy thresholds by introducing breaks in the global haplotype contigs whenever the confidence score of a local block was below a threshold . depending on the threshold , the n50 lengths of haplotype blocks varied between 130 and 750 kbp ; the percentage of snvs phased varied between 96.0% and 99.4% ( fig . 2 and supplementary tables 5 , 6 ) . in particular , at the 0.9 confidence score threshold , about 99% of all snvs were phased in blocks with n50 lengths of 560650 kbp ( supplementary tables 7 , 8) . about 89% of 23,645 genes derived from the ucsc dataset were fully contained in a single haplotype block ( table 2 and supplementary table 9 ) . the haplotype blocks also contained more than 73% of novel snvs and indels ( supplementary table 9 ) , although the accuracy over these variants was lower than average , partly because of the increased difficulty in genotyping such rare variants . the majority of phased genes contained compound heterozygous snvs ( about 75% of all genes with snvs ) ; hla - c , a gene whose haplotypes are used to predict immune response during organ transplantation is an example of a compound heterozygous gene ( supplementary fig . 2 ) . phased genes also contained about 2,500 snvs that were found to be damaging by the sift software package . about 1,500 genes were affected by these mutations , and about 500 were found to have at least one damaging snv on both the maternal and the paternal copy ( table 2 ) . to assess the accuracy of slrh , we compared the above haplotypes to ones derived from applying mendelian inheritance rules to our three samples . even the long statistically assembled haplotypes obtained from a 0.5 probability threshold were highly accurate : for sample na12878 , slrh produced long blocks with n50 lengths of 1.1 mbp that contained long switching events at an average rate of 0.85 per mbp ( supplementary tables 5 , 6 ) . breaking these haplotypes at low - confidence positions further improved their precision : in every sample , long switch errors occurred at an average rate between 0.2 and 0.9 per mbp , depending on the chosen accuracy threshold ( fig . 2 and supplementary tables 5 , 6 ) . at the 0.9 threshold , long switch events occurred at a rate of 0.47 per mbp for sample na12878 ( supplementary tables 7 , 8) . at a small number of positions , we observed short ( one - base ) discordances with parental haplotyping . this affected about 0.15% of heterozygous positions ( supplementary tables 5 , 6 ) , which were often associated with centromeres and copy number variations . finally , the absolute accuracy of the haplotype blocks ( i.e. without correcting for haplotype switching ) varied between 93% and 96% , depending on the sample . next , to demonstrate the low sequencing requirements of slrh , we ran the prism algorithm separately on each individual phasing library . we observed only a small loss of haplotyping performance , highlighting the robustness of our statistically aided approach . overall , we obtained haplotype blocks that were almost as accurate and only 100 kbp shorter than ones derived from 60 gbp of sequencing ( fig . 9899% of all snvs were phased in blocks with n50 lengths of 400500 kbp , depending on the sample ( supplementary fig . 3 and supplementary tables 10 , 11 ) . long switching events always occurred at rates below one switch per mbp , and at the 0.9 threshold , we measured 0.50.8 switches per mbp . this corresponds to accuracies of 99.8799.90% , a drop of only about 0.01% with respect to two phasing libraries per sample . finally , results for the two replicate libraries of the hapmap sample na12878 were highly concordant ( fig . 3 and supplementary table 12 ) . the two libraries were prepared with exactly the same input parameters ( e.g. fragment length , number of fragments per well ) ; their performance metrics differed by less than 1% and the two replicates assigned the same phase to most snvs ( online methods ) . next , as a demonstration of scientific applications based on the phased genome made possible by slrh , we performed an analysis of differential dna methylation across the genome of the hapmap sample na12878 ( lymphoblastoid cell line gm12878 ) based on its haplotype information . we thus obtained a detailed map of allele - specific methylation patterns within a human genome ; such maps are useful for understanding biological mechanisms such as genomic imprinting . in brief , we performed methylc - seq experiment on gm12878 cell line and assigned methylated short reads to their closest haplotype . with the reads coverage and bisulfite conversion rates on both alleles , we then quantified allele - specific dna methylation ( asm ) using fisher 's exact test ( online methods ) . we found 216,034 statistically significant asm events that clustered in 992 differentially methylated regions ( dmrs ) ranging in size from 6 to 3,181 bp ( median size 190 bp , online methods ) . ten of the dmrs were located at previously studied areas of the genome , such as in the upstream region of the h19 gene ( fig . the full list of dmrs and their associated genes is available in the supplementary material . to gather more insight into how differential methylation may affect gene expression , we determined the overlap between the dmrs and transcription start sites ( tsss ) , transcription end sites ( tess ) , exons and intergenic regions defined by genecode v14 . consistently with previous findings , the dmrs were significantly enriched at gene promoters ( p < 2.2e-16 , binomial test ) . about 20% of the dmrs were located at gene tsss , and an additional 42% were located within annotated genes ( which include tess , introns and exons ) ; the remaining 38% were found at distal intergenic regions ( supplementary fig . we further explored the regulatory role of the majority of dmrs that are not in gene promoters by assessing the overlap between the dmrs and dnase i hypersensitive sites and tf binding sites identified by encode . we found that about 55% of the dmrs overlapped with tf binding sites and 82% overlapped with dnasei hypersensitive sites ( supplementary figs . 4 and 5 ) . overall , the above findings support the fact that differential methylation plays a role in gene regulation , particularly in the differential expression of genes . we compared the asm events we found with a previous study that studied methylation patterns within the hapmap sample na12878 using reduced - representation bisulfite sequencing ( rrbs ) . we discovered substantially more asm events ( 216,034 , compared to 2,998 ) than were previously found using rrbs , a method that targets only gc - enriched regions . since methylc - seq can detect dna methylation in the whole genome while rrbs only detect dna methylation in gc- enriched regions , our results suggest the prevalence of asm events outside of cpg islands captured by rrbs technology . to our surprise , although 326 cytosines that were identified as asm in the rrbs study also passed the criteria for testing in our study , only 96 were significantly ( p<0.05 , fisher 's exact test ) differentially methylated between the two alleles . we suspect the rrbs technology may introduce high bias from the amplification that leads to high false positive rates . both pcr and the nextera transposase introduce errors in the haplotyping process ; we assessed the significance of these errors by running prism on a high - quality synthetic data set obtained by sampling 7 kbp reads uniformly at random from the trio - phased genome of na12878 ( online methods ) . analysis of the synthetic data resulted in more complete haplotypes with a 0.4% higher snv phasing rate . a further analysis of pcr amplification bias ( online methods ) suggested that some areas of the genome exhibit a systematically lower amplification rate , and are covered by fewer long fragments . the long - range switch accuracy on both datasets was similar , but the short switch accuracy was much higher on the synthetic dataset . this suggests that pcr and nextera mainly introduce gaps in the phased haplotypes as well as point errors at individual variants ; however , their impact on long - rage phase information appears to be small . the wealth of information one can obtain from a haplotype - resolved genome promises new advances in both biology and medicine . compared with existing dilution haplotyping methods , slrh produces haplotypes of equal or greater quality using substantially less sequencing effort ( supplementary table 1 ) . whereas existing methods require from 110 gbp to 496 gbp of sequencing , slrh requires as little as 30 gbp . moreover , our method phases up to 99% of all snvs , whereas others exhibit phasing rates of at most 97% , and typically less than 95% . slrh haplotypes also retain long - range phase information , with n50 lengths of 450560 kbp ; alternative methods have n50 lengths from 350 kbp to 600kbp . notably , slrh achieves this performance without sacrificing accuracy : long - range switching events occur less than once per mbp on average ( 99.9099.92% long switch accuracy ) . for applications demanding an even higher level of precision , slrh provides confidence scores that may be used to trade - off haplotype completeness for increased accuracy . at the most stringent thresholds , the method yields short and highly accurate regions that may be valuable in clinical applications the two components of slrh that enable these advances are a low - bias pcr - based amplification step , and the prism statistical phasing algorithm , which compensates for having relatively little input data . the two components naturally complement each other : although long fragments can not span across long regions of low heterozygosity , such regions typically exhibit high linkage disequilibrium , and are more amenable to statistical phasing . the limitations of slrh include the need to use a compute cluster for statistical phasing , and a lower phasing accuracy in statistically - assembled regions ( supplementary table 13 ) . the statistical component of slrh also can not be applied to species other than human due to the lack of a suitable reference panel . however , we expect that the molecular component can be applied to species with genomes of at least 100 mbp , which are large enough for long fragments to be sufficiently diluted . finally , compared to a recently introduced proximity ligation - based method ( haploseq ) , our approach produces shorter , but more complete haplotype blocks . while haploseq phases 81% of snvs in a human genome , slrh phases 99% . the errors of haploseq mostly affect individual positions without altering the global haplotype structure ; slrh produces much fewer errors , but some of them may introduce long - range switching events . overall , the two methods appear to have complementary strengths and weaknesses . as an example of the scientific studies that are made possible by slrh , we determined the allele - specific methylation patterns across a phased human genome . we observed many methylation events , and found that the drms are often associated with cis - regulatory regions . in previous studies , differential methylation patterns were determined either by purely statistical methods or from mendelian inheritance rules . such methods may be inaccurate and may not scale to large studies owing to the need to sequence the parents of every subject . here , we were able to reproduce the work of previous studies ? without relying on parental information or large amounts of sequencing . besides differential methylation studies , haplotype information has applications in many areas of genomics , including : ( i ) the analysis of disorders affected by compound heterozygocity , such as blistering skin , cerebral palsy , deafness and others ; ( ii ) population genetics , where population - specific haplotype blocks are currently resolved using lower - accuracy statistical methods ; ( iii ) the detection of structural variations , which has been shown to benefit from phase information ; ( iv ) the matching of hosts and donors in organ transplantation based on the hla region of the genome ; ( v ) the evolution of genomes across species . the wide range of these fields highlights the importance of phase information in human genetics . tools that facilitate access to this information such as ones we presented here will lay the foundation for further advances throughout genomics .

rapid growth of sequencing technologies has greatly contributed to increasing our understanding of human genetics . yet , in spite of this growth , mainstream technologies have been largely unsuccessful in resolving the diploid nature of the human genome . here we describe statistically aided long read haplotyping ( slrh ) , a rapid , accurate method based on a simple experimental protocol that requires potentially as little as 30 gbp of sequencing in addition to a standard ( 50x coverage ) whole - genome analysis for human samples . using this technology , we phase 99% of single - nucleotide variants in three human genomes into long haplotype blocks of 200 kbp to 1 mbp in length . as a demonstration of the potential applications of our method , we determine allele - specific methylation patterns in a human genome and identify hundreds of differentially methylated regions that were previously unknown . such information may offer insight into the mechanisms behind differential gene expression .

Results Statistically aided long read haplotyping Phasing three genomes using SLRH Whole-genome phasing from 30 Gbp of sequencing Determining allele-specific DNA methylation Effects of PCR and Nextera on haplotyping performance Discussion Supplementary Material

essentially , slrh is a form of dilution haplotyping similar to previous approaches based on fosmid clones or long fragment reads ( lfr ) . each pool has a unique barcode that identifies its fragments , which are then recovered from short - read sequences and assembled into long haplotype blocks using a phasing algorithm ( fig . fragments are then diluted into a 384-well plate containing 3,0006,000 molecules per well and pcr - amplified using adapter - specific primers ( supplementary fig . a single phasing library typically corresponds to about 30 gbp of 101-base , paired - end illumina reads . the sequenced reads are then aligned to the reference genome and mapped back to their original wells as specified by the barcode adapters . mapped reads within each variants in each fragment are called based on the subject 's whole - genome genotyping ; in our experiments , the subject 's genotypes were determined from a 50x read coverage on the illumina platform ( online methods ) . at least half of all sequenced bases are within fragments of at least 7 - 9kbp ) and cover the genome to a depth of about 48x ( supplementary tables 3 and 4 ) . 1b and supplementary data ) , which augments dilution haplotyping with statistical techniques . in brief , first , in a local_stage , it assembles fragments into haplotype blocks by connecting them together at their overlapping heterozygous snvs . then , in a global stage , prism exploits linkage disequilibrium patterns to assemble local blocks into long and accurate global haplotype contigs . such contigs can phase up to 99% of heterozygous snvs and up to 95% of heterozygous variants . the global phasing stage is an essential component of the slrh pipeline . simply connecting long dna fragments at heterozygous snvs results in local blocks of about 60 kbp in length ( supplementary the global phasing performed by prism increases the lengths of these blocks by more than ten - fold ; it also increases snv phasing rates from 94% to more than 99% . at a high level , the hmm tries to represent the partially phased haplotypes of the sample as an imperfect mosaic of haplotypes from a pre - phased reference panel from the 1000 genomes project . confidence scores between adjacent blocks are derived from the forwards - backwards variables of the hidden markov model . unlike existing packages , our method is able to use partially phased information obtained from long reads , which allows us to achieve greater accuracy than traditional statistical methods . as a demonstration of the ability of slrh to accurately phase human genomes , we prepared libraries for three hapmap samples : na12878 , na12891 and na12892 ( supplementary table 2 ) . depending on the threshold , the n50 lengths of haplotype blocks varied between 130 and 750 kbp ; the percentage of snvs phased varied between 96.0% and 99.4% ( fig . in particular , at the 0.9 confidence score threshold , about 99% of all snvs were phased in blocks with n50 lengths of 560650 kbp ( supplementary tables 7 , 8) . about 89% of 23,645 genes derived from the ucsc dataset were fully contained in a single haplotype block ( table 2 and supplementary table 9 ) . the haplotype blocks also contained more than 73% of novel snvs and indels ( supplementary table 9 ) , although the accuracy over these variants was lower than average , partly because of the increased difficulty in genotyping such rare variants . the majority of phased genes contained compound heterozygous snvs ( about 75% of all genes with snvs ) ; hla - c , a gene whose haplotypes are used to predict immune response during organ transplantation is an example of a compound heterozygous gene ( supplementary fig . phased genes also contained about 2,500 snvs that were found to be damaging by the sift software package . to assess the accuracy of slrh , we compared the above haplotypes to ones derived from applying mendelian inheritance rules to our three samples . even the long statistically assembled haplotypes obtained from a 0.5 probability threshold were highly accurate : for sample na12878 , slrh produced long blocks with n50 lengths of 1.1 mbp that contained long switching events at an average rate of 0.85 per mbp ( supplementary tables 5 , 6 ) . at a small number of positions , we observed short ( one - base ) discordances with parental haplotyping . this affected about 0.15% of heterozygous positions ( supplementary tables 5 , 6 ) , which were often associated with centromeres and copy number variations . finally , the absolute accuracy of the haplotype blocks ( i.e. next , to demonstrate the low sequencing requirements of slrh , we ran the prism algorithm separately on each individual phasing library . we observed only a small loss of haplotyping performance , highlighting the robustness of our statistically aided approach . overall , we obtained haplotype blocks that were almost as accurate and only 100 kbp shorter than ones derived from 60 gbp of sequencing ( fig . long switching events always occurred at rates below one switch per mbp , and at the 0.9 threshold , we measured 0.50.8 switches per mbp . this corresponds to accuracies of 99.8799.90% , a drop of only about 0.01% with respect to two phasing libraries per sample . finally , results for the two replicate libraries of the hapmap sample na12878 were highly concordant ( fig . next , as a demonstration of scientific applications based on the phased genome made possible by slrh , we performed an analysis of differential dna methylation across the genome of the hapmap sample na12878 ( lymphoblastoid cell line gm12878 ) based on its haplotype information . we thus obtained a detailed map of allele - specific methylation patterns within a human genome ; such maps are useful for understanding biological mechanisms such as genomic imprinting . in brief , we performed methylc - seq experiment on gm12878 cell line and assigned methylated short reads to their closest haplotype . with the reads coverage and bisulfite conversion rates on both alleles , we then quantified allele - specific dna methylation ( asm ) using fisher 's exact test ( online methods ) . we found 216,034 statistically significant asm events that clustered in 992 differentially methylated regions ( dmrs ) ranging in size from 6 to 3,181 bp ( median size 190 bp , online methods ) . ten of the dmrs were located at previously studied areas of the genome , such as in the upstream region of the h19 gene ( fig . to gather more insight into how differential methylation may affect gene expression , we determined the overlap between the dmrs and transcription start sites ( tsss ) , transcription end sites ( tess ) , exons and intergenic regions defined by genecode v14 . about 20% of the dmrs were located at gene tsss , and an additional 42% were located within annotated genes ( which include tess , introns and exons ) ; the remaining 38% were found at distal intergenic regions ( supplementary fig . we further explored the regulatory role of the majority of dmrs that are not in gene promoters by assessing the overlap between the dmrs and dnase i hypersensitive sites and tf binding sites identified by encode . we found that about 55% of the dmrs overlapped with tf binding sites and 82% overlapped with dnasei hypersensitive sites ( supplementary figs . we compared the asm events we found with a previous study that studied methylation patterns within the hapmap sample na12878 using reduced - representation bisulfite sequencing ( rrbs ) . we discovered substantially more asm events ( 216,034 , compared to 2,998 ) than were previously found using rrbs , a method that targets only gc - enriched regions . to our surprise , although 326 cytosines that were identified as asm in the rrbs study also passed the criteria for testing in our study , only 96 were significantly ( p<0.05 , fisher 's exact test ) differentially methylated between the two alleles . both pcr and the nextera transposase introduce errors in the haplotyping process ; we assessed the significance of these errors by running prism on a high - quality synthetic data set obtained by sampling 7 kbp reads uniformly at random from the trio - phased genome of na12878 ( online methods ) . analysis of the synthetic data resulted in more complete haplotypes with a 0.4% higher snv phasing rate . a further analysis of pcr amplification bias ( online methods ) suggested that some areas of the genome exhibit a systematically lower amplification rate , and are covered by fewer long fragments . essentially , slrh is a form of dilution haplotyping similar to previous approaches based on fosmid clones or long fragment reads ( lfr ) . each pool has a unique barcode that identifies its fragments , which are then recovered from short - read sequences and assembled into long haplotype blocks using a phasing algorithm ( fig . fragments are then diluted into a 384-well plate containing 3,0006,000 molecules per well and pcr - amplified using adapter - specific primers ( supplementary fig . a single phasing library typically corresponds to about 30 gbp of 101-base , paired - end illumina reads . the sequenced reads are then aligned to the reference genome and mapped back to their original wells as specified by the barcode adapters . mapped reads within each variants in each fragment are called based on the subject 's whole - genome genotyping ; in our experiments , the subject 's genotypes were determined from a 50x read coverage on the illumina platform ( online methods ) . at least half of all sequenced bases are within fragments of at least 7 - 9kbp ) and cover the genome to a depth of about 48x ( supplementary tables 3 and 4 ) . 1b and supplementary data ) , which augments dilution haplotyping with statistical techniques . in brief , first , in a local_stage , it assembles fragments into haplotype blocks by connecting them together at their overlapping heterozygous snvs . then , in a global stage , prism exploits linkage disequilibrium patterns to assemble local blocks into long and accurate global haplotype contigs . such contigs can phase up to 99% of heterozygous snvs and up to 95% of heterozygous variants . the global phasing stage is an essential component of the slrh pipeline . simply connecting long dna fragments at heterozygous snvs results in local blocks of about 60 kbp in length ( supplementary the global phasing performed by prism increases the lengths of these blocks by more than ten - fold ; it also increases snv phasing rates from 94% to more than 99% . at a high level , the hmm tries to represent the partially phased haplotypes of the sample as an imperfect mosaic of haplotypes from a pre - phased reference panel from the 1000 genomes project . confidence scores between adjacent blocks are derived from the forwards - backwards variables of the hidden markov model . unlike existing packages , our method is able to use partially phased information obtained from long reads , which allows us to achieve greater accuracy than traditional statistical methods . as a demonstration of the ability of slrh to accurately phase human genomes , we prepared libraries for three hapmap samples : na12878 , na12891 and na12892 ( supplementary table 2 ) . depending on the threshold , the n50 lengths of haplotype blocks varied between 130 and 750 kbp ; the percentage of snvs phased varied between 96.0% and 99.4% ( fig . in particular , at the 0.9 confidence score threshold , about 99% of all snvs were phased in blocks with n50 lengths of 560650 kbp ( supplementary tables 7 , 8) . about 89% of 23,645 genes derived from the ucsc dataset were fully contained in a single haplotype block ( table 2 and supplementary table 9 ) . the haplotype blocks also contained more than 73% of novel snvs and indels ( supplementary table 9 ) , although the accuracy over these variants was lower than average , partly because of the increased difficulty in genotyping such rare variants . the majority of phased genes contained compound heterozygous snvs ( about 75% of all genes with snvs ) ; hla - c , a gene whose haplotypes are used to predict immune response during organ transplantation is an example of a compound heterozygous gene ( supplementary fig . phased genes also contained about 2,500 snvs that were found to be damaging by the sift software package . to assess the accuracy of slrh , we compared the above haplotypes to ones derived from applying mendelian inheritance rules to our three samples . even the long statistically assembled haplotypes obtained from a 0.5 probability threshold were highly accurate : for sample na12878 , slrh produced long blocks with n50 lengths of 1.1 mbp that contained long switching events at an average rate of 0.85 per mbp ( supplementary tables 5 , 6 ) . at a small number of positions , we observed short ( one - base ) discordances with parental haplotyping . this affected about 0.15% of heterozygous positions ( supplementary tables 5 , 6 ) , which were often associated with centromeres and copy number variations . finally , the absolute accuracy of the haplotype blocks ( i.e. next , to demonstrate the low sequencing requirements of slrh , we ran the prism algorithm separately on each individual phasing library . we observed only a small loss of haplotyping performance , highlighting the robustness of our statistically aided approach . overall , we obtained haplotype blocks that were almost as accurate and only 100 kbp shorter than ones derived from 60 gbp of sequencing ( fig . long switching events always occurred at rates below one switch per mbp , and at the 0.9 threshold , we measured 0.50.8 switches per mbp . this corresponds to accuracies of 99.8799.90% , a drop of only about 0.01% with respect to two phasing libraries per sample . finally , results for the two replicate libraries of the hapmap sample na12878 were highly concordant ( fig . next , as a demonstration of scientific applications based on the phased genome made possible by slrh , we performed an analysis of differential dna methylation across the genome of the hapmap sample na12878 ( lymphoblastoid cell line gm12878 ) based on its haplotype information . we thus obtained a detailed map of allele - specific methylation patterns within a human genome ; such maps are useful for understanding biological mechanisms such as genomic imprinting . in brief , we performed methylc - seq experiment on gm12878 cell line and assigned methylated short reads to their closest haplotype . with the reads coverage and bisulfite conversion rates on both alleles , we then quantified allele - specific dna methylation ( asm ) using fisher 's exact test ( online methods ) . we found 216,034 statistically significant asm events that clustered in 992 differentially methylated regions ( dmrs ) ranging in size from 6 to 3,181 bp ( median size 190 bp , online methods ) . ten of the dmrs were located at previously studied areas of the genome , such as in the upstream region of the h19 gene ( fig . to gather more insight into how differential methylation may affect gene expression , we determined the overlap between the dmrs and transcription start sites ( tsss ) , transcription end sites ( tess ) , exons and intergenic regions defined by genecode v14 . about 20% of the dmrs were located at gene tsss , and an additional 42% were located within annotated genes ( which include tess , introns and exons ) ; the remaining 38% were found at distal intergenic regions ( supplementary fig . we further explored the regulatory role of the majority of dmrs that are not in gene promoters by assessing the overlap between the dmrs and dnase i hypersensitive sites and tf binding sites identified by encode . we found that about 55% of the dmrs overlapped with tf binding sites and 82% overlapped with dnasei hypersensitive sites ( supplementary figs . we compared the asm events we found with a previous study that studied methylation patterns within the hapmap sample na12878 using reduced - representation bisulfite sequencing ( rrbs ) . we discovered substantially more asm events ( 216,034 , compared to 2,998 ) than were previously found using rrbs , a method that targets only gc - enriched regions . to our surprise , although 326 cytosines that were identified as asm in the rrbs study also passed the criteria for testing in our study , only 96 were significantly ( p<0.05 , fisher 's exact test ) differentially methylated between the two alleles . both pcr and the nextera transposase introduce errors in the haplotyping process ; we assessed the significance of these errors by running prism on a high - quality synthetic data set obtained by sampling 7 kbp reads uniformly at random from the trio - phased genome of na12878 ( online methods ) . analysis of the synthetic data resulted in more complete haplotypes with a 0.4% higher snv phasing rate . a further analysis of pcr amplification bias ( online methods ) suggested that some areas of the genome exhibit a systematically lower amplification rate , and are covered by fewer long fragments . whereas existing methods require from 110 gbp to 496 gbp of sequencing , slrh requires as little as 30 gbp . moreover , our method phases up to 99% of all snvs , whereas others exhibit phasing rates of at most 97% , and typically less than 95% . slrh haplotypes also retain long - range phase information , with n50 lengths of 450560 kbp ; alternative methods have n50 lengths from 350 kbp to 600kbp . at the most stringent thresholds , the method yields short and highly accurate regions that may be valuable in clinical applications the two components of slrh that enable these advances are a low - bias pcr - based amplification step , and the prism statistical phasing algorithm , which compensates for having relatively little input data . however , we expect that the molecular component can be applied to species with genomes of at least 100 mbp , which are large enough for long fragments to be sufficiently diluted . finally , compared to a recently introduced proximity ligation - based method ( haploseq ) , our approach produces shorter , but more complete haplotype blocks . while haploseq phases 81% of snvs in a human genome , slrh phases 99% . as an example of the scientific studies that are made possible by slrh , we determined the allele - specific methylation patterns across a phased human genome . in previous studies , differential methylation patterns were determined either by purely statistical methods or from mendelian inheritance rules . here , we were able to reproduce the work of previous studies ? without relying on parental information or large amounts of sequencing . besides differential methylation studies , haplotype information has applications in many areas of genomics , including : ( i ) the analysis of disorders affected by compound heterozygocity , such as blistering skin , cerebral palsy , deafness and others ; ( ii ) population genetics , where population - specific haplotype blocks are currently resolved using lower - accuracy statistical methods ; ( iii ) the detection of structural variations , which has been shown to benefit from phase information ; ( iv ) the matching of hosts and donors in organ transplantation based on the hla region of the genome ; ( v ) the evolution of genomes across species . the wide range of these fields highlights the importance of phase information in human genetics .

global estimates indicate that there are 15.9 million ( range 11.0 - 21.2 million ) people who inject drugs ( pwid ) across 151 countries . of these , furthermore , it is estimated that 10 per cent of all new hiv infections can be attributed to injecting drugs , rising to 30 per cent when sub - saharan africa is excluded . within what the world health organization ( who ) define as their south - east asia region ( sear ) there are an estimated 400,000 - 500,000 pwid , primarily located in countries that experience a high or medium burden of illicit drug injecting ( who office for sear covers 11 countries : bangladesh , bhutan , dpr korea , india , indonesia , maldives , myanmar , nepal , sri lanka , thailand and timor - leste ) . data from several sources show that high risk behaviours among pwid are common and provision of comprehensive hiv prevention services varies ( table 1 ) . hiv prevalence among pwid in south - east asia was commonly identified at 20 - 25% and has remained consistently high in indonesia , nepal , thailand , myanmar and some regions of india . whilst a decline was recorded in hiv prevalence among nepalese pwid , from 68% in 2002 to 21% in 2009 and 6% in 2011 , hiv prevalence rose in bangladeshi pwid , from 4% in 2003 - 2004 to 7% in 2007 , and in punjab ( india ) from 13% in 2006 to 21% in 2011 . high - risk behaviours among pwid , such as the sharing of contaminated needles and syringes , remain a major determining factor in the course of the hiv epidemic throughout asia . the ongoing hiv epidemic among pwid in south - east asia has prompted many countries to develop hiv policies linked to national hiv strategic plans in which harm reduction interventions are increasingly viewed with understanding and acceptance . unaids undoc and who outline nine interventions as part of a comprehensive package that has the greatest impact on hiv prevention , treatment and care for pwid . meta - analysis and systematic reviews show conclusively that providing opioid substitution therapy ( ost ) together with needle and syringe programmes and antiretroviral treatment targeted towards pwid have the greatest effect in preventing hiv infection . in this article the focus is upon one essential harm reduction intervention , ost , and its use in countries that fall under the administrative purview of who s sear , with specific focus on bangladesh , india , indonesia , maldives , myanmar , nepal and thailand . we excluded timor - leste , bhutan and the democratic people s republic of korea due to limited data . methadone has been prescribed for the treatment of opioid dependency for over four decades and buprenorphine has been available since the early 2000s . both were added to the who list of essential medicines in 2005 , and are usually consumed orally or sublingually as maintenance treatment to relieve opioid withdrawal and to reduce cravings . research has shown them to be effective in the reduction of hiv risk behaviours and other comorbidities . due to the chronic nature of opioid dependency , treatment outcomes are better in long - term therapy incorporating adequate dosing ( at least 60 mg of methadone and 8 - 6 mg buprenorphine per day ) . despite the strong evidence for various benefits resulting from the use of ost improved retention in treatment for hiv , hcv and opioid use , reduced illicit opioid use , reduced risk of hiv , reduced criminality , better psychosocial outcomes and decreased risk to pregnant drug - dependent women ( during the intra - uterine , partum and postpartum period ) fewer than three per cent of pwid in the south - east asia region have access to ost . in this article we explore the historical adoption of ost , the current situation , some of the major challenges , and suggest ways to scale up ost in the sear . we reviewed published and grey literature located using medline and pubmed , who program documents for the countries of the sear , the joint united nations programme on hiv / aids ( unaids ) , the united nations office on drugs and crime ( unodc ) documents , and generic internet search engines between october 2011 and june 2013 . direct email requests were made to who hiv country focal points and official government counterparts for additional data and comments about ost in the sear between september 2010 and march 2013 . the amount and quality of the data available on the commencement of ost programmes , responsible agencies , the number of pwid receiving ost , and the linkages with other health and non - health systems vary considerably among the countries in south - east asia ( table 2 ) . nepal started a government - approved methadone - based ost programme in 1994 . despite satisfactory outcomes , the programme was suspended in 1998 , and then restarted as an emergency response for hiv prevention in september 2007 . in india , sublingual buprenorphine treatment commenced in 1993 as a pilot project and has been licensed for treatment of opioid dependence in drug treatment centres since 1999 . limited methadone treatment became available in 1979 , primarily as a detoxification program in approved clinics . in 1989 a national clinical trial of mmt was initiated . mmt started in bangkok as an hiv prevention measure in 1990 , and gradually extended to a few clinics outside the capital . buprenorphine was approved for opioid treatment in 2007 , and a trial of buprenorphine - naloxone was initiated in chiang mai during the same year , but it is not routinely used in opioid treatment programs . buprenorphine was introduced in indonesia in 2001 , and the manufacturers of the drug encouraged private practitioners to prescribe the medication to drug - dependent clients . in 2003 the indonesian ministry of health established mmt ( with technical and financial assistance from who ) in two pilot projects , one in jakarta and one in a joint pilot within a bali prison and a hospital . these two pilots continued until the end of 2005 , serving a population of approximately 300 drug users . preparation for the introduction of methadone began in myanmar in 2004 , with the ministry of health commencing the delivery of mmt over february and march 2006 in four drug treatment centres . around 2900 clients were receiving mmt by 2013 . in november 2008 , mmt was introduced into the maldives as part of a pilot study ; buprenorphine remained unavailable due to its classification as an illegal substance . in nepal , as of june 2011 , 380 clients were receiving methadone from three sites in the cities of kathmandu , patan and pokhara ( kramarz p. , 2011 ; personal communication ) . the demand for methadone was reported to be high , with a long waiting list to join the programme . in pokhara , 88 pwid were enrolled to receive methadone and 62 were active recipients . in 2009 , 30 nepalese pwid received buprenorphine administered as part of a three - month tapering reduction dose rather than long - term maintenance therapy . at the end of 2010 , ngos were dispensing buprenorphine from six sites to 175 clients . the three - month duration of treatment of 2 - 4 mg per day remained unchanged . take - home / home delivery of buprenorphine was only granted under exceptional circumstances ( pandey b. , 2010 ; personal communication ) . the provision of buprenorphine is not currently an official part of the government ost programme . initial indian targets were for 40,000 pwid to receive ost by 2012 , with delivery through 320 sites , primarily ngos or government - run treatment centres . by may 2011 , buprenorphine was available from 52 sites and 4810 clients were in treatment ; data from december 2012 show that 120 buprenorphine sites were delivering treatment to 7500 regular patients ( in india regular patients are defined as those accessing ost 24 days or more per month ; those that received less frequent treatment are not included ) . apart from small pilot studies methadone has not been available for clinical use in india following its removal from the indian pharmacopoeia in 1982 . however , in early 2010 the government approved the use of methadone in a pilot study intended to involve six sites in late 2010 . by 2012 four methadone dispensing sites were servicing 350 patients ( kumar r. 2013 ; personal communication ) . a buprenorphine project commenced inside tihar prison , new delhi in late 2008 , but client numbers remain small . in 2009 the bangkok metropolitan administration funded 20 methadone clinics at two hospitals and 18 health centres , and across thailand an estimated 4000 - 5000 patients were receiving methadone . in 2010 , 2200 patients received methadone treatment , but probable duplication of some client records makes this number uncertain , and whether they were receiving long - term treatment is undetermined ( thanpresertsuk s. , 2011 ; personal communication ) . the reasons for the apparent fall in client numbers between 2009 and 2010 are unknown ( the 2009 figures are estimates only ) , although the general shift from opioid use to stimulant use in thailand is likely to have contributed . in 2012 methadone is the national health security office s approved ost medication and was placed on the thai essential drug list in 2008 ; it is available as part of the universal health coverage package for all thai citizens . in 2009 , an estimated 150 clients were reported to be receiving buprenorphine as part of an hiv prevention trial at chiang mai university , which has since been completed . in indonesia , methadone expansion began in 2006 with seven clinics serving 1000 clients by the end of that year . by april 2010 methadone was available in ten provinces from 51 clinics ( five specialist hospitals ; 10 general hospitals ; 30 primary health centres ; and six prison and detention centres ) ( sarasvita r. , 2010 ; personal communication ) . in june 2011 , indonesia had 68 government - funded clinics with 2540 active patients . in addition , more than 50 medical prescribers in private practices were dispensing buprenorphine to an estimated 3000 patients . in myanmar in january 2010 , 821 patients were receiving methadone from eight sites in four states , and by june 2011 patient numbers had increased to 1087 . in late 2012 methadone was dispensed from drug treatment centres as part of global fund round . in the maldives , 47 patients had been enrolled to receive methadone in late 2009 , but by april 2010 only 17 patients remained , decreasing to 11 by august 2010 ( mohamed a.j . , 2010 ; personal communication ) ; by july 2011 patient numbers had rebounded to 57 ( aminath z. , 2011 ; personal communication ) . barriers to methadone programme enrolment in the maldives include the need to obtain clearance from five different government departments to ensure no criminal cases are pending . it is reported that the methadone programme functions well , but scope for improvement remains . in july 2010 , after several years of advocacy to initiate ost , bangladesh commence methadone provision , starting with one site in dhaka . by early 2013 an additional site had opened and 188 clients were receiving methadone ( azim t. , 2013 ; personal communication ) . various common challenges and barriers exist to the implementation and expansion of ost services . throughout the region drug users continue to experience high levels of stigma and discrimination due to their substance use . this impacts upon the delivery of general health services , including access to ost . in the uk it has been noted that key government policies to assist pwid reintegration and recovery through the use of ost were highly unlikely to succeed while stigmatising attitudes towards pwid remain pervasive . stigma and discrimination remain major barriers across south - east asia . acknowledging drug use and responding to hiv risk - taking behaviour was largely delayed across asia in comparison to many western countries , and the criminalisation of drug use still commonly overshadows a public health response to address this problem . official reports state that 235,000 people were detained in over 1000 compulsory drug detention centres in east and south east asia alone in 2012 . since 2012 , many united nation agencies have called for their closure for reasons including human rights abuses and a lack of evidence of their effectiveness . as found in other regions of the world , the attitude that the abstinence model of drug treatment is of paramount importance persists and rejection of the concept of ost maintenance is widespread , despite opioid dependence being acknowledged as a chronic medical condition . as a result , it could be argued that this has been the major reason for reduced political will , financial under - investment and lack of commitment toward the scaling up of essential harm reduction interventions , including ost . advocacy for and funding of ost scale - up in the countries under review remains largely driven by un agencies and the international donor community . initial funding of and sustainable investment in harm reduction services , including ost , remains a perennial problem . recent global studies have revealed over 90% of hiv - related harm reduction funds in low and middle income countries stem from international donors , and total funding remains inadequate . the cost of ost for individual recipients is prohibitive in many cases , and this greatly reduces the numbers of pwid in treatment . in indonesia , while methadone could cost a client 1 - 2 ud dollars ( $ ) per dose through a government service , an 8 - 12 mg dose of buprenorphine commonly costs 11 - 17 $ through a private medical practitioner . as a result , many pwid can only afford to purchase an inadequate dose that proves ineffective and contributes towards ongoing injecting of illicit drugs . despite these problems , it is important to highlight progressive steps from asian countries such as malaysia and china , where governments strongly endorse ost and are expanding the ost programme nationwide . by 2009 , china had 680 methadone clinics in 27 provinces with 242,000 patients ( ever ) enrolled , while malaysia had 59 government facilities and nine private clinics dispensing methadone to 7065 registered clients , with an additional 10,000 clients receiving methadone through private practitioners . in 2011 malaysia further expanded its methadone programme , with substantially more distribution sites : 168 public health clinics , 48 public hospitals , 24 general practitioners ( gps ) , 32 national anti - drug agency sites , 18 prisons and two others . a cumulative 20,955 pwid have been registered in public facilities since 2006 and another 23,573 pwids are receiving mmt from gps . due to the separate registration systems of private and public services , it is not possible to identify whether patients receiving methadone from private gps were also registered with public facilities ( naning h. , 2012 ; personal communication ) . in all countries under review , the focus remains largely on the traditional treatment model of detoxification and rehabilitation despite strong evidence of recidivism , with rates often ranging from 60 - 100% . despite the international evidence highlighting the broadranging health , social and economic benefits of ost , a lack of indepth knowledge , awareness and education about ost among policymakers , community members , drug users and the health sector constrains the scaling up of ost . as found worldwide , one of the greatest obstacles to ost acceptance in the sear is limited acceptance and practice of evidence - based medicine associated with ost in the medical workforce . research has shown that it is commonly perceived that providing ost is simply replacing one drug with another , despite evidence that methadone is superior to non - pharmacological methods in terms of client retention in treatment , reduced self - reported heroin use and both urine and hair analysis . in some parts of the world , including in some asian countries , providing ost can be viewed as a failure to deal with the criminal behaviour of drug users . consequently , the implementation of an abstinence - based model of treatment is still strongly advocated as the primary approach to address drug dependency . in addition , tensions continue between supply / demand reduction and harm reduction approaches , and impacts upon expansion of ost services . highlighting an effective ost treatment model that builds upon local evidence can contribute towards expansion of drug treatment services for pwid . one aspect of effectiveness is to achieve an optimal dose of ost ( usually greater than 60 mg of methadone ) that can lead to a decrease in the use of illicit drugs and ensure a higher retention rate and decrease in hiv risk behaviours . however , it is not uncommon practice for a lower dose to be prescribed for clients receiving ost in south - east asia . for example , in indonesia an integrated biological survey among pwid noted that most of those who received methadone in the past year were still injecting illicit opioids in the past week , and it was speculated that one reason for this was inadequate dosage . doses of methadone of more than 60 mg daily are recommended , with most clients requiring 60 - 120 mg daily . in some countries under review , recent buprenorphine guidelines in india ( 2009 ) recommend 4 - 8 mg as the optimal buprenorphine dose , despite international evidence recommending that people be stabilised on 8 - 16 mg daily . overall poor monitoring and evaluation of ost programmes exists in the countries under review and this does not allow local experiences to be translated into coherently managed knowledge about a program . as a consequence this impacts upon seeking ways to improve and expand coverage of the ost service . the amount and quality of the data available on the commencement of ost programmes , responsible agencies , the number of pwid receiving ost , and the linkages with other health and non - health systems vary considerably among the countries in south - east asia ( table 2 ) . nepal started a government - approved methadone - based ost programme in 1994 . despite satisfactory outcomes , the programme was suspended in 1998 , and then restarted as an emergency response for hiv prevention in september 2007 . in india , sublingual buprenorphine treatment commenced in 1993 as a pilot project and has been licensed for treatment of opioid dependence in drug treatment centres since 1999 . limited methadone treatment became available in 1979 , primarily as a detoxification program in approved clinics . in 1989 a national clinical trial of mmt was initiated . mmt started in bangkok as an hiv prevention measure in 1990 , and gradually extended to a few clinics outside the capital . buprenorphine was approved for opioid treatment in 2007 , and a trial of buprenorphine - naloxone was initiated in chiang mai during the same year , but it is not routinely used in opioid treatment programs . buprenorphine was introduced in indonesia in 2001 , and the manufacturers of the drug encouraged private practitioners to prescribe the medication to drug - dependent clients . in 2003 the indonesian ministry of health established mmt ( with technical and financial assistance from who ) in two pilot projects , one in jakarta and one in a joint pilot within a bali prison and a hospital . these two pilots continued until the end of 2005 , serving a population of approximately 300 drug users . preparation for the introduction of methadone began in myanmar in 2004 , with the ministry of health commencing the delivery of mmt over february and march 2006 in four drug treatment centres . around 2900 clients were receiving mmt by 2013 . in november 2008 , mmt was introduced into the maldives as part of a pilot study ; buprenorphine remained unavailable due to its classification as an illegal substance . in nepal , as of june 2011 , 380 clients were receiving methadone from three sites in the cities of kathmandu , patan and pokhara ( kramarz p. , 2011 was reported to be high , with a long waiting list to join the programme . in pokhara , 88 pwid were enrolled to receive methadone and 62 were active recipients . in 2009 , 30 nepalese pwid received buprenorphine administered as part of a three - month tapering reduction dose rather than long - term maintenance therapy . at the end of 2010 , ngos were dispensing buprenorphine from six sites to 175 clients . the three - month duration of treatment of 2 - 4 mg per day remained unchanged . take - home / home delivery of buprenorphine was only granted under exceptional circumstances ( pandey b. , 2010 ; personal communication ) . the provision of buprenorphine is not currently an official part of the government ost programme . initial indian targets were for 40,000 pwid to receive ost by 2012 , with delivery through 320 sites , primarily ngos or government - run treatment centres . by may 2011 , buprenorphine was available from 52 sites and 4810 clients were in treatment ; data from december 2012 show that 120 buprenorphine sites were delivering treatment to 7500 regular patients ( in india regular patients are defined as those accessing ost 24 days or more per month ; those that received less frequent treatment are not included ) . apart from small pilot studies methadone has not been available for clinical use in india following its removal from the indian pharmacopoeia in 1982 . however , in early 2010 the government approved the use of methadone in a pilot study intended to involve six sites in late 2010 . by 2012 four methadone dispensing sites were servicing 350 patients ( kumar r. 2013 ; personal communication ) . a buprenorphine project commenced inside tihar prison , new delhi in late 2008 , but client numbers remain small . in 2009 the bangkok metropolitan administration funded 20 methadone clinics at two hospitals and 18 health centres , and across thailand an estimated 4000 - 5000 patients were receiving methadone . in 2010 , 2200 patients received methadone treatment , but probable duplication of some client records makes this number uncertain , and whether they were receiving long - term treatment is undetermined ( thanpresertsuk s. , 2011 ; personal communication ) . the reasons for the apparent fall in client numbers between 2009 and 2010 are unknown ( the 2009 figures are estimates only ) , although the general shift from opioid use to stimulant use in thailand is likely to have contributed . in 2012 methadone is the national health security office s approved ost medication and was placed on the thai essential drug list in 2008 ; it is available as part of the universal health coverage package for all thai citizens . in 2009 , an estimated 150 clients were reported to be receiving buprenorphine as part of an hiv prevention trial at chiang mai university , which has since been completed . in indonesia , methadone expansion began in 2006 with seven clinics serving 1000 clients by the end of that year . by april 2010 methadone was available in ten provinces from 51 clinics ( five specialist hospitals ; 10 general hospitals ; 30 primary health centres ; and six prison and detention centres ) ( sarasvita r. , 2010 ; personal communication ) . in june 2011 in addition , more than 50 medical prescribers in private practices were dispensing buprenorphine to an estimated 3000 patients . in myanmar in january 2010 , 821 patients were receiving methadone from eight sites in four states , and by june 2011 patient numbers had increased to 1087 . in late 2012 methadone was dispensed from drug treatment centres as part of global fund round . in the maldives , 47 patients had been enrolled to receive methadone in late 2009 , but by april 2010 only 17 patients remained , decreasing to 11 by august 2010 ( mohamed a.j . , 2010 ; personal communication ) ; by july 2011 patient numbers had rebounded to 57 ( aminath z. , 2011 ; personal communication ) . barriers to methadone programme enrolment in the maldives include the need to obtain clearance from five different government departments to ensure no criminal cases are pending . it is reported that the methadone programme functions well , but scope for improvement remains . in july 2010 , after several years of advocacy to initiate ost , bangladesh commence methadone provision , starting with one site in dhaka . by early 2013 an additional site had opened and 188 clients were receiving methadone ( azim t. , 2013 ; personal communication ) . various common challenges and barriers exist to the implementation and expansion of ost services . throughout the region drug users continue to experience high levels of stigma and discrimination due to their substance use . this impacts upon the delivery of general health services , including access to ost . in the uk it has been noted that key government policies to assist pwid reintegration and recovery through the use of ost were highly unlikely to succeed while stigmatising attitudes towards pwid remain pervasive . stigma and discrimination remain major barriers across south - east asia . acknowledging drug use and responding to hiv risk - taking behaviour was largely delayed across asia in comparison to many western countries , and the criminalisation of drug use still commonly overshadows a public health response to address this problem . official reports state that 235,000 people were detained in over 1000 compulsory drug detention centres in east and south east asia alone in 2012 . since 2012 , many united nation agencies have called for their closure for reasons including human rights abuses and a lack of evidence of their effectiveness . as found in other regions of the world , the attitude that the abstinence model of drug treatment is of paramount importance persists and rejection of the concept of ost maintenance is widespread , despite opioid dependence being acknowledged as a chronic medical condition . as a result , it could be argued that this has been the major reason for reduced political will , financial under - investment and lack of commitment toward the scaling up of essential harm reduction interventions , including ost . advocacy for and funding of ost scale - up in the countries under review remains largely driven by un agencies and the international donor community . initial funding of and sustainable investment in harm reduction services , including ost , remains a perennial problem . recent global studies have revealed over 90% of hiv - related harm reduction funds in low and middle income countries stem from international donors , and total funding remains inadequate . the cost of ost for individual recipients is prohibitive in many cases , and this greatly reduces the numbers of pwid in treatment . in indonesia , while methadone could cost a client 1 - 2 ud dollars ( $ ) per dose through a government service , an 8 - 12 mg dose of buprenorphine commonly costs 11 - 17 $ through a private medical practitioner . as a result , many pwid can only afford to purchase an inadequate dose that proves ineffective and contributes towards ongoing injecting of illicit drugs . despite these problems , it is important to highlight progressive steps from asian countries such as malaysia and china , where governments strongly endorse ost and are expanding the ost programme nationwide . by 2009 , china had 680 methadone clinics in 27 provinces with 242,000 patients ( ever ) enrolled , while malaysia had 59 government facilities and nine private clinics dispensing methadone to 7065 registered clients , with an additional 10,000 clients receiving methadone through private practitioners . in 2011 malaysia further expanded its methadone programme , with substantially more distribution sites : 168 public health clinics , 48 public hospitals , 24 general practitioners ( gps ) , 32 national anti - drug agency sites , 18 prisons and two others . a cumulative 20,955 pwid have been registered in public facilities since 2006 and another 23,573 pwids are receiving mmt from gps . due to the separate registration systems of private and public services , it is not possible to identify whether patients receiving methadone from private gps were also registered with public facilities ( naning h. , 2012 ; personal communication ) . in all countries under review , the focus remains largely on the traditional treatment model of detoxification and rehabilitation despite strong evidence of recidivism , with rates often ranging from 60 - 100% . despite the international evidence highlighting the broadranging health , social and economic benefits of ost , a lack of indepth knowledge , awareness and education about ost among policymakers , community members , drug users and the health sector constrains the scaling up of ost . as found worldwide , one of the greatest obstacles to ost acceptance in the sear is limited acceptance and practice of evidence - based medicine associated with ost in the medical workforce . research has shown that it is commonly perceived that providing ost is simply replacing one drug with another , despite evidence that methadone is superior to non - pharmacological methods in terms of client retention in treatment , reduced self - reported heroin use and both urine and hair analysis . in some parts of the world , including in some asian countries , providing ost can be viewed as a failure to deal with the criminal behaviour of drug users . consequently , the implementation of an abstinence - based model of treatment is still strongly advocated as the primary approach to address drug dependency . in addition , tensions continue between supply / demand reduction and harm reduction approaches , and impacts upon expansion of ost services . highlighting an effective ost treatment model that builds upon local evidence can contribute towards expansion of drug treatment services for pwid . one aspect of effectiveness is to achieve an optimal dose of ost ( usually greater than 60 mg of methadone ) that can lead to a decrease in the use of illicit drugs and ensure a higher retention rate and decrease in hiv risk behaviours . however , it is not uncommon practice for a lower dose to be prescribed for clients receiving ost in south - east asia . for example , in indonesia an integrated biological survey among pwid noted that most of those who received methadone in the past year were still injecting illicit opioids in the past week , and it was speculated that one reason for this was inadequate dosage . doses of methadone of more than 60 mg daily are recommended , with most clients requiring 60 - 120 mg daily . in some countries under review , low - dose buprenorphine was common practice ( < 4 mg daily ) . recent buprenorphine guidelines in india ( 2009 ) recommend 4 - 8 mg as the optimal buprenorphine dose , despite international evidence recommending that people be stabilised on 8 - 16 mg daily . overall poor monitoring and evaluation of ost programmes exists in the countries under review and this does not allow local experiences to be translated into coherently managed knowledge about a program . as a consequence this impacts upon seeking ways to improve and expand coverage of the ost service . in 2005 , who added methadone and buprenorphine to the who model list of essential medicines , and this endorsement provided high - level official support to advocate with the appropriate government ministries for the introduction and wider use of ost in treatment programmes . however , despite the official use of ost in all countries under review , specific policies for ost either do not exist or are too underdeveloped to have the desired impact . many countries in the region need greater official acknowledgement of pwid and the role they play in the hiv epidemic and respond according to the epidemiological and national context . a national policy would assist in establishing a set of standards for ost provision and address the needs of politicians , health administrators , program managers , clinicians and ost recipients . such policies should reflect a national agreement about the important strategic role of ost in reducing the adverse health , social and economic consequences of drug use , and outline key objectives and procedures . evidence of the benefits of ost in all countries under review warrants increased highlevel support for and expansion of ost , as has occurred in china . criminalisation of drug use continues to restrict the wider utilisation of ost . as a result it is critically important to increase the law enforcement sector s understanding of ost , and to advocate for it to endorse the expansion of ost . national working groups or task forces on harm reduction , with a high priority on ost , are similarly important , requiring participation of senior officials from health , police , academia and representatives of civil society for maximum benefit . despite an incremental growth in knowledge of ost since its introduction in various countries of south - east asia the skills and knowledge of staff working on drug dependency and ost is often lacking , and consequently regular training programmes are necessary . poly - drug use is widespread , with opioids often being mixed with benzodiazapines and other pharmaceuticals ; many clinical staff are uncertain as to how to address these issues . training programmes facilitate sustainable expansion and improved quality of ost services , and encourage long - term commitment towards ost treatment patients and pwid in general . of equal importance is the need to conduct regular monitoring and evaluation of ost programmes at national and local level . data identifying both effective approaches and difficulties inform policymakers and other stakeholders about how to improve quality and continue expansion of ost services . in 2005 , who added methadone and buprenorphine to the who model list of essential medicines , and this endorsement provided high - level official support to advocate with the appropriate government ministries for the introduction and wider use of ost in treatment programmes . however , despite the official use of ost in all countries under review , specific policies for ost either do not exist or are too underdeveloped to have the desired impact . many countries in the region need greater official acknowledgement of pwid and the role they play in the hiv epidemic and respond according to the epidemiological and national context . a national policy would assist in establishing a set of standards for ost provision and address the needs of politicians , health administrators , program managers , clinicians and ost recipients . such policies should reflect a national agreement about the important strategic role of ost in reducing the adverse health , social and economic consequences of drug use , and outline key objectives and procedures . evidence of the benefits of ost in all countries under review warrants increased highlevel support for and expansion of ost , as has occurred in china . criminalisation of drug use continues to restrict the wider utilisation of ost . as a result it is critically important to increase the law enforcement sector s understanding of ost , and to advocate for it to endorse the expansion of ost . national working groups or task forces on harm reduction , with a high priority on ost , are similarly important , requiring participation of senior officials from health , police , academia and representatives of civil society for maximum benefit . despite an incremental growth in knowledge of ost since its introduction in various countries of south - east asia the skills and knowledge of staff working on drug dependency and ost is often lacking , and consequently regular training programmes are necessary . poly - drug use is widespread , with opioids often being mixed with benzodiazapines and other pharmaceuticals ; many clinical staff are uncertain as to how to address these issues . training programmes facilitate sustainable expansion and improved quality of ost services , and encourage long - term commitment towards ost treatment patients and pwid in general . of equal importance is the need to conduct regular monitoring and evaluation of ost programmes at national and local level . data identifying both effective approaches and difficulties inform policymakers and other stakeholders about how to improve quality and continue expansion of ost services .

the south - east asia region contains an estimated 400,000 - 500,000 people who inject drugs ( pwid ) . hiv prevalence among pwid is commonly 20% or higher in indonesia , thailand , myanmar and some regions of india . opioid substitution therapy ( ost ) is an important hiv prevention intervention in this part of the world . however , key challenges and barriers to scale up of ost exist , including : pervasive stigma and discrimination towards pwid ; criminalisation of drug use overshadowing a public health response ; lack of political will and national commitment ; low financial investment ; focus towards traditional treatment models of detoxification and rehabilitation ; inadequate dosing of ost ; and poor monitoring and evaluation of programmes . our review of local evidence highlights that ost can be successful within the asian context . such evidence should be utilised more widely to advocate for policy change and increased political commitment to ensure ost reaches substantially more drug users.significance for public healthseveral countries in the world health organization south - east asia region can be commended for introducing opioid substitution therapy ( ost ) to address the ongoing hiv epidemic among people who inject drugs ( pwid ) . local evidence shows ost is an effective drug treatment approach in the asian context given sufficient technical and institutional support . however , despite much progress , the number of ost dispensing sites and recipients remains totally inadequate in terms of impact upon the current hiv epidemic among pwid . ongoing advocacy is needed if countries are to achieve the who s target of 40% of pwid being dosed with ost . greater political commitment a strengthened policy environment , capacity building for ost clinics , lessening the criminalisation of drug use and promoting a public health response will give many more pwid access to ost and slow the advance of the hiv epidemic .

Background Methods Results Implementation of opioid substitution therapy Opioid substitution therapy availability in 2009-2012 Challenges and barriers to scale up of opioid substitution therapy Discussion Moving forward with scale-up of opioid substitution therapy

global estimates indicate that there are 15.9 million ( range 11.0 - 21.2 million ) people who inject drugs ( pwid ) across 151 countries . within what the world health organization ( who ) define as their south - east asia region ( sear ) there are an estimated 400,000 - 500,000 pwid , primarily located in countries that experience a high or medium burden of illicit drug injecting ( who office for sear covers 11 countries : bangladesh , bhutan , dpr korea , india , indonesia , maldives , myanmar , nepal , sri lanka , thailand and timor - leste ) . data from several sources show that high risk behaviours among pwid are common and provision of comprehensive hiv prevention services varies ( table 1 ) . hiv prevalence among pwid in south - east asia was commonly identified at 20 - 25% and has remained consistently high in indonesia , nepal , thailand , myanmar and some regions of india . whilst a decline was recorded in hiv prevalence among nepalese pwid , from 68% in 2002 to 21% in 2009 and 6% in 2011 , hiv prevalence rose in bangladeshi pwid , from 4% in 2003 - 2004 to 7% in 2007 , and in punjab ( india ) from 13% in 2006 to 21% in 2011 . high - risk behaviours among pwid , such as the sharing of contaminated needles and syringes , remain a major determining factor in the course of the hiv epidemic throughout asia . the ongoing hiv epidemic among pwid in south - east asia has prompted many countries to develop hiv policies linked to national hiv strategic plans in which harm reduction interventions are increasingly viewed with understanding and acceptance . unaids undoc and who outline nine interventions as part of a comprehensive package that has the greatest impact on hiv prevention , treatment and care for pwid . meta - analysis and systematic reviews show conclusively that providing opioid substitution therapy ( ost ) together with needle and syringe programmes and antiretroviral treatment targeted towards pwid have the greatest effect in preventing hiv infection . in this article the focus is upon one essential harm reduction intervention , ost , and its use in countries that fall under the administrative purview of who s sear , with specific focus on bangladesh , india , indonesia , maldives , myanmar , nepal and thailand . despite the strong evidence for various benefits resulting from the use of ost improved retention in treatment for hiv , hcv and opioid use , reduced illicit opioid use , reduced risk of hiv , reduced criminality , better psychosocial outcomes and decreased risk to pregnant drug - dependent women ( during the intra - uterine , partum and postpartum period ) fewer than three per cent of pwid in the south - east asia region have access to ost . in this article we explore the historical adoption of ost , the current situation , some of the major challenges , and suggest ways to scale up ost in the sear . we reviewed published and grey literature located using medline and pubmed , who program documents for the countries of the sear , the joint united nations programme on hiv / aids ( unaids ) , the united nations office on drugs and crime ( unodc ) documents , and generic internet search engines between october 2011 and june 2013 . the amount and quality of the data available on the commencement of ost programmes , responsible agencies , the number of pwid receiving ost , and the linkages with other health and non - health systems vary considerably among the countries in south - east asia ( table 2 ) . despite satisfactory outcomes , the programme was suspended in 1998 , and then restarted as an emergency response for hiv prevention in september 2007 . buprenorphine was introduced in indonesia in 2001 , and the manufacturers of the drug encouraged private practitioners to prescribe the medication to drug - dependent clients . these two pilots continued until the end of 2005 , serving a population of approximately 300 drug users . the provision of buprenorphine is not currently an official part of the government ost programme . in 2012 methadone is the national health security office s approved ost medication and was placed on the thai essential drug list in 2008 ; it is available as part of the universal health coverage package for all thai citizens . in 2009 , an estimated 150 clients were reported to be receiving buprenorphine as part of an hiv prevention trial at chiang mai university , which has since been completed . in indonesia , methadone expansion began in 2006 with seven clinics serving 1000 clients by the end of that year . by april 2010 methadone was available in ten provinces from 51 clinics ( five specialist hospitals ; 10 general hospitals ; 30 primary health centres ; and six prison and detention centres ) ( sarasvita r. , 2010 ; personal communication ) . in addition , more than 50 medical prescribers in private practices were dispensing buprenorphine to an estimated 3000 patients . in late 2012 methadone was dispensed from drug treatment centres as part of global fund round . barriers to methadone programme enrolment in the maldives include the need to obtain clearance from five different government departments to ensure no criminal cases are pending . various common challenges and barriers exist to the implementation and expansion of ost services . throughout the region drug users continue to experience high levels of stigma and discrimination due to their substance use . this impacts upon the delivery of general health services , including access to ost . in the uk it has been noted that key government policies to assist pwid reintegration and recovery through the use of ost were highly unlikely to succeed while stigmatising attitudes towards pwid remain pervasive . stigma and discrimination remain major barriers across south - east asia . acknowledging drug use and responding to hiv risk - taking behaviour was largely delayed across asia in comparison to many western countries , and the criminalisation of drug use still commonly overshadows a public health response to address this problem . since 2012 , many united nation agencies have called for their closure for reasons including human rights abuses and a lack of evidence of their effectiveness . as found in other regions of the world , the attitude that the abstinence model of drug treatment is of paramount importance persists and rejection of the concept of ost maintenance is widespread , despite opioid dependence being acknowledged as a chronic medical condition . as a result , it could be argued that this has been the major reason for reduced political will , financial under - investment and lack of commitment toward the scaling up of essential harm reduction interventions , including ost . advocacy for and funding of ost scale - up in the countries under review remains largely driven by un agencies and the international donor community . the cost of ost for individual recipients is prohibitive in many cases , and this greatly reduces the numbers of pwid in treatment . in indonesia , while methadone could cost a client 1 - 2 ud dollars ( $ ) per dose through a government service , an 8 - 12 mg dose of buprenorphine commonly costs 11 - 17 $ through a private medical practitioner . despite these problems , it is important to highlight progressive steps from asian countries such as malaysia and china , where governments strongly endorse ost and are expanding the ost programme nationwide . in 2011 malaysia further expanded its methadone programme , with substantially more distribution sites : 168 public health clinics , 48 public hospitals , 24 general practitioners ( gps ) , 32 national anti - drug agency sites , 18 prisons and two others . in all countries under review , the focus remains largely on the traditional treatment model of detoxification and rehabilitation despite strong evidence of recidivism , with rates often ranging from 60 - 100% . despite the international evidence highlighting the broadranging health , social and economic benefits of ost , a lack of indepth knowledge , awareness and education about ost among policymakers , community members , drug users and the health sector constrains the scaling up of ost . as found worldwide , one of the greatest obstacles to ost acceptance in the sear is limited acceptance and practice of evidence - based medicine associated with ost in the medical workforce . research has shown that it is commonly perceived that providing ost is simply replacing one drug with another , despite evidence that methadone is superior to non - pharmacological methods in terms of client retention in treatment , reduced self - reported heroin use and both urine and hair analysis . in some parts of the world , including in some asian countries , providing ost can be viewed as a failure to deal with the criminal behaviour of drug users . consequently , the implementation of an abstinence - based model of treatment is still strongly advocated as the primary approach to address drug dependency . highlighting an effective ost treatment model that builds upon local evidence can contribute towards expansion of drug treatment services for pwid . one aspect of effectiveness is to achieve an optimal dose of ost ( usually greater than 60 mg of methadone ) that can lead to a decrease in the use of illicit drugs and ensure a higher retention rate and decrease in hiv risk behaviours . however , it is not uncommon practice for a lower dose to be prescribed for clients receiving ost in south - east asia . for example , in indonesia an integrated biological survey among pwid noted that most of those who received methadone in the past year were still injecting illicit opioids in the past week , and it was speculated that one reason for this was inadequate dosage . overall poor monitoring and evaluation of ost programmes exists in the countries under review and this does not allow local experiences to be translated into coherently managed knowledge about a program . the amount and quality of the data available on the commencement of ost programmes , responsible agencies , the number of pwid receiving ost , and the linkages with other health and non - health systems vary considerably among the countries in south - east asia ( table 2 ) . despite satisfactory outcomes , the programme was suspended in 1998 , and then restarted as an emergency response for hiv prevention in september 2007 . in india , sublingual buprenorphine treatment commenced in 1993 as a pilot project and has been licensed for treatment of opioid dependence in drug treatment centres since 1999 . mmt started in bangkok as an hiv prevention measure in 1990 , and gradually extended to a few clinics outside the capital . buprenorphine was introduced in indonesia in 2001 , and the manufacturers of the drug encouraged private practitioners to prescribe the medication to drug - dependent clients . preparation for the introduction of methadone began in myanmar in 2004 , with the ministry of health commencing the delivery of mmt over february and march 2006 in four drug treatment centres . in november 2008 , mmt was introduced into the maldives as part of a pilot study ; buprenorphine remained unavailable due to its classification as an illegal substance . the provision of buprenorphine is not currently an official part of the government ost programme . however , in early 2010 the government approved the use of methadone in a pilot study intended to involve six sites in late 2010 . by 2012 four methadone dispensing sites were servicing 350 patients ( kumar r. 2013 ; personal communication ) . in 2012 methadone is the national health security office s approved ost medication and was placed on the thai essential drug list in 2008 ; it is available as part of the universal health coverage package for all thai citizens . in 2009 , an estimated 150 clients were reported to be receiving buprenorphine as part of an hiv prevention trial at chiang mai university , which has since been completed . in indonesia , methadone expansion began in 2006 with seven clinics serving 1000 clients by the end of that year . in late 2012 methadone was dispensed from drug treatment centres as part of global fund round . barriers to methadone programme enrolment in the maldives include the need to obtain clearance from five different government departments to ensure no criminal cases are pending . various common challenges and barriers exist to the implementation and expansion of ost services . throughout the region drug users continue to experience high levels of stigma and discrimination due to their substance use . this impacts upon the delivery of general health services , including access to ost . in the uk it has been noted that key government policies to assist pwid reintegration and recovery through the use of ost were highly unlikely to succeed while stigmatising attitudes towards pwid remain pervasive . stigma and discrimination remain major barriers across south - east asia . acknowledging drug use and responding to hiv risk - taking behaviour was largely delayed across asia in comparison to many western countries , and the criminalisation of drug use still commonly overshadows a public health response to address this problem . as found in other regions of the world , the attitude that the abstinence model of drug treatment is of paramount importance persists and rejection of the concept of ost maintenance is widespread , despite opioid dependence being acknowledged as a chronic medical condition . as a result , it could be argued that this has been the major reason for reduced political will , financial under - investment and lack of commitment toward the scaling up of essential harm reduction interventions , including ost . advocacy for and funding of ost scale - up in the countries under review remains largely driven by un agencies and the international donor community . the cost of ost for individual recipients is prohibitive in many cases , and this greatly reduces the numbers of pwid in treatment . in indonesia , while methadone could cost a client 1 - 2 ud dollars ( $ ) per dose through a government service , an 8 - 12 mg dose of buprenorphine commonly costs 11 - 17 $ through a private medical practitioner . in 2011 malaysia further expanded its methadone programme , with substantially more distribution sites : 168 public health clinics , 48 public hospitals , 24 general practitioners ( gps ) , 32 national anti - drug agency sites , 18 prisons and two others . in all countries under review , the focus remains largely on the traditional treatment model of detoxification and rehabilitation despite strong evidence of recidivism , with rates often ranging from 60 - 100% . despite the international evidence highlighting the broadranging health , social and economic benefits of ost , a lack of indepth knowledge , awareness and education about ost among policymakers , community members , drug users and the health sector constrains the scaling up of ost . as found worldwide , one of the greatest obstacles to ost acceptance in the sear is limited acceptance and practice of evidence - based medicine associated with ost in the medical workforce . research has shown that it is commonly perceived that providing ost is simply replacing one drug with another , despite evidence that methadone is superior to non - pharmacological methods in terms of client retention in treatment , reduced self - reported heroin use and both urine and hair analysis . in some parts of the world , including in some asian countries , providing ost can be viewed as a failure to deal with the criminal behaviour of drug users . consequently , the implementation of an abstinence - based model of treatment is still strongly advocated as the primary approach to address drug dependency . in addition , tensions continue between supply / demand reduction and harm reduction approaches , and impacts upon expansion of ost services . highlighting an effective ost treatment model that builds upon local evidence can contribute towards expansion of drug treatment services for pwid . one aspect of effectiveness is to achieve an optimal dose of ost ( usually greater than 60 mg of methadone ) that can lead to a decrease in the use of illicit drugs and ensure a higher retention rate and decrease in hiv risk behaviours . however , it is not uncommon practice for a lower dose to be prescribed for clients receiving ost in south - east asia . for example , in indonesia an integrated biological survey among pwid noted that most of those who received methadone in the past year were still injecting illicit opioids in the past week , and it was speculated that one reason for this was inadequate dosage . overall poor monitoring and evaluation of ost programmes exists in the countries under review and this does not allow local experiences to be translated into coherently managed knowledge about a program . as a consequence this impacts upon seeking ways to improve and expand coverage of the ost service . in 2005 , who added methadone and buprenorphine to the who model list of essential medicines , and this endorsement provided high - level official support to advocate with the appropriate government ministries for the introduction and wider use of ost in treatment programmes . however , despite the official use of ost in all countries under review , specific policies for ost either do not exist or are too underdeveloped to have the desired impact . many countries in the region need greater official acknowledgement of pwid and the role they play in the hiv epidemic and respond according to the epidemiological and national context . a national policy would assist in establishing a set of standards for ost provision and address the needs of politicians , health administrators , program managers , clinicians and ost recipients . such policies should reflect a national agreement about the important strategic role of ost in reducing the adverse health , social and economic consequences of drug use , and outline key objectives and procedures . evidence of the benefits of ost in all countries under review warrants increased highlevel support for and expansion of ost , as has occurred in china . criminalisation of drug use continues to restrict the wider utilisation of ost . as a result it is critically important to increase the law enforcement sector s understanding of ost , and to advocate for it to endorse the expansion of ost . despite an incremental growth in knowledge of ost since its introduction in various countries of south - east asia the skills and knowledge of staff working on drug dependency and ost is often lacking , and consequently regular training programmes are necessary . poly - drug use is widespread , with opioids often being mixed with benzodiazapines and other pharmaceuticals ; many clinical staff are uncertain as to how to address these issues . training programmes facilitate sustainable expansion and improved quality of ost services , and encourage long - term commitment towards ost treatment patients and pwid in general . of equal importance is the need to conduct regular monitoring and evaluation of ost programmes at national and local level . in 2005 , who added methadone and buprenorphine to the who model list of essential medicines , and this endorsement provided high - level official support to advocate with the appropriate government ministries for the introduction and wider use of ost in treatment programmes . however , despite the official use of ost in all countries under review , specific policies for ost either do not exist or are too underdeveloped to have the desired impact . many countries in the region need greater official acknowledgement of pwid and the role they play in the hiv epidemic and respond according to the epidemiological and national context . a national policy would assist in establishing a set of standards for ost provision and address the needs of politicians , health administrators , program managers , clinicians and ost recipients . such policies should reflect a national agreement about the important strategic role of ost in reducing the adverse health , social and economic consequences of drug use , and outline key objectives and procedures . evidence of the benefits of ost in all countries under review warrants increased highlevel support for and expansion of ost , as has occurred in china . criminalisation of drug use continues to restrict the wider utilisation of ost . as a result it is critically important to increase the law enforcement sector s understanding of ost , and to advocate for it to endorse the expansion of ost . despite an incremental growth in knowledge of ost since its introduction in various countries of south - east asia the skills and knowledge of staff working on drug dependency and ost is often lacking , and consequently regular training programmes are necessary . poly - drug use is widespread , with opioids often being mixed with benzodiazapines and other pharmaceuticals ; many clinical staff are uncertain as to how to address these issues . of equal importance is the need to conduct regular monitoring and evaluation of ost programmes at national and local level .

the discovery of fullerenes and other forms of elemental carbon with curved surfaces introduced a novel aspect of supramolecular assembly based on the relatively weak dispersion forces between the convex surfaces of the conjugated carbon networks and the appropriate molecular receptors . buckybowls , curved - surface polycyclic aromatic hydrocarbons ( pah ) structurally related to fullerenes , appear to be good candidates for receptors due to the complementarity of their accessible concave surfaces with the convex surfaces of the fullerenes . while supramolecular assemblies of fullerenes with the smallest buckybowl corannulene ( 1 ) have not been detected in solution , we have shown that the efficient molecular receptors for both c60 and c70 can be constructed if at least two corannulene pincers are preorganized on a proper tether . for example , buckycatcher c60h28 ( 2 ) consisting of two corannulene subunits on a tetrabenzocyclooctatetraene tether was shown to form 1:1 inclusion complexes with fullerenes in both the solid state and in toluene solutions . the c60@2 inclusion complex has become a prototypical system for large dispersion - driven supramolecular systems and , as such , has been the subject of several computational studies performed at various levels of theory . recently , inclusion complexes for both c60 and c70 with 2 were incorporated into the s12l test set of noncovalently bound complexes used to evaluate computational methods performance in modeling the dispersion interactions . the reported theoretical gas - phase binding energies of the c60@2 complex vary dramatically , thus emphasizing the difficulty of accurately computing the energetics of dispersion forces . fock based calculations as well as several commonly employed dft functionals predict either repulsion or negligible binding energies for the assembly , while the dispersion - sensitive dft functionals predict strong gas - phase binding energies in the range 20 to 44 kcal mol . obviously , there is a need for reliable experimental data to assess the quality of the computational results . to date , the only reported thermodynamic results for the association of buckycatcher ( 2 ) with fullerenes are the ambient temperature gibbs enthalpies determined in toluene - d8 by h nmr titration ( 5.3 and 5.1 kcal mol for c60 and c70 , respectively ) . however , since the gas - phase experimental data for the thermodynamics of these inclusion complexes are not available , it is necessary to develop reliable computational models capable of assessing the solvent contributions to the enthalpy and entropy effects on the association thermodynamics in solution . in the first such attempt , zhao and truhlar calculated the entropy contribution to the gas - phase formation of c60@2 based on the rigid rotator - harmonic oscillator model and concluded that while the calculated binding energy of the assembly is 26.4 kcal mol ( e = 26.4 kcal mol ) the gas - phase gibbs free energy of association is only ca . in addition , the association of c60 with 2 in solution results in a considerable loss of the solvent - accessible surfaces which further reduces the exergonicity of the process . in a more comprehensive study , grimme applied a similar approach combining dispersion - corrected dft calculations for the gas - phase binding energies with the cosmo - rs continuum solvation model for the solvation free enthalpy assessment and evaluating the remaining rotational vibrational enthalpic / entropic contributions based on the harmonic frequency calculations . a series of inclusion complexes ( including the c60@2 and c70@2 complexes ) were studied , and the calculated g values in solutions were reported to differ on average by only 2 kcal / mol from the available experimental data . considering the simplicity of the model , the accuracy of the results is quite impressive , but a closer inspection of the results reveals some limitations to this approach . as an example , grimme s model predicts overbinding of both c60 and c70 by buckycatcher ( 2 ) in toluene by ca . 34 kcal mol , significantly more than the average error for the studied pool of inclusion complexes . obviously , a larger set of precise experimental thermodynamic data is needed to assess the accuracy of the computational methods as well as to improve the theoretical models used to describe solvation in weakly bound inclusion complexes . herein , we report the results of our study of the energetics of complexation of c60 and c70 with buckycatcher by both isothermal titration calorimetry ( itc ) and h nmr titration . the use of the itc method allowed us to obtain a complete set of thermodynamic parameters ( ka ( or g ) , h , and ts ) for the formation of c60@2 and c70@2 complexes in a number of solvents and at a number of different temperatures . we also repeated some of the earlier nmr titrations at lower concentration and at three temperatures for a better comparison with the itc results . the heat capacity changes , cp , for formation of the c60@2 and c70@2 inclusion complexes in toluene were also obtained from the temperature dependence of the calorimetric enthalpy changes . the buckycatcher ( 2 ) was synthesized in our laboratory according to the procedure we previously reported . anhydrous toluene , chlorobenzene , and o - dichlorobenzene were obtained from sigma - aldrich ( st . louis , mo ) . toluene - d8 and chlorobenzene - d5 were obtained from cambridge isotope laboratories ( tewksbury , ma ) . h nmr titrations were performed according to the procedure reported previously but at lower concentrations of fullerenes and 2 and with careful temperature control . the spectra were recorded on bruker ( billerica , ma ) avance iii 600 and 850 mhz spectrometers in toluene - d8 and chlorobenzene - d5 at 288 , 298 , and 308 k. several proton signals on the corannulene subunits of 2 exhibited measurable changes in chemical shift upon complexation with either c60 or c70 . if necessary , the overlapping peaks of some of these protons were deconvoluted using spinworks 3 nmr software ( kirk marat , university of manitoba ) in order to extract the precise chemical shift values . the association constant ka was determined using eq 11where x = [ fullerene]total , y = total , and l = max ( i.e. , at 100% complexation ) . values of ka and l were obtained from the nonlinear regression using the curve - fitting tools of origin v.8.5 ( northampton , ma ) . itc experiments were performed using a microcal - ge ( northampton , ma ) vp - itc . titrations were typically done at temperatures ranging from 278 to 323 k and involved overfilling the itc cell with 1.5 ml of fullerene solution ( c60 or c70 ) and adding as many as 20 injections ( 14 l each ) of the titrant solution of 2 . typically , three replicate measurements were performed . the raw calorimetric data were corrected for the heat of dilution of 2 and fullerenes by subtracting the heats from the appropriate blank titrations even though these heats were negligible in comparison to the binding interaction heats . the corrected itc titration results were fit with a nonlinear regression algorithm using the chasm itc data analysis program developed in our laboratory and assuming a 1:1 inclusion complex model . appi - ms experiments were carried out on a bruker ( billerica , ma ) micro - tof - q mass spectrometer . the fullerene solutions were prepared at a concentration of approximately 100 m , while the solutions of the buckycatcher were prepared at a concentration as high as 300 m . the appi - ms samples were prepared by mixing the solutions to yield a mixture containing a 2-fold excess of 2 . the ms capillary voltage was set to + 4500 v , dry n2 gas flow was adjusted to 12 l min at 453 k , and the samples were directly infused into the ms by using a kd scientific syringe pump set to a flow rate of 200 l / h . upon the addition of c60 or c70 to a solution of the buckycatcher , several proton nmr peaks of 2 exhibit measurable changes of their chemical shifts . the job plot constructed for one of the corannulene pincer protons is shown in figure 1 . following the changes in chemical shift and using the method of continuous variation , the maximum change in chemical shifts is observed at or near a mole fraction , / ( + [ c60 ] ) , of 0.5 . this is consistent with a saturation stoichiometry of 1:1 for formation of the c60@2 complex . similar results were obtained for other protons exhibiting measurable chemical shift changes upon titration . using the same job plot analysis , the 1:1 stoichiometry was also determined for the formation of the c70@2 complex in deuterated toluene and chlorobenzene . values of the [ molar ratio ] are plotted vs the mole fraction of 2 at 288 k ( ) , 298 k ( ) , and 308 k ( ) . 1:1 complex stoichiometry was also detected in the gas phase by appi mass spectrometry experiments . figure 2 shows the appi mass spectra obtained for each of the following chemical species in toluene : c60 , c70 , the buckycatcher 2 , and the c60@2 and c70@2 1:1 inclusion complexes . appi mass spectra for toluene solutions containing c60 ( a ) , 2 ( b ) , c70 ( c ) , and the mixtures of 2 with c60 ( d ) and c70 ( e ) . panels a , b , and c of figure 2 show the appi mass spectra for solutions containing the single chemical species , c60 , 2 , and c70 , respectively . panels a and c show only a single peak , e.g. , the c60 isothermal titration calorimetry experiments were performed , wherein a dilute solution of the titrant ( 2 ) was added to a dilute solution of the fullerene titrate . a typical itc thermogram for the addition of 2 to c60 in toluene at 298 k is shown in figure 3 . the solid line through the data points represents a nonlinear regression fit of the data to a thermodynamic model for the formation of a 1:1 inclusion complex . this analysis of the itc data yields a complete set of thermodynamic parameters ( ka ( or g ) , h , and ts ) for the formation of the fullerene@2 complexes . the left panel shows the baseline - corrected raw itc signal for a typical titration experiment in which 20 separate injections of dilute 2 titrant solution ( = 0.7 mm in toluene , injection volume = 14 l ) were made into the itc cell filled with the a dilute c60 solution ( [ c60 ] = 70 m in toluene ) . the right panel shows h for each injection ( ) along with the best - fit nonlinear regression line ( ) for a 1:1 inclusion complex model . the thermodynamic data for the formation of the c60@2 and c70@2 complexes in toluene , chlorobenzene , and o - dichlorobenzene at 298 k are listed in table 1 . similar thermodynamic data for the formation of these complexes in other solvents and at other temperatures are given in the supporting information ( see tables s1 , s2 , s3 , and s4 ) . the association constants at 298 k as determined in the itc experiments are relatively weak , ranging from ka = 4600 m for the formation of c70@2 in toluene to ka = 200 m for the formation of c70@2 complex in o - dichlorobenzene . first , the association constants for c70 with buckycatcher ( 2 ) are typically greater than those for formation of the c60@2 complexes . second , complex formation becomes less favorable as the solvent becomes a better solvent for either the fullerene or the buckycatcher ( 2 ) , resulting in a significant reduction in ka for the formation of the fullerene@2 complex in o - dichlorobenzene as compared to chlorobenzene and toluene . as seen in table 1 , at 298 k , the favorable free energy change , g , for complex formation is principally the result of a favorable change in enthalpy , h . with only one exception , c70@2 in toluene at 278 k , the entropy term , ts , for formation of the fullerene@2 complexes is smaller than the enthalpy change in every instance ( see the supporting information , tables s1 , s2 , s3 , and s4 ) . the values of the entropy term ( ts ) for the formation of the c60@2 complex in toluene and for the formation of the c60@2 and c70@2 complexes in chlorobenzene are close to zero . however , the values of the entropy term ( ts ) for the formation of the c70@2 complex in toluene and for the formation of the c60@2 and c70@2 complexes in o - dichlorobenzene range from 1.15 to 2.04 kcal mol . unexpectedly , the entropy changes for the formation of the c60@2 and c70@2 complexes are generally either zero or favorable for complex formation with notable exceptions for both complexes in 1,1,2,2-tetrachloroethane and c60@2 in anisole . values for g , h , and ts have units of kcal mol , and the errors listed are the standard deviations for a minimum of three replicate itc titrations . the association constants , ka , for formation of the c60@2 and c70@2 complexes in toluene - d8 and chlorobenzene - d5 at 288 , 298 , and 308 k , determined by h nmr titration , are compared with the respective itc determined constants in nondeuterated solvents in table 2 . the ka values determined by two different methods are in good to excellent agreement with one another in both toluene and chlorobenzene over the temperature range of the study . the differences between the nmr and itc determined g values for the formation of c60@2 complexes at 288 , 298 , and 308 k , respectively , are 0.01 , + 0.08 , and 0.06 kcal mol in toluene and + 0.11 , + 0.26 and + 0.46 kcal mol , respectively , in chlorobenzene . similarly , the differences between the nmr and itc based g values for the complexation of c70 with 2 are 0.06 , + 0.22 , and + 0.26 in toluene and 0.03 , + 0.01 , and + 0.06 kcal / mol in chlorobenzene at 288 , 298 , and 308 k , respectively . in order to gain a deeper insight into the solvent effects on the complexation , we performed the additional itc titrations in anisole , 1,1,2,2-tetrachloroethane , and o - dichlorobenzene at temperatures ranging from 278 to 323 k. the data from these itc experiments can be found in the supporting information ( see table s4 ) . in addition , the calorimetric enthalpy changes , hcal , for formation of the c60@2 and c70@2 complexes in toluene at 278 , 288 , 298 , and 308 k were plotted versus temperature to yield an estimate of the heat capacity change , cp , for the formation of the two fullerene2 complexes ( see figure s1 , supporting information ) . the enthalpy changes for formation of both the c60 and c70 buckyball@buckycatcher ( 2 ) complexes are linearly dependent on t over the experimental temperature range . the estimated heat capacity changes for formation of the two host guest complexes are 0.045 0.005 and 0.028 0.005 kcal mol k for the c60@2 and c70@2 complexes , respectively . it is clear that the two cp values observed in toluene are significantly different . in this study , we have used itc methods to develop a complete thermodynamic description ( ka or g , h , and ts ) for the formation of c60 and c70 fullerenebuckycatcher ( 2 ) complexes . in general , the itc values for ka were in good to excellent agreement with the nmr ka data . in addition to providing values for ka , g , h , and ts for formation of these dispersion complexes , the itc experiments provided estimates for the cp values for complex formation , and evidence for an unexpected enthalpy entropy compensation effect in the temperature dependence of the free energy change . it is important to note that the itc experiments reported here were only possible for these relatively weak complexes because the complex stoichiometry was determined in complementary nmr experiments and because we were able to work at reasonably high concentrations for both the fullerenes and the buckycatcher . in other words , we designed itc experiments wherein we were able to measure the heats for the formation of the complex and were able to determine the ka values from the curvature in the titration data . these conditions were met even for the systems exhibiting ka values as low as 200 m. stoichiometric information obtained from job plot analysis of the nmr titrations clearly suggests a saturation stoichiometry of 1:1 for both the c60 and c70 inclusion complexes these results are in agreement with the previously reported crystallographic structure for the 1:1 inclusion complex of c60@2 formed in the solid state . also , the expected 1:1 inclusion complexes of the fullerenes and buckycatcher were observed in the appi mass spectrometry experiments . although formally possible , and predicted by mm calculations to be quite stable ( at least in the gas phase ) , the 2:1 complex 3 was not detected in the appi ms experiments . in addition , job plots based on the nmr titration indicate that complex 3 does not exist in measurable amounts in toluene or chlorobenzene solutions , at least in the studied concentration ranges . in addition to the expected 1:1 inclusion complexes of 2 with fullerenes , appi experiments indicate the presence of small amounts of homodimers of the buckycatcher ( figure 2 ) . indeed , the dimeric head - to - head structure 4 was recently found in the crystals of buckycatcher grown by high - vacuum sublimation and a very substantial gas - phase binding energy for the dimer was calculated by the dispersion - corrected dft methods . however , as described in the methods section , the itc measured heat of dilution of 2 was negligible in comparison to the heats of binding to either fullerene . also , we did not observe any measurable change in the h nmr chemical shifts of 2 upon dilution in the concentration ranges studied in both deuterated toluene and chlorobenzene . we therefore conclude that the thermodynamics of association represents the formation of the solvated 1:1 fullerene@2 complex from the solvated fullerene and solvated 2 . the reaction scheme for formation of the inclusion complex is shown as eq 2 below:2it is important to note that the solvation of the ( fullerene@2 ) complex refers to the number of solvent molecules associated with the complex ( z ) which would be expected to be different than the total number of solvent molecules involved in the solvation of the free fullerene and buckycatcher molecules ( x + y ) . the last term in the equation , ( solvent)(x+yz ) , reflects the net number of solvent molecules lost , ( x + y z ) > 0 , upon fullerene@2 complex formation . in previous studies , we reported nmr titration derived ka values for the association of both c60 and c70 with the buckycatcher ( 2 ) in toluene - d8 at ambient temperatures . the reported ka values were 8600 500 m for formation of c60@2 complex and 6800 400 m for formation of c70@2 complex , relating to g of 5.3 and 5.1 kcal / mol , respectively . the analogous itc determined g values reported in this study are 4.8 and 5.0 kcal mol , respectively ( table 1 ) . since the difference in the case of c60@2 is larger than the expected error in the itc experiments , we decided to repeat the nmr titrations . we speculated that any real differences in the g values obtained in the h nmr and itc titrations could be attributed to the very low solubility of the fullerene@2 inclusion complexes in toluene . in an attempt to resolve the differences between the results of the previous h nmr results and the current itc results , the h nmr was repeated in toluene - d8 at lower concentrations for both fullerenes and 2 . the latest nmr derived ka values for the formation of c60@2 and c70@2 at 298 k in toluene are 2780 80 and 3030 330 m , respectively , in a much better agreement with the itc ka values . more importantly , we now find that in toluene the buckycatcher binds c70 with a slightly higher affinity than it binds c60 ( see table 1 ) . however , it must be noted that the small preference for binding of c70 in toluene ( 0.2 to 0.3 kcal mol , depending on temperature ) practically disappears in the other solvents used in this study , typically exhibiting a difference in g of less than 0.1 kcal mol for formation of the c60@2 and c70@2 complexes . as noted in the results section above , the entropy term , ts , for formation of the fullerene2 complexes was typically observed to be either negligibly small ( e.g. , c60 and c70 in chlorobenzene ) or favorable for complex formation ( e.g. , 2.9 kcal mol for c70 in toluene at 278 k to 1.9 kcal mol for c70 in toluene at 308 k ) . this is rather surprising considering the strongly destabilizing entropy contributions predicted by the computational models for the association both in the gas phase and in toluene solution . a few unfavorable or positive values for ts for formation of the fullerene2 complexes were observed ( e.g. , for both fullerenes in 1,1,2,2-tetrachloroethane ( ca . + 1.3 kcal mol ) and for c60 in anisole ( + 1.8 kcal mol ) ) . a recent paper by barnes et al . reported small positive ts values of + 0.6 to + 2.5 kcal mol for the formation of pah@exbox inclusion complexes in acetonitrile . the differences between stoddard s work and the present study can be attributed to differences in the structure of the guest molecules ( e.g. , fullerenes vs pahs ) , differences in the structure and charge of the host molecules ( 2 vs exbox ) , and of course the solvent , acetonitrile . entropy changes observed for complex formation ( sexp ) are often decomposed into a change in the configurational entropy ( sconf , associated with the host guest motions only ) and a change in solvation entropy ( ssolv ) , as shown in eq 3 . the latter term is related to motions of the solvent averaged over all possible host guest conformations.3the configurational entropy term can be estimated by summing the rotational and vibrational gas phase entropic contributions . on the basis of harmonic frequency calculations , grimme predicted that the sconf should be very unfavorable for formation of the c60@2 and c70@2 complexes at 298 k ( with ts values of + 14.8 and + 15.6 kcal mol for c60 and c70 , respectively ) . similar entropy destabilization of the c60@2 complex in the gas phase had previously been predicted by zhao and truhlar . using the cosmo - rs solvation model to provide solvation free enthalpies , the solvation entropy , tssolv , contributions to the overall entropy term free energy were estimated to be 9.9 and 10.3 kcal mol for the formation of c60@2 and c70@2 complexes at 298 k , not exothermic enough to override the strongly endothermic sconf contribution . there are at least two limitations to this approach for calculating the overall entropy change , sexp , for formation of these complexes . first , the cosmo - rs solvation model does not implicitly include solvent molecules , and even with implicit solvent molecules and molecular dynamic calculations , a quantitative assessment of solvation effects is by no means routine ( e.g. , see moghaddam et al . ) . second , as reported by grimme , this model yields free solvation energies , and the corresponding enthalpies and entropies calculated from their temperature dependence are sometimes used for analysis purposes but they do not represent the fundamental quantities . the total entropy changes , ts , as calculated by grimme s model for the formation of the c60@2 and c70@2 complexes are + 4.9 and + 5.2 kcal mol , suggesting a substantial destabilization entropy for both complexes in toluene at 298 k. in contrast , the itc determined ts values for the formation of both complexes in toluene at 298 k are both negative ( 0.2 and 2.0 kcal mol , respectively , see table 1 ) . these experimental ts values indicate at least modest entropic stabilization of the fullerene@2 complexes in toluene at 298 k. a reasonable assumption is that the calculated gas - phase sconf values are accurately estimated but the ssolv contributions are substantially underestimated by the cosmo - rs continuum solvation model . grimme also speculated that the calculated free energy changes , g values , for formation of the c60@2 and c70@2 complexes in toluene are more negative than observed experimentally due to the poor performance of the cosmo - rs solvation model in predicting ssolv for a nonpolar solute in a nonpolar solvent . the heat capacity changes , cp , for formation of fullerene@2 complexes in toluene were determined from the slope of the linear regression lines in plots of hcal versus temperature from 278 to 308 k ( see figure s1 , supporting information ) . the cp values for formation of both the c60@2 and c70@2 complexes are 0.045 and 0.028 kcal mol k. these small negative values for cp indicate that the fullerene2 complexes are somewhat less structured than the free fullerene and free 2 . the observation of a negative heat capacity change is typically attributed to the release of solvent molecules upon complex formation . in the fullerene buckycatcher system , some solvent molecules must be expelled from the interacting surfaces of the fullerene and the cleft of the buckycatcher with the net negative change in cp resulting from the net loss of solvent from the complex vs the free fullerene and free buckycatcher molecules ( eq 2 ) . similar heat capacity effects were observed earlier for the complexation of various guests by macrocyclic cyclophane hosts in cdcl3 . the more negative cp value for formation of the c60@2 complex ( 0.045 kcal mol k ) vs the cp value for formation of the c70@2 complex ( 0.028 kcal mol k ) in toluene suggests that c60 may fit better into the buckycatcher pocket and that more toluene is released in the formation of the c60 complex than for formation of the c70 complex . thermodynamic data obtained from fitting itc experiments for the addition of 2 into either c60 or c70 solutions performed at several different temperature ranging from 278 to 308 k in toluene are plotted in figure 4 . normalized values for the thermodynamic parameters ( g gave ) ( ) , ( h have ) ( ) , and ( ts + tsave ) ( ) for the formation of the fullerene 2 complexes in toluene plotted at four different temperatures 278 , 288 , 298 , and 308 k. panel a shows the thermodynamic data for formation of the c60@2 complex . the changes in the free energy change , g , for fullerene@2 complex formation over the temperature range 278308 k are very small . for example , the change in the free energy change , g , for the formation of the c60@2 complex at 308 k vs 273 k is less than + 0.1 kcal mol and less than + 0.2 kcal mol for formation of the c70@2 complex at 308 k vs 273 k. variations in the enthalpy and entropy changes for the formation of the fullerene2 complexes are 510 times larger ( ca . the changes in h and in ts have opposite signs and approximately compensate one another over this temperature range , resulting in a g/t value of almost zero . entropy compensation as brought about by changes in temperature has only infrequently been observed or reported for reactions taking place in organic solvents . referring to the extensive enthalpy entropy compensation literature for reactions taking place in aqueous solution , we are not surprised by this result , since the origin of the compensation phenomenon is typically attributed to changes in solvation . the formation of fullerene@2 complexes must involve solvent removal from the interacting surfaces of the associated fullerene guest and the buckycatcher host pocket as well as solvent reorganization around the complex . it was noted in the results section that fullerene@2 complex formation becomes less favorable in solvents where the solubility of the fullerene or 2 is greater , presumably underlining the importance of any desolvation penalty . to further explore the nature of this observation , itc results obtained in five different solvents ( toluene , anisole , chlorobenzene , 1,1,2,2-tetrachloroethane , and o - dichlorobenzene ) at 298 k are compared . these thermodynamic data which can be found in table 1 and in the supporting information ( see table s4 ) are plotted as a function of solvent dielectric constant in figure 5 . again , we observe enthalpy entropy compensation in which the free energy change for complex formation is less dependent on the solvent properties ( e.g. , polarity , hydrogen bonding , dielectric constant , etc . ) than is either the enthalpy or entropy change . in fact , while the free energy changes for formation of the fullerene@2 complexes are observed to vary linearly with the solvent dielectric constant , becoming 1.52 kcal mol less favorable in o - dichlorobenzene than in toluene , both the enthalpy and entropy changes vary unpredictably while exhibiting a high degree of compensation . in effect , every change in h is opposed by a compensating change in ts . the explanation for this phenomenon must reside in the fact that complex formation proceeds with the release of solvent from the fullerene@2 complex . entropy compensation for the formation of the ( a ) c60@2 and ( b ) c70@2 complexes , respectively . the thermodynamic parameters for fullerene@2 formation , g ( ) , h ( ) , and ts ( ) , are plotted as a function of solvent dielectric constant for five different organic solvents at 298 k. while the mechanism of enthalpy entropy compensation remains uncertain , it is obvious that there must be a linear relationship between h and ts for those systems where this phenomenon is observed . linear equations , like eq 4 , have been used to evaluate the degree of compensation:4the slope , in eq 4 , approaches a value of 1.0 for perfect compensation , and c represents the inherent stability of the complex , i.e. , g for the reaction with h = 0 . plot of the ts value vs the h value for formation of the c60@2 complex in five different solvents at 298 k. the data points from left to right correspond to anisole , 1,1,2,2-tetrachloroethane , toluene , chlorobenzene , and o - dichlorobenzene . the broken line shows the correlation for the four solvents with the toluene data omitted . as shown from the linear regression fit of the thermodynamic data in figure 6 , there is a reasonable linear correlation between h and ts ( r = 0.94 ) , with a slope ( ) of 0.660 and an intercept ( ts0 ) of 2.66 kcal mol for the formation of the c60@2 complex in the five solvents sampled . if the toluene point is not included in the fit , the slope remains unchanged ( = 0.661 ) , the value for ts0 changes from 2.66 to 2.53 kcal mol , and the correlation coefficient for the linear fit is improved , r = 0.998 . the slope indicates that 66% of the change in enthalpy is canceled out ( or compensated ) by an opposite change in the entropy term . the value of ( = 0.66 ) determined here for the formation of the c60@2 inclusion complexes is very similar to the values found by inoue and wada for the quinine@porphyrin receptor ( 0.60 ) and pyridine@metalloporphirin ( 0.61 ) inclusion complexes in organic solvents . these moderate values of have been interpreted to indicate that only moderate conformational changes are taking place in the host molecule . the positive ts0 value ( 2.7 kcal / mol ) indicates that the s term for desolvation is favorable for formation of the inclusion complexes in the studied solvents . this seems to be consistent with desolvation of the buckycatcher pocket ( the loss of 12 molecules of solvent , see our x - ray studies of the solvates of 2 ) and the removal of some of the solvent molecules from the first coordination ( solvation ) sphere of the fullerene ( probably another 24 molecules ) . the negative cp values discussed earlier for the formation of the fullerene@2 complexes are consistent with the loss of 47 solvent molecules . detailed nmr and itc titration studies provided a set of thermodynamic data for the formation of c60@2 and c70@2 inclusion complexes over a 30 k temperature range and in five different solvents . the formation of these host@guest inclusion complexes is typically enthalpically driven . in contrast with the predictions based on the existing solvation models , the entropy contributions are typically either stabilizing or close to zero , with the notable exception for both fullerenes in 1,1,2,2-tetrachloroethane and for c60 in anisole . entropy compensation effects were observed at different temperatures and in different solvents . better solvents for fullerenes significantly decrease their association with the buckycatcher , an effect which is not predicted by the cosmo - rs solvation model . relatively small but significant heat capacity effects were found with cp for formation of c60@2 and c70@2 complexes , 45 and 28 cal mol k. the thermodynamic data for these prototypical large dispersion - driven supramolecular systems should be invaluable to the further development and fine - tuning of computational methods and models for estimating the energetics of interacting systems in solution . these data will be particularly important in predicting dispersion - driven complex formation in aromatic or bonding solvents .

1h nmr and isothermal titration calorimetry ( itc ) experiments were employed to obtain reliable thermodynamic data for the formation of the 1:1 inclusion complexes of fullerenes c60 and c70 with the buckycatcher ( c60h28 ) . nmr measurements were done in toluene - d8 and chlorobenzene - d5 at 288 , 298 , and 308 k , while the itc titrations were performed in toluene , chlorobenzene , o - dichlorobenzene , anisole , and 1,1,2,2-tetrachloroethane at temperatures from 278 to 323 k. the association constants , ka , obtained with both techniques are in very good agreement . the thermodynamic data obtained by itc indicate that generally the host guest association is enthalpy - driven . interestingly , the entropy contributions are , with rare exceptions , slightly stabilizing or close to zero . neither h nor s is constant over the temperature range studied , and these thermodynamic functions exhibit classical enthalpy / entropy compensation . the cp values calculated from the temperature dependence of the calorimetric h values are negative for the association of both fullerenes with the buckycatcher in toluene . the negative cp values are consistent with some desolvation of the host - cavity and the guest in the inclusion complexes , c60@c60h28 and c70@c60h28 .

Introduction Materials and Methods Results Discussion Conclusion

to date , the only reported thermodynamic results for the association of buckycatcher ( 2 ) with fullerenes are the ambient temperature gibbs enthalpies determined in toluene - d8 by h nmr titration ( 5.3 and 5.1 kcal mol for c60 and c70 , respectively ) . in the first such attempt , zhao and truhlar calculated the entropy contribution to the gas - phase formation of c60@2 based on the rigid rotator - harmonic oscillator model and concluded that while the calculated binding energy of the assembly is 26.4 kcal mol ( e = 26.4 kcal mol ) the gas - phase gibbs free energy of association is only ca . a series of inclusion complexes ( including the c60@2 and c70@2 complexes ) were studied , and the calculated g values in solutions were reported to differ on average by only 2 kcal / mol from the available experimental data . as an example , grimme s model predicts overbinding of both c60 and c70 by buckycatcher ( 2 ) in toluene by ca . herein , we report the results of our study of the energetics of complexation of c60 and c70 with buckycatcher by both isothermal titration calorimetry ( itc ) and h nmr titration . the use of the itc method allowed us to obtain a complete set of thermodynamic parameters ( ka ( or g ) , h , and ts ) for the formation of c60@2 and c70@2 complexes in a number of solvents and at a number of different temperatures . the heat capacity changes , cp , for formation of the c60@2 and c70@2 inclusion complexes in toluene were also obtained from the temperature dependence of the calorimetric enthalpy changes . anhydrous toluene , chlorobenzene , and o - dichlorobenzene were obtained from sigma - aldrich ( st . toluene - d8 and chlorobenzene - d5 were obtained from cambridge isotope laboratories ( tewksbury , ma ) . the spectra were recorded on bruker ( billerica , ma ) avance iii 600 and 850 mhz spectrometers in toluene - d8 and chlorobenzene - d5 at 288 , 298 , and 308 k. several proton signals on the corannulene subunits of 2 exhibited measurable changes in chemical shift upon complexation with either c60 or c70 . titrations were typically done at temperatures ranging from 278 to 323 k and involved overfilling the itc cell with 1.5 ml of fullerene solution ( c60 or c70 ) and adding as many as 20 injections ( 14 l each ) of the titrant solution of 2 . using the same job plot analysis , the 1:1 stoichiometry was also determined for the formation of the c70@2 complex in deuterated toluene and chlorobenzene . values of the [ molar ratio ] are plotted vs the mole fraction of 2 at 288 k ( ) , 298 k ( ) , and 308 k ( ) . figure 2 shows the appi mass spectra obtained for each of the following chemical species in toluene : c60 , c70 , the buckycatcher 2 , and the c60@2 and c70@2 1:1 inclusion complexes . , the c60 isothermal titration calorimetry experiments were performed , wherein a dilute solution of the titrant ( 2 ) was added to a dilute solution of the fullerene titrate . the solid line through the data points represents a nonlinear regression fit of the data to a thermodynamic model for the formation of a 1:1 inclusion complex . this analysis of the itc data yields a complete set of thermodynamic parameters ( ka ( or g ) , h , and ts ) for the formation of the fullerene@2 complexes . the thermodynamic data for the formation of the c60@2 and c70@2 complexes in toluene , chlorobenzene , and o - dichlorobenzene at 298 k are listed in table 1 . similar thermodynamic data for the formation of these complexes in other solvents and at other temperatures are given in the supporting information ( see tables s1 , s2 , s3 , and s4 ) . the association constants at 298 k as determined in the itc experiments are relatively weak , ranging from ka = 4600 m for the formation of c70@2 in toluene to ka = 200 m for the formation of c70@2 complex in o - dichlorobenzene . first , the association constants for c70 with buckycatcher ( 2 ) are typically greater than those for formation of the c60@2 complexes . second , complex formation becomes less favorable as the solvent becomes a better solvent for either the fullerene or the buckycatcher ( 2 ) , resulting in a significant reduction in ka for the formation of the fullerene@2 complex in o - dichlorobenzene as compared to chlorobenzene and toluene . with only one exception , c70@2 in toluene at 278 k , the entropy term , ts , for formation of the fullerene@2 complexes is smaller than the enthalpy change in every instance ( see the supporting information , tables s1 , s2 , s3 , and s4 ) . the values of the entropy term ( ts ) for the formation of the c60@2 complex in toluene and for the formation of the c60@2 and c70@2 complexes in chlorobenzene are close to zero . however , the values of the entropy term ( ts ) for the formation of the c70@2 complex in toluene and for the formation of the c60@2 and c70@2 complexes in o - dichlorobenzene range from 1.15 to 2.04 kcal mol . unexpectedly , the entropy changes for the formation of the c60@2 and c70@2 complexes are generally either zero or favorable for complex formation with notable exceptions for both complexes in 1,1,2,2-tetrachloroethane and c60@2 in anisole . the association constants , ka , for formation of the c60@2 and c70@2 complexes in toluene - d8 and chlorobenzene - d5 at 288 , 298 , and 308 k , determined by h nmr titration , are compared with the respective itc determined constants in nondeuterated solvents in table 2 . the ka values determined by two different methods are in good to excellent agreement with one another in both toluene and chlorobenzene over the temperature range of the study . the differences between the nmr and itc determined g values for the formation of c60@2 complexes at 288 , 298 , and 308 k , respectively , are 0.01 , + 0.08 , and 0.06 kcal mol in toluene and + 0.11 , + 0.26 and + 0.46 kcal mol , respectively , in chlorobenzene . similarly , the differences between the nmr and itc based g values for the complexation of c70 with 2 are 0.06 , + 0.22 , and + 0.26 in toluene and 0.03 , + 0.01 , and + 0.06 kcal / mol in chlorobenzene at 288 , 298 , and 308 k , respectively . in order to gain a deeper insight into the solvent effects on the complexation , we performed the additional itc titrations in anisole , 1,1,2,2-tetrachloroethane , and o - dichlorobenzene at temperatures ranging from 278 to 323 k. the data from these itc experiments can be found in the supporting information ( see table s4 ) . in addition , the calorimetric enthalpy changes , hcal , for formation of the c60@2 and c70@2 complexes in toluene at 278 , 288 , 298 , and 308 k were plotted versus temperature to yield an estimate of the heat capacity change , cp , for the formation of the two fullerene2 complexes ( see figure s1 , supporting information ) . the enthalpy changes for formation of both the c60 and c70 buckyball@buckycatcher ( 2 ) complexes are linearly dependent on t over the experimental temperature range . the estimated heat capacity changes for formation of the two host guest complexes are 0.045 0.005 and 0.028 0.005 kcal mol k for the c60@2 and c70@2 complexes , respectively . in this study , we have used itc methods to develop a complete thermodynamic description ( ka or g , h , and ts ) for the formation of c60 and c70 fullerenebuckycatcher ( 2 ) complexes . in addition to providing values for ka , g , h , and ts for formation of these dispersion complexes , the itc experiments provided estimates for the cp values for complex formation , and evidence for an unexpected enthalpy entropy compensation effect in the temperature dependence of the free energy change . in other words , we designed itc experiments wherein we were able to measure the heats for the formation of the complex and were able to determine the ka values from the curvature in the titration data . these conditions were met even for the systems exhibiting ka values as low as 200 m. stoichiometric information obtained from job plot analysis of the nmr titrations clearly suggests a saturation stoichiometry of 1:1 for both the c60 and c70 inclusion complexes these results are in agreement with the previously reported crystallographic structure for the 1:1 inclusion complex of c60@2 formed in the solid state . also , the expected 1:1 inclusion complexes of the fullerenes and buckycatcher were observed in the appi mass spectrometry experiments . in addition to the expected 1:1 inclusion complexes of 2 with fullerenes , appi experiments indicate the presence of small amounts of homodimers of the buckycatcher ( figure 2 ) . the reaction scheme for formation of the inclusion complex is shown as eq 2 below:2it is important to note that the solvation of the ( fullerene@2 ) complex refers to the number of solvent molecules associated with the complex ( z ) which would be expected to be different than the total number of solvent molecules involved in the solvation of the free fullerene and buckycatcher molecules ( x + y ) . in previous studies , we reported nmr titration derived ka values for the association of both c60 and c70 with the buckycatcher ( 2 ) in toluene - d8 at ambient temperatures . we speculated that any real differences in the g values obtained in the h nmr and itc titrations could be attributed to the very low solubility of the fullerene@2 inclusion complexes in toluene . in an attempt to resolve the differences between the results of the previous h nmr results and the current itc results , the h nmr was repeated in toluene - d8 at lower concentrations for both fullerenes and 2 . the latest nmr derived ka values for the formation of c60@2 and c70@2 at 298 k in toluene are 2780 80 and 3030 330 m , respectively , in a much better agreement with the itc ka values . the total entropy changes , ts , as calculated by grimme s model for the formation of the c60@2 and c70@2 complexes are + 4.9 and + 5.2 kcal mol , suggesting a substantial destabilization entropy for both complexes in toluene at 298 k. in contrast , the itc determined ts values for the formation of both complexes in toluene at 298 k are both negative ( 0.2 and 2.0 kcal mol , respectively , see table 1 ) . the heat capacity changes , cp , for formation of fullerene@2 complexes in toluene were determined from the slope of the linear regression lines in plots of hcal versus temperature from 278 to 308 k ( see figure s1 , supporting information ) . in the fullerene buckycatcher system , some solvent molecules must be expelled from the interacting surfaces of the fullerene and the cleft of the buckycatcher with the net negative change in cp resulting from the net loss of solvent from the complex vs the free fullerene and free buckycatcher molecules ( eq 2 ) . the more negative cp value for formation of the c60@2 complex ( 0.045 kcal mol k ) vs the cp value for formation of the c70@2 complex ( 0.028 kcal mol k ) in toluene suggests that c60 may fit better into the buckycatcher pocket and that more toluene is released in the formation of the c60 complex than for formation of the c70 complex . thermodynamic data obtained from fitting itc experiments for the addition of 2 into either c60 or c70 solutions performed at several different temperature ranging from 278 to 308 k in toluene are plotted in figure 4 . normalized values for the thermodynamic parameters ( g gave ) ( ) , ( h have ) ( ) , and ( ts + tsave ) ( ) for the formation of the fullerene 2 complexes in toluene plotted at four different temperatures 278 , 288 , 298 , and 308 k. panel a shows the thermodynamic data for formation of the c60@2 complex . for example , the change in the free energy change , g , for the formation of the c60@2 complex at 308 k vs 273 k is less than + 0.1 kcal mol and less than + 0.2 kcal mol for formation of the c70@2 complex at 308 k vs 273 k. variations in the enthalpy and entropy changes for the formation of the fullerene2 complexes are 510 times larger ( ca . the formation of fullerene@2 complexes must involve solvent removal from the interacting surfaces of the associated fullerene guest and the buckycatcher host pocket as well as solvent reorganization around the complex . to further explore the nature of this observation , itc results obtained in five different solvents ( toluene , anisole , chlorobenzene , 1,1,2,2-tetrachloroethane , and o - dichlorobenzene ) at 298 k are compared . in fact , while the free energy changes for formation of the fullerene@2 complexes are observed to vary linearly with the solvent dielectric constant , becoming 1.52 kcal mol less favorable in o - dichlorobenzene than in toluene , both the enthalpy and entropy changes vary unpredictably while exhibiting a high degree of compensation . entropy compensation for the formation of the ( a ) c60@2 and ( b ) c70@2 complexes , respectively . plot of the ts value vs the h value for formation of the c60@2 complex in five different solvents at 298 k. the data points from left to right correspond to anisole , 1,1,2,2-tetrachloroethane , toluene , chlorobenzene , and o - dichlorobenzene . as shown from the linear regression fit of the thermodynamic data in figure 6 , there is a reasonable linear correlation between h and ts ( r = 0.94 ) , with a slope ( ) of 0.660 and an intercept ( ts0 ) of 2.66 kcal mol for the formation of the c60@2 complex in the five solvents sampled . if the toluene point is not included in the fit , the slope remains unchanged ( = 0.661 ) , the value for ts0 changes from 2.66 to 2.53 kcal mol , and the correlation coefficient for the linear fit is improved , r = 0.998 . the value of ( = 0.66 ) determined here for the formation of the c60@2 inclusion complexes is very similar to the values found by inoue and wada for the quinine@porphyrin receptor ( 0.60 ) and pyridine@metalloporphirin ( 0.61 ) inclusion complexes in organic solvents . this seems to be consistent with desolvation of the buckycatcher pocket ( the loss of 12 molecules of solvent , see our x - ray studies of the solvates of 2 ) and the removal of some of the solvent molecules from the first coordination ( solvation ) sphere of the fullerene ( probably another 24 molecules ) . the negative cp values discussed earlier for the formation of the fullerene@2 complexes are consistent with the loss of 47 solvent molecules . detailed nmr and itc titration studies provided a set of thermodynamic data for the formation of c60@2 and c70@2 inclusion complexes over a 30 k temperature range and in five different solvents . in contrast with the predictions based on the existing solvation models , the entropy contributions are typically either stabilizing or close to zero , with the notable exception for both fullerenes in 1,1,2,2-tetrachloroethane and for c60 in anisole . relatively small but significant heat capacity effects were found with cp for formation of c60@2 and c70@2 complexes , 45 and 28 cal mol k. the thermodynamic data for these prototypical large dispersion - driven supramolecular systems should be invaluable to the further development and fine - tuning of computational methods and models for estimating the energetics of interacting systems in solution .

the rho family of small ( 2040 kda ) gtpases , which are monomeric g - proteins , belong to the ras superfamily of gtpases containing more than 150 proteins . the ras superfamily has been classified into 5 subfamilies based on their sequence similarity which are ras , rho , rab , ran , and arf [ 1 , 2 ] . the rho family of gtpases are cell membrane - associated gtp - binding proteins that actively participate in cell signaling networks , which regulate actin organization , cell cycle progression and gene expression [ 3 , 4 ] . up to date , 20 members of rho gtpases have been found in four main subclasses , namely , rho , rnd , rac , and cdc42 [ 2 , 5 ] . rho gtpases are the most fundamental regulators of the actin cytoskeleton , along with other crucial properties in the cell , such as cell adhesion , gene transcription and cell proliferation , cell motility , vesicular trafficking , phagocytosis , and cytokinesis [ 2 , 49 ] . similar to other g - proteins and gtpases , rho family proteins can serve as molecular switches , by binding to either gdp or gtp . gtpases are active and are capable of transmitting cell signals to downstream proteins when they are bound to gtp and inactive when they are bound to gdp [ 10 , 11 ] . since nucleotide association and dissociation are normally slow , some regulators within the cell catalyze the process of cycling between gdp- and gtp - bound states of rho gtpases [ 12 , 13 ] . these regulators are guanine nucleotide exchange factors ( gefs ) , gtpase - activating proteins ( gaps ) , and guanine nucleotide dissociation inhibitors ( gdis ) . gefs stimulate the substitution of gdp for gtp to activate rho gtpases , whereas gaps inactivate rho gtpases by stimulating the substitution of gtp for gdp . gdis avoid the dissociation of gdp from gtpases and retain them in their nonsignaling state [ 15 , 16 ] . nucleotide exchange occurs due to the conformational changes in switch i and switch ii regions of gtpases , upon their contact with gefs / gaps [ 5 , 11 ] . owing to the important role of rho gtpases in cell signaling events and several cellular functions , they are favored by many bacterial pathogens as targets to deliver cytotoxins . bacterial effector proteins invade host cells via a specialized secretion system and regulate rho gtpases by mimicking either gef or gap activity [ 10 , 19 ] . examples of such bacteria are burkholderia , chlamydia , salmonella , shigella , and yersinia . these bacteria use type iii secretion system to inject their effector proteins directly into the host cell via a needle complex extending from the bacterial membrane and cytosol . bacterial proteins disorganize the actin cytoskeleton by depolymerization of actin stress fibers of the host cell . by rearranging actin dynamics additionally , bacterial effector proteins can manipulate gtpase signaling mechanism and can transmit signals to downstream effector proteins [ 21 , 22 ] . as a result , bacterial effector proteins can lead to many diseases , including infection and cancer . yope also has been reported to weaken the immune system of the host cell by affecting cytokine production and cause bubonic plague . in this work , the bacterial protein toxin yersinia outer protein e ( yope ) , which has been found to exert gap activity towards rhoa , rac1 , and cdc42 of gtpases in vitro , yope has been reported to disrupt actin cytoskeleton , prevent phagocytosis , and weaken host cell 's immune system by affecting cytokine production [ 2831 ] . yope , shown in figure 1 , is a monomeric protein of 219 amino acids , with a four antiparallel -helix bundle , four small helices , and two -strands . c - terminal domain of yope between residues 90219 is essential for its virulence since it comprises the bacterial gtpase activating protein ( gap ) domain . similar to other gap proteins , yope has an arginine residue ( arg144 ) that is reported to be essential for gap activity . in addition to arg144 , residues 182184 are reported to be conserved among other bacterial gap proteins , which are exos of pseudomonas aeruginosa and sptp of salmonella spp . yope interactions with g - proteins are investigated and important yope residues that govern its activity are reported by several studies [ 1 , 3 , 32 ] . residues ile106 , leu109 , thr138 , gly139 , ser140 , and gln149 are observed to interact with switch ii region of the gtpases . the key residue arg144 along with thr183 , ile184 , and gly185 is reported to contact the nucleotide and both of the switch regions . residues thr148 , gln151 , gln155 , pro177 , ser179 , and gln180 are found to interact with nucleotide and switch i region of rho gtpases . up to date , no cocrystallized or experimentally discovered ligands of yope have been reported . however , inhibition of type iii secretion system has been investigated by several studies [ 3337 ] , and 23 compounds that belong to a class of acylated hydrazones of different salicylaldehydes that prevent yope secretion in vitro have been identified . salicylidene acylhydrazides , which have been denoted as a class of antivirulence compounds , were also reported to obstruct type iii secretion of other gram - negative bacteria other than yersinia . although there were a number of mechanisms postulated , the inhibition mechanism and the target proteins of these salicylidene acylhydrazides were the focus of several studies [ 38 , 40 , 41 ] . recently , yersinia pseudotuberculosis proteins tpx and wrba , which take role in yope secretion , were recognized as targets of these compounds , . nevertheless , other potential targets of salicylidene acylhydrazides remain unknown , whose identification is essential for the investigation and design of new therapeutic molecules against bacterial secretion mechanism . here , we represent a hybrid virtual screening approach to identify molecules with inhibition potential against yope , using computational drug discovery tools . pharmacophore building was carried out via phase , based on the three - dimensional structures of 23 salicylidene acylhydrazides . small molecules selected from zinc database were screened and filtered with the pharmacophore model . multistep docking of filtered molecules was carried out with glide , taking ligand flexibility into account . virtual screening hits were clustered and further evaluated for their interactions with the target and pharmacokinetic properties . all virtual screening applications were performed using schrdinger suite 2011 ( llc , new york , ny , usa ) on linux platform using hp xw6600 workstation . the following schrdinger modules were used : protein preparation wizard [ 4244 ] , ligprep , confgen , phase , sitemap , qikprop , and glide [ 5052 ] . the 3d structures of the small molecules from the big vendors of zinc database ( see table s1 in supplementary material available online at http://dx.doi.org/10.1155/2013/640518 ) were prepared via ligprep and confgen scripts and merged using phase database generation module . the parameters used in the preparation step are the following : ( a ) possible ionization and tautomer states were generated at ph 7.0 2.0 ; ( b ) chiralities were obtained from the 3d geometry of the structures ; ( c ) for each compound , maximum 4 low energy isomers , 1 ring conformation , and 10 conformations per rotatable bond were generated ; ( d ) 50 steps of energy minimization were carried out for the conformers with the truncated newton ( tcng ) method using the opls_2005 force field and a distance dependent dielectric constant of 4 ; ( e ) conformers with high - energy ionization / tautomer states were automatically removed and the number of conformers was limited to 100 per compound , which is the default value for conformer generation in phase database generation module ; ( f ) a built - in qikprop script predicted the adme and druglikeness properties of the molecules ; ( g ) molecules that violated lipinski 's rule ( molecular weight < 500 , hydrogen bond donor < 5 , hydrogen bond acceptor < 10 , and partition coefficient < 5 ) or had reactive functional groups were removed . coordinates of yope ( pdb i d : 1hy5 , ) were obtained from rcsb protein data bank . the biological assembly is known to be a monomer , and therefore , one yope chain from the crystal dimer structure was prepared and refined using the protein preparation wizard . charges and bond orders were assigned , hydrogens were added to the heavy atoms , selenomethionines were converted to methionines , and all waters were deleted . reorientation of certain hydroxyl and thiol groups , amide groups of asparagines , glutamines and imidazole ring of histidines , protonation states of histidines , aspartic acids , and glutamic acids was optimized at neutral ph . using force field opls_2005 , after preparation , receptor grids were generated with glide by specifying the binding site with a 3d cubic box . sitemap was used to estimate the location of the active site by searching regions near the protein surface , generating hydrophobic and hydrophilic contour maps of the protein , and calculating energy potentials . enclosing box of 14 side lengths was placed by centering the critical arg144 residue , depending on the sitemap prediction ( figure s1 ) . based upon the fact that the binding site is not shallow , the nonpolar atoms were slightly scaled back , by choosing the van der waals radius scaling factor of 0.75 for nonpolar parts , so that nonnative ligands would dock to the receptor better . pharmacophore model was developed using the 23 experimentally determined inhibitors of yope secretion ( table 1 ) via phase module . the small molecules from the literature were drawn in maestro workspace , and these 2d structures were converted to all - atom 3d structures using embedded ligprep script . up to 32 stereoisomers were generated per ligand by determining chiralities from 3d structures , and all possible ionization states of ligands were generated at target ph of 7 . after converting to 3d , conformation search was carried out to generate conformers and search for low energy structures using opls_2005 force field and other default parameters with confgen script . maximum number of conformers per rotatable bond and number of conformers per structure were kept at 100 and 1000 , respectively . each compound , along with its different states and conformers , was represented by a set of points in space . when these set of points were aligned , some of them were found to coincide with each other , which indicates a structural feature or pharmacophore site . six built - in sets of pharmacophore features were searched : hydrogen bond acceptor ( a ) , hydrogen bond donor ( d ) , hydrophobic group ( h ) , negatively charged group ( n ) , positively charged group ( p ) , and aromatic ring ( r ) . a common set of pharmacophore features that are observed consistently in inhibitors with similar spatial arrangements were identified and grouped . then , all groups were individually investigated and tree - based partitioning technique was applied . if the grouped pharmacophore points do not coincide with at least one arbitrary pharmacophore site of each compound , they were eliminated . the remaining pharmacophore groups , which then are called pharmacophore hypotheses , were scored according to their alignment to the input molecules . overall quality of each hypothesis was measured from its survival score , and aaadr.21 was selected . the database was filtered based on its 3d similarity to the selected pharmacophore hypothesis , such that the minimum fitness value was 1.4 . molecules obtained by filtering were used for multistep receptor docking workflow . glide standard precision ( sp ) docking was performed with these molecules , and hits above 4 kcal / mol based on docking score were redocked to yope in xp mode , keeping all docking parameters as default . using monte carlo random search algorithm , ligand poses were generated for each input molecule , and binding affinity of these molecules to yope was predicted in terms of glide docking score . potential energies of the docked molecules were also predicted with empirical e - model scoring function . postdocking minimization was performed with opls_2005 force field , and one pose per ligand was saved . strain energies of bound and free forms of ligands were calculated , and hits with more than 4 kcal / mol energy difference between the two forms received a penalty equal to the quarter of their strain energy difference , which is added to the docking score . investigation of novel inhibitors targeting the bacterial yope was performed with the help of computational drug design tools . database generation , pharmacophore modeling and screening , and molecular docking and scoring were carried out to propose a set of biochemically active molecules with inhibition potential against yope . 23 chemically synthesized compounds belonging to a class of acylated hydrazones of salicylidene acylhydrazides that inhibit yope secretion in vitro were utilized ( table s2 ) for pharmacophore development . the ability of these salicylidene acylhydrazides to inhibit yope and to make important interactions upon direct contact , similar to tpx and wrba , was sought . for this purpose , prior to pharmacophore development , extra precision glide docking was carried out with these 23 compounds , where docking scores were observed to be between 2.0 and 5.9 kcal / mol ( data not shown ) . the binding orientations of these compounds were found to be in close proximity to the critical arginine residue , occupying the cleft between arg144 and a bulge formed by residues thr183-gly185 . the most common interactions between yope and 23 inhibitors were observed in residues arg144 , gln151 , and thr183 . the compounds also interacted with other important residues reported upon gtpase contact , some of which are thr138 , gly139 , thr148 , ser179 , gln180 , gly182 , ile184 , and gly185 . the binding modes and numerous interactions between yope and the salicylidene acylhydrazides supported the choice of using these compounds for pharmacophore development . six out of 23 compounds that have the critical arg144 interaction and docking scores above 4 kcal / mol were selected ( table 1 ) . it was visually observed that , below this score , the likelihood of encountering favorable interactions diminishes . interaction with the arg144 of yope was regarded as necessary since this residue was shown to interact with both of the switch regions of g - proteins , and its importance in catalytic activity was reported by mutation analyses [ 27 , 55 ] . it was observed that arg144 made hydrogen bonds either with the formic hydrazide group ( nnc = o ) or the hydroxybenzene groups of the compounds . the pharmacophore model was developed based on the structures of the six selected compounds using phase . after hypotheses were constructed , they were scored and ranked according to their coordination to the ligands , which can be seen in table 2 . the quality of each hypothesis was measured by its survival score , which is a weighted combination of site , vector , volume , and selectivity scores . site score indicates root mean squared deviation of compounds from the hypothesis site positions , whereas vector score is the average cosine of the angles formed by corresponding pairs of site features in the aligned structures and volume score measures how a compound overlays with pharmacophore sites based on van der waals spheres . site , volume , and vector scores range between 0 and 1 , and high values indicate better alignment of ligands on hypothesis . selectivity is an empirical prediction defined on log scale , which calculates the fraction of molecules that could match the hypothesis , whether it is an active or inactive ligand . for example , a selectivity value of 2 means that 1 molecule out of 100 ( log 10 ) arbitrary ligand molecules would match the hypothesis , regardless of their activity value . therefore , higher selectivity is favored , since it implies uniqueness to the ligand set . however , site , vector and , volume scores are underlined in the literature due to their geometric significance . overall , five - point hypothesis aaadr.21 , with three hydrogen acceptors , one hydrogen donor , and one ring group , gave the best three - dimensional alignment to the selected six compounds in terms of site , volume , selectivity , and total survival scores . aaadr.21 site points matched with compound 15 are represented in figure 2 along with pharmacophore site - site distances . two hydrogen acceptors and one hydrogen donor were observed to coincide with the atoms of arg144 interacting formic hydrazide group , whereas the other acceptor was aligned with the hydroxyl group of the benzene ring . pharmacophore model was used to search 3d database ( small molecule database generated from zinc ) to identify the molecules that satisfy the hypothesis . the molecules obtained by filtering with hypothesis aaadr.21 were docked to the receptor yope with glide software to predict binding affinity of molecules to the target and to investigate their ligand - protein interactions . the binding orientation and features of each database molecule relative to the receptor protein were determined and scored with internal scoring function glidescore . glide standard precision ( sp ) docking was first conducted with 18230 hits using the 3d structure of yope prepared for docking calculations , as described in the receptor protein preparation and receptor grid generation sections . top 30% hits of glide sp in terms of docking score , which comprises 5604 molecules , were redocked to yope in glide extra precision ( xp ) mode with the identical internal docking parameters . docking scores varied between 7.2 and 0.5 kcal / mol in glide xp mode . a pose filter was carried out and virtual screening hits that interact with the critical arg144 residue were determined . the number of remaining hits was further reduced to 185 by filtering with a docking score threshold of 4 kcal / mol . structures of the remaining molecules were analyzed and clustered by their 2d structural similarities via chemmine web tool . hierarchy of clusters based on pairwise compound similarity was defined using the tanimoto similarity coefficient and structural descriptors such as atom pairs . the hits were clustered using the tanimoto coefficient threshold as 0.50 . based on this similarity criterion , the majority of the hits formed individual cluster or only small groups of two or three members . only three more populated groups , with 8 , 26 , and 11 members , were formed according to the chemmine similarity clustering . database titles and docking scores of each member of these large clusters are tabulated in table s4 . three top scoring members of aforementioned clusters were chosen for visual inspection and detailed analysis . the 2d structures and docking results of the hits are given in tables 3 and 4 . the docking scores of the selected hits were found to be higher than the initial six compounds that were used in pharmacophore development . three molecules from each cluster were analyzed based on the docking score as well as additional criteria , such as absorption , distribution , metabolism and excretion ( adme ) considerations , druglikeness of ligands , and strain energy differences of ligands . therefore , ligands were allowed to be strained during docking , in order to fit the ligands to the protein binding site . however , too much strain may indicate false positives , and therefore , the strain in the molecules were determined . to this end , energies of free and docked conformations of hits were calculated and strain penalties were determined as a postdocking analysis , as described in glide docking section . another scoring function represented in table 4 , namely , e - model , also includes penalty terms for internal strain energy of the generated poses . only zinc01703513 , zinc19800113 , and zinc05297691 received a small strain penalty , which was considered as insignificant . pharmacokinetic properties , druglikeness as well as other significant descriptors , such as molecular weight , h - bond donors , h - bond acceptors , solvent accessible surface area , log herg ( blockage of k+ channels ) , log s ( aqueous solubility ) , log p ( octanol / water ) , and human oral absorption , for the selected hits were determined by qikprop ( table 5 ) . druglikeness , as predicted by the lipinski rule , was investigated along with the predicted adme and molecular properties . according to this rule , in order for a compound to be drug - like and orally active , it should have a molecular weight less than 500 da , hydrogen bond donor equal to or less than five , hydrogen bond acceptor equal to or less than 10 , and partition coefficient ( qp log po / w ) less than five . molecular weight , donor and acceptor atom numbers of the selected molecules were within the allowed values . qp log po / w ( or log p ) gives an estimate of compound 's lipophilicity . up to a certain limit , compounds with higher lipophilicity have higher ability to permeate across biological membranes , which is necessary for a drug candidate . in this study , all nine hits had acceptable values for the analyzed properties and exhibited drug - like characteristics based on the rule of 5 . the complete list of predicted physiochemical descriptors and adme properties is given in table s6 . superposition of the proposed molecules yope showed that their binding orientations are similar ( figure 3 ) . the interactions of the nine selected hits ( l1l9 , table 4 ) were analyzed to verify that the ligands made contacts with previously identified important residues of yope . all of the selected hits were found to reside in the cavity surrounded by the critical arg144 residue and other residues that are known to react with the switch regions of the g - proteins . the 2d representation of the selected hits and their receptor hydrogen bond and hydrophobic interactions is shown in figure 4 . the ligand - protein interaction diagrams were generated in ligplot by supplying the receptor - ligand complexes to pdbsum in pdb format . hydrogen bond interactions and their atomic distances ( in ) are shown in dashed lines , whereas hydrophobic contacts are shown in red crescents . all proposed molecules favoring the hydrogen bond with critical arg144 residue , which is known to be essential for gap function of yope . l1 , which has the highest docking score , had multiple hydrogen bond interactions with yope residues that interact with the switch regions in addition to arg144 . gln151 and ser179 are known to bond with both nucleotide and switch i region of rho gtpases , whereas thr183 interacts with the two switch regions . similar interactions were also observed in the majority of hits , which can be seen in figure 4 . fewer interactions were made with ala137 , gly139 , gln180 , and ile184 of yope . gly139 and gln180 are also among the reported switch i interacting residues , and ile184 , similar to thr183 , has the ability to make contacts with both of the switch regions . multiple hydrophobic contacts were also observed in each ligand - protein complex , with ile147 and gly182 being the most significant . overall , the numbers of bonded interactions and hydrophobic contacts were observed to be high , suggesting a strong binding between the proposed hits and the target protein . in addition , the binding orientation of the hits was observed to occlude the cleft comprising arg144 and other switch - interacting residues , and hence , presence of these compounds could possibly block the interaction between yope and gtpases . the hits were visually inspected and their similarity to the salicylidene acylhydrazides used in pharmacophore development was determined . the nine selected hits satisfied all of the pharmacophore sites with a 2 tolerance , suggesting that the pharmacophore building and docking results converged . the superposition of the selected hits on the pharmacophore hypothesis aaadr.21 is given in figure s3 . the hits from the third cluster ( table 4 ) were found to overlay very well with the pharmacophore sites . these hits also showed close structural resemblance to each other as well as to the initial six salicylidene acylhydrazides . members of the third cluster , l7 , l8 , and l9 , include a 5-hydroxypyrazole ring in their structure . similar to salicylidene acylhydrazides , they also have two ring conformations on the sides connected to a formic hydrazide group ( nnc = o ) . the ligand interaction maps reveal that the formic hydrazide substructure has multiple contacts with the yope binding site residues ( figures 4(g ) , 4(h ) , and 4(i ) ) . the first two clusters , on the other hand , do not share a notable structural similarity with salicylidene acylhydrazides although they align with pharmacophore site points to a certain extent . the first cluster , which includes l1 , l2 , and l3 , has a common erythritol ( r - butane-1,2,3,4-tetraol ) substructure ( figures 4(a ) , 4(b ) , and 4(c ) ) , whereas hits from the second cluster , l4 , l5 , and l6 , have tetrahydrofuran-2,3,4-triol ring in their structure ( figures 4(d ) , 4(e ) , and 4(f ) ) . both of these substructures also account for the vast majority of interactions between yope and ligands . hence , the scaffolds that have been observed within each cluster may lead to the indication of potent functional groups upon yope binding . the p. aeruginosa cytotoxins exosgap [ 13 , 60 ] and s. enterica sptpgap are the homolog gap proteins of yope with 22% and 29% amino acid sequence identity , respectively . although their sequence identity is remarkably low , their structure alignment on the backbone shows considerable resemblance . after superposition based on the yope c coordinates , the root mean square deviations between the c coordinates of yope with exosgap and with sptpgap are 1.26 and 1.36 , respectively . the key arginine finger of yope ( arg144 ) was also conserved in these gap proteins , as arg146exosgap and arg209sptpgap . their 3d structures were taken from the protein data bank ( pdb i d : 1g4u for sptpgap and pdb i d : 1he1 for exosgap ) prepared in maestro workspace , and their binding site grids were generated for all - atom docking calculations , as described in receptor protein preparation and receptor grid generation sections . by centering the key arginine residues and keeping the previous docking parameters identical , glide xp docking was performed on exosgap and sptpgap . the results reveal that the selected nine hits have higher average ligand strain and lower binding affinity toward exosgap and sptpgap in terms of docking scores ( table 6 ) . furthermore , only l2 and l7 interacted with arg146 of exosgap and only l5 interacted with arg209 of sptpgap . the absence of the critical arginine interaction as well as the low scores suggests that the proposed molecules may not bind the homologous proteins and that they are selective toward yopegap . in this work , the aim was to discover small molecules with inhibition potential against yope , which is a bacterial cytotoxin that inhibits small rho gtpases by mimicking their regulator proteins within the host cell . proper functioning of gtpases is crucial for the regulation of signaling events within the cell , and therefore , drug design against gtpase inhibitors , such as yope , is an important area of research . here , we used virtual screening to investigate potent drug - like inhibitors of yope . 23 small compounds , which were previously shown to inhibit the yope secretion mechanism , were utilized to develop a pharmacophore hypothesis . 500,000 unique small drug - like molecules selected from the zinc database were filtered based on 3d similarity to the hypothesis aaadr . binding orientations and features of these molecules were investigated with multistep molecular docking approach using glide software , allowing ligand flexibility . virtual screening hits that exhibit a certain binding affinity to yope in terms of docking score ( 4 kcal / mol ) were clustered based on their structural similarity using a tanimoto coefficient threshold of 0.5 . the three top scoring representative members from the most populated clusters were pooled and further analyzed . one cluster was found to be structurally similar to the salicylidene acylhydrazides , which can inhibit the bacterial activity of type iii secretion systems . thi acts as an immunosuppressant inhibitor of sphingosine-1-phosphate lyase ( s1pl ) , whose reduced activity is targeted for autoimmune disorder treatment . the other cluster , having tetrahydrofuran-2,3,4-triol ring as a common substructure , shares similarity to nelarabine , a drug used in the treatment of t - cell lymphoblastic leukemia . druglikeness of the clusters was also predicted , and molecular descriptors and pharmacokinetic and adme properties of the nine hits were found to be in accordance with known chemically and biologically active compounds . the aim of this virtual screening study was to find potent yope inhibitors that would hinder the interaction between their gap domain and gtpases . the binding site was selected such that the inhibitors would occlude the vicinity of the arg144 residues that contact the switch regions of gtpases . indeed , the molecules made contacts with the critical arginine finger as well as the other residues that were reported to interact with both switch i and switch ii regions . the binding modes of the hits showed that the molecules occupied the cleft formed in the vicinity of arg144 of yope . selectivity against yope was verified by docking the nine hits to the known yope homologs , namely , exosgap and sptpgap . docking results showed that these hits have lower binding affinity toward the homologous proteins , and the critical arginine bonding was not observed in the majority of hits . the proposed set of ligands has shown a promising inhibitory potential toward yope in silico and hence , can be used for further scientific studies , and the results can be extended to experimental validation .

gram - negative bacteria yersinia secrete virulence factors that invade eukaryotic cells via type iii secretion system . one particular virulence member , yersinia outer protein e ( yope ) , targets rho family of small gtpases by mimicking regulator gap protein activity , and its secretion mainly induces cytoskeletal disruption and depolymerization of actin stress fibers within the host cell . in this work , potent drug - like inhibitors of yope are investigated with virtual screening approaches . more than 500,000 unique small molecules from zinc database were screened with a five - point pharmacophore , comprising three hydrogen acceptors , one hydrogen donor , and one ring , and derived from different salicylidene acylhydrazides . binding modes and features of these molecules were investigated with a multistep molecular docking approach using glide software . virtual screening hits were further analyzed based on their docking score , chemical similarity , pharmacokinetic properties , and the key arg144 interaction along with other active site residue interactions with the receptor . as a final outcome , a diverse set of ligands with inhibitory potential were proposed .

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

the rho family of small ( 2040 kda ) gtpases , which are monomeric g - proteins , belong to the ras superfamily of gtpases containing more than 150 proteins . the ras superfamily has been classified into 5 subfamilies based on their sequence similarity which are ras , rho , rab , ran , and arf [ 1 , 2 ] . the rho family of gtpases are cell membrane - associated gtp - binding proteins that actively participate in cell signaling networks , which regulate actin organization , cell cycle progression and gene expression [ 3 , 4 ] . rho gtpases are the most fundamental regulators of the actin cytoskeleton , along with other crucial properties in the cell , such as cell adhesion , gene transcription and cell proliferation , cell motility , vesicular trafficking , phagocytosis , and cytokinesis [ 2 , 49 ] . these regulators are guanine nucleotide exchange factors ( gefs ) , gtpase - activating proteins ( gaps ) , and guanine nucleotide dissociation inhibitors ( gdis ) . bacterial effector proteins invade host cells via a specialized secretion system and regulate rho gtpases by mimicking either gef or gap activity [ 10 , 19 ] . examples of such bacteria are burkholderia , chlamydia , salmonella , shigella , and yersinia . these bacteria use type iii secretion system to inject their effector proteins directly into the host cell via a needle complex extending from the bacterial membrane and cytosol . bacterial proteins disorganize the actin cytoskeleton by depolymerization of actin stress fibers of the host cell . yope also has been reported to weaken the immune system of the host cell by affecting cytokine production and cause bubonic plague . in this work , the bacterial protein toxin yersinia outer protein e ( yope ) , which has been found to exert gap activity towards rhoa , rac1 , and cdc42 of gtpases in vitro , yope has been reported to disrupt actin cytoskeleton , prevent phagocytosis , and weaken host cell 's immune system by affecting cytokine production [ 2831 ] . yope , shown in figure 1 , is a monomeric protein of 219 amino acids , with a four antiparallel -helix bundle , four small helices , and two -strands . c - terminal domain of yope between residues 90219 is essential for its virulence since it comprises the bacterial gtpase activating protein ( gap ) domain . yope interactions with g - proteins are investigated and important yope residues that govern its activity are reported by several studies [ 1 , 3 , 32 ] . the key residue arg144 along with thr183 , ile184 , and gly185 is reported to contact the nucleotide and both of the switch regions . up to date , no cocrystallized or experimentally discovered ligands of yope have been reported . however , inhibition of type iii secretion system has been investigated by several studies [ 3337 ] , and 23 compounds that belong to a class of acylated hydrazones of different salicylaldehydes that prevent yope secretion in vitro have been identified . salicylidene acylhydrazides , which have been denoted as a class of antivirulence compounds , were also reported to obstruct type iii secretion of other gram - negative bacteria other than yersinia . although there were a number of mechanisms postulated , the inhibition mechanism and the target proteins of these salicylidene acylhydrazides were the focus of several studies [ 38 , 40 , 41 ] . recently , yersinia pseudotuberculosis proteins tpx and wrba , which take role in yope secretion , were recognized as targets of these compounds , . here , we represent a hybrid virtual screening approach to identify molecules with inhibition potential against yope , using computational drug discovery tools . pharmacophore building was carried out via phase , based on the three - dimensional structures of 23 salicylidene acylhydrazides . small molecules selected from zinc database were screened and filtered with the pharmacophore model . virtual screening hits were clustered and further evaluated for their interactions with the target and pharmacokinetic properties . the following schrdinger modules were used : protein preparation wizard [ 4244 ] , ligprep , confgen , phase , sitemap , qikprop , and glide [ 5052 ] . the 3d structures of the small molecules from the big vendors of zinc database ( see table s1 in supplementary material available online at http://dx.doi.org/10.1155/2013/640518 ) were prepared via ligprep and confgen scripts and merged using phase database generation module . the parameters used in the preparation step are the following : ( a ) possible ionization and tautomer states were generated at ph 7.0 2.0 ; ( b ) chiralities were obtained from the 3d geometry of the structures ; ( c ) for each compound , maximum 4 low energy isomers , 1 ring conformation , and 10 conformations per rotatable bond were generated ; ( d ) 50 steps of energy minimization were carried out for the conformers with the truncated newton ( tcng ) method using the opls_2005 force field and a distance dependent dielectric constant of 4 ; ( e ) conformers with high - energy ionization / tautomer states were automatically removed and the number of conformers was limited to 100 per compound , which is the default value for conformer generation in phase database generation module ; ( f ) a built - in qikprop script predicted the adme and druglikeness properties of the molecules ; ( g ) molecules that violated lipinski 's rule ( molecular weight < 500 , hydrogen bond donor < 5 , hydrogen bond acceptor < 10 , and partition coefficient < 5 ) or had reactive functional groups were removed . the biological assembly is known to be a monomer , and therefore , one yope chain from the crystal dimer structure was prepared and refined using the protein preparation wizard . reorientation of certain hydroxyl and thiol groups , amide groups of asparagines , glutamines and imidazole ring of histidines , protonation states of histidines , aspartic acids , and glutamic acids was optimized at neutral ph . sitemap was used to estimate the location of the active site by searching regions near the protein surface , generating hydrophobic and hydrophilic contour maps of the protein , and calculating energy potentials . pharmacophore model was developed using the 23 experimentally determined inhibitors of yope secretion ( table 1 ) via phase module . the small molecules from the literature were drawn in maestro workspace , and these 2d structures were converted to all - atom 3d structures using embedded ligprep script . up to 32 stereoisomers were generated per ligand by determining chiralities from 3d structures , and all possible ionization states of ligands were generated at target ph of 7 . each compound , along with its different states and conformers , was represented by a set of points in space . six built - in sets of pharmacophore features were searched : hydrogen bond acceptor ( a ) , hydrogen bond donor ( d ) , hydrophobic group ( h ) , negatively charged group ( n ) , positively charged group ( p ) , and aromatic ring ( r ) . overall quality of each hypothesis was measured from its survival score , and aaadr.21 was selected . glide standard precision ( sp ) docking was performed with these molecules , and hits above 4 kcal / mol based on docking score were redocked to yope in xp mode , keeping all docking parameters as default . using monte carlo random search algorithm , ligand poses were generated for each input molecule , and binding affinity of these molecules to yope was predicted in terms of glide docking score . potential energies of the docked molecules were also predicted with empirical e - model scoring function . postdocking minimization was performed with opls_2005 force field , and one pose per ligand was saved . strain energies of bound and free forms of ligands were calculated , and hits with more than 4 kcal / mol energy difference between the two forms received a penalty equal to the quarter of their strain energy difference , which is added to the docking score . database generation , pharmacophore modeling and screening , and molecular docking and scoring were carried out to propose a set of biochemically active molecules with inhibition potential against yope . the ability of these salicylidene acylhydrazides to inhibit yope and to make important interactions upon direct contact , similar to tpx and wrba , was sought . the binding orientations of these compounds were found to be in close proximity to the critical arginine residue , occupying the cleft between arg144 and a bulge formed by residues thr183-gly185 . the most common interactions between yope and 23 inhibitors were observed in residues arg144 , gln151 , and thr183 . the compounds also interacted with other important residues reported upon gtpase contact , some of which are thr138 , gly139 , thr148 , ser179 , gln180 , gly182 , ile184 , and gly185 . the binding modes and numerous interactions between yope and the salicylidene acylhydrazides supported the choice of using these compounds for pharmacophore development . interaction with the arg144 of yope was regarded as necessary since this residue was shown to interact with both of the switch regions of g - proteins , and its importance in catalytic activity was reported by mutation analyses [ 27 , 55 ] . the pharmacophore model was developed based on the structures of the six selected compounds using phase . the quality of each hypothesis was measured by its survival score , which is a weighted combination of site , vector , volume , and selectivity scores . site score indicates root mean squared deviation of compounds from the hypothesis site positions , whereas vector score is the average cosine of the angles formed by corresponding pairs of site features in the aligned structures and volume score measures how a compound overlays with pharmacophore sites based on van der waals spheres . site , volume , and vector scores range between 0 and 1 , and high values indicate better alignment of ligands on hypothesis . overall , five - point hypothesis aaadr.21 , with three hydrogen acceptors , one hydrogen donor , and one ring group , gave the best three - dimensional alignment to the selected six compounds in terms of site , volume , selectivity , and total survival scores . two hydrogen acceptors and one hydrogen donor were observed to coincide with the atoms of arg144 interacting formic hydrazide group , whereas the other acceptor was aligned with the hydroxyl group of the benzene ring . the molecules obtained by filtering with hypothesis aaadr.21 were docked to the receptor yope with glide software to predict binding affinity of molecules to the target and to investigate their ligand - protein interactions . the binding orientation and features of each database molecule relative to the receptor protein were determined and scored with internal scoring function glidescore . glide standard precision ( sp ) docking was first conducted with 18230 hits using the 3d structure of yope prepared for docking calculations , as described in the receptor protein preparation and receptor grid generation sections . top 30% hits of glide sp in terms of docking score , which comprises 5604 molecules , were redocked to yope in glide extra precision ( xp ) mode with the identical internal docking parameters . a pose filter was carried out and virtual screening hits that interact with the critical arg144 residue were determined . the number of remaining hits was further reduced to 185 by filtering with a docking score threshold of 4 kcal / mol . hierarchy of clusters based on pairwise compound similarity was defined using the tanimoto similarity coefficient and structural descriptors such as atom pairs . only three more populated groups , with 8 , 26 , and 11 members , were formed according to the chemmine similarity clustering . database titles and docking scores of each member of these large clusters are tabulated in table s4 . three molecules from each cluster were analyzed based on the docking score as well as additional criteria , such as absorption , distribution , metabolism and excretion ( adme ) considerations , druglikeness of ligands , and strain energy differences of ligands . however , too much strain may indicate false positives , and therefore , the strain in the molecules were determined . to this end , energies of free and docked conformations of hits were calculated and strain penalties were determined as a postdocking analysis , as described in glide docking section . only zinc01703513 , zinc19800113 , and zinc05297691 received a small strain penalty , which was considered as insignificant . pharmacokinetic properties , druglikeness as well as other significant descriptors , such as molecular weight , h - bond donors , h - bond acceptors , solvent accessible surface area , log herg ( blockage of k+ channels ) , log s ( aqueous solubility ) , log p ( octanol / water ) , and human oral absorption , for the selected hits were determined by qikprop ( table 5 ) . druglikeness , as predicted by the lipinski rule , was investigated along with the predicted adme and molecular properties . according to this rule , in order for a compound to be drug - like and orally active , it should have a molecular weight less than 500 da , hydrogen bond donor equal to or less than five , hydrogen bond acceptor equal to or less than 10 , and partition coefficient ( qp log po / w ) less than five . molecular weight , donor and acceptor atom numbers of the selected molecules were within the allowed values . in this study , all nine hits had acceptable values for the analyzed properties and exhibited drug - like characteristics based on the rule of 5 . the interactions of the nine selected hits ( l1l9 , table 4 ) were analyzed to verify that the ligands made contacts with previously identified important residues of yope . all of the selected hits were found to reside in the cavity surrounded by the critical arg144 residue and other residues that are known to react with the switch regions of the g - proteins . l1 , which has the highest docking score , had multiple hydrogen bond interactions with yope residues that interact with the switch regions in addition to arg144 . gln151 and ser179 are known to bond with both nucleotide and switch i region of rho gtpases , whereas thr183 interacts with the two switch regions . fewer interactions were made with ala137 , gly139 , gln180 , and ile184 of yope . gly139 and gln180 are also among the reported switch i interacting residues , and ile184 , similar to thr183 , has the ability to make contacts with both of the switch regions . overall , the numbers of bonded interactions and hydrophobic contacts were observed to be high , suggesting a strong binding between the proposed hits and the target protein . in addition , the binding orientation of the hits was observed to occlude the cleft comprising arg144 and other switch - interacting residues , and hence , presence of these compounds could possibly block the interaction between yope and gtpases . the hits were visually inspected and their similarity to the salicylidene acylhydrazides used in pharmacophore development was determined . the nine selected hits satisfied all of the pharmacophore sites with a 2 tolerance , suggesting that the pharmacophore building and docking results converged . similar to salicylidene acylhydrazides , they also have two ring conformations on the sides connected to a formic hydrazide group ( nnc = o ) . the ligand interaction maps reveal that the formic hydrazide substructure has multiple contacts with the yope binding site residues ( figures 4(g ) , 4(h ) , and 4(i ) ) . the first two clusters , on the other hand , do not share a notable structural similarity with salicylidene acylhydrazides although they align with pharmacophore site points to a certain extent . the first cluster , which includes l1 , l2 , and l3 , has a common erythritol ( r - butane-1,2,3,4-tetraol ) substructure ( figures 4(a ) , 4(b ) , and 4(c ) ) , whereas hits from the second cluster , l4 , l5 , and l6 , have tetrahydrofuran-2,3,4-triol ring in their structure ( figures 4(d ) , 4(e ) , and 4(f ) ) . both of these substructures also account for the vast majority of interactions between yope and ligands . the p. aeruginosa cytotoxins exosgap [ 13 , 60 ] and s. enterica sptpgap are the homolog gap proteins of yope with 22% and 29% amino acid sequence identity , respectively . after superposition based on the yope c coordinates , the root mean square deviations between the c coordinates of yope with exosgap and with sptpgap are 1.26 and 1.36 , respectively . the key arginine finger of yope ( arg144 ) was also conserved in these gap proteins , as arg146exosgap and arg209sptpgap . by centering the key arginine residues and keeping the previous docking parameters identical , glide xp docking was performed on exosgap and sptpgap . in this work , the aim was to discover small molecules with inhibition potential against yope , which is a bacterial cytotoxin that inhibits small rho gtpases by mimicking their regulator proteins within the host cell . proper functioning of gtpases is crucial for the regulation of signaling events within the cell , and therefore , drug design against gtpase inhibitors , such as yope , is an important area of research . here , we used virtual screening to investigate potent drug - like inhibitors of yope . 500,000 unique small drug - like molecules selected from the zinc database were filtered based on 3d similarity to the hypothesis aaadr . binding orientations and features of these molecules were investigated with multistep molecular docking approach using glide software , allowing ligand flexibility . virtual screening hits that exhibit a certain binding affinity to yope in terms of docking score ( 4 kcal / mol ) were clustered based on their structural similarity using a tanimoto coefficient threshold of 0.5 . the three top scoring representative members from the most populated clusters were pooled and further analyzed . one cluster was found to be structurally similar to the salicylidene acylhydrazides , which can inhibit the bacterial activity of type iii secretion systems . the other cluster , having tetrahydrofuran-2,3,4-triol ring as a common substructure , shares similarity to nelarabine , a drug used in the treatment of t - cell lymphoblastic leukemia . druglikeness of the clusters was also predicted , and molecular descriptors and pharmacokinetic and adme properties of the nine hits were found to be in accordance with known chemically and biologically active compounds . the binding modes of the hits showed that the molecules occupied the cleft formed in the vicinity of arg144 of yope . docking results showed that these hits have lower binding affinity toward the homologous proteins , and the critical arginine bonding was not observed in the majority of hits . the proposed set of ligands has shown a promising inhibitory potential toward yope in silico and hence , can be used for further scientific studies , and the results can be extended to experimental validation .

the effect of chronic cr to modulate cellular insulin signaling and transcriptional regulation was assessed in skeletal muscle obtained from nonhuman primates subjected to a 4-year period of 30% cr compared with ad libitum ( al ) feeding conditions . the metabolic and physiological changes observed in this cohort with cr were previously reported ( 14 ) . specifically , 32 feral adult male cynomolgus monkeys ( macaca fascicularis ) ( mean sem age 8.2 1.2 years ) were part of a randomized trial in which the independent effect of cr and its interaction with insulin resistance and atherosclerotic lesion extent and composition were evaluated ( fig . demonstration of the study design for the 4-year caloric restriction trial . as shown , after pretrial evaluations the animals were acquired directly from the institute pertanian ( bogar , indonesia ) and quarantined for 3 months . animals were housed socially in pairs except when separated at mealtime by sliding a partition to separate them ( 14 ) . beginning in the 4th month and throughout the pretrial ( months 46 ) , all animals were fed a moderately atherogenic diet ( 0.25 mg cholesterol / calorie ) containing 30% of calories from fat . caloric intake for each animal was assessed by feeding a known allotment and weighing the uneaten food ( 14 ) . after pretrial evaluations , the animals were assigned to al or cr diet groups using a stratified randomization . the cr diet was introduced over a 3-month transition period ( 90% of al intake during the 1st month , 80% during the 2nd month , and 70% during the 3rd month and thereafter ) . additional vitamin and mineral mixture , -sitosterol , and crystalline cholesterol were added to the cr diet so that the same amount of these components was ingested regardless of the randomized group ( 14 ) . insulin sensitivity was assessed at 6-month intervals using a modified minimal model and at the end of the study using a hyperinsulinemic - euglycemic clamp as described ( 14 ) . at the baseline of the clamp , after the insulin infusion , repeat biopsies were obtained at 5 , 20 , and 40 min of the clamp . muscle samples ( 200 mg wet wt ) were immediately placed into liquid nitrogen and then stored at 80c . there was no difference between the steady state plasma glucose ( 5.38 0.10 vs. 5.42 0.10 mmol / l ) or plasma insulin levels observed during the clamp ( 1,472 145 vs. 1,535 155 muscle tissue lysates were prepared by dissection and homogenized in buffer a ( 25 mmol / l hepes , ph 7.4 , 1% nonidet p-40 , 137 mmol / l nacl , 1 mmol / l phenylmethylsulfonyl fluoride , 10 g / ml aprotinin , 1 g / ml pepstatin , and 5 g / ml leupeptin ) , using a pro 200 homogenizer ( pro scientific , oxford , ct ) . the samples were centrifuged at 14,000 g for 20 min at 4c , and protein content of the supernatant was determined ( bio - rad protein assay kit ; bio - rad laboratories , hercules , ca ) . supernatants ( 50 g ) were resolved by sds - page and subjected to immunoblotting using chemiluminescence reagent ( perkinelmer life science , boston , ma ) and quantified as described ( 22 ) . the 19s proteasome base anti - s5a / rpn10 antibodies were ordered from calbiochem ( gibbstown , nj ) . antibodies for phospho insulin receptor substrate ( irs)-1 ( tyr612 ) , phospho insulin receptor ( ir ) ( tyr1150/1151 ) , phosphoinositol ( pi ) 3-kinase protein 85 ( p85 of pi 3-kinase ) , phospho - akt ( ser473 ) , irs-1 and irs-2 , 20s proteasome subunit 2i , akt , serum- and glucocorticoid - inducible kinase 1 ( sgk1 ) , signal transducer and activator of transcription 3 ( stat3 ) , and sirt1 antibodies were obtained from upstate biotech ( lake placid , ny ) . anti-19s proteasome lid subunits s9/rpn6 and s14/rpn12 antibodies were ordered from biomol international ( plymouth meeting , pa ) . lipoprotein lipase ( lpl ) antibody was purchased from genetex ( san antonio , tx ) and -actin from affinity bioreagents ( golden , co ) . ir tyrosine kinase activity was assessed as described by le marchand - brustel et al . briefly , 500 g of muscle lysate at each time point was added to 50 l of agarose bound wheat germ agglutinin incubated at room temperature for 2 h. after washing with buffer a , bound receptor was eluted by 3 ir kinase activity was initialed by adding 40 l of reaction solution consisting of 1 mmol / l dtt , 10 mmol / l mgcl2 , 3 mmol / l mncl2 , and 5 mol / l [ r - p]atp . reactions were carried out for 30 min at 22c and terminated by adding 2 l of 0.5 mol / l edta and 10 l of 500 mol / l atp . then 2 g of ir -subunit antibody and 50 l of protein a agarose beads were added to the reaction mixture and incubated at room temperature for 30 min . after extensive washing with buffer a , 40 l of electrophoresis sample buffer was added and heated at 95c for 4 min . following sds - page ir -subunit tyrosine phosphorylation and ir -subunit abundance were measured by western blot techniques ( 24,25 ) . briefly , 50 g of lysates prepared as described above was subjected to 8% sds - page and transferred to nitrocellulose membrane . ir -subunit phosphorylation was detected with anti phospho - ir ( tyr1150/1151 ) antibody . the bands were detected with anti phospho - irs-1 ( tyr612 ) antibody ( 26 ) . after measuring phospho - irs-1 abundance , the membrane was striped with strip buffer and reprobed with anti total irs-1 antibody to obtain abundance . irs-1associated pi 3-kinase activities of the muscle at each time point were assessed ( 2527 ) . we confirm that wortmannin treatment could completely inhibit irs-1associated pi 3-kinase activity of muscle tissues ( data not shown ) . total akt and pakt ( ser473 ) at each time point were similarly assessed with western blot techniques . protein content for irs-2 , glut4 , pi 3-kinase ( p85 ) , lpl , sgk1 , sirt1 , and stat3 in the lysates was measured also using western blot analysis . results of scanning for each gel were normalized by -actin , and the data are presented as mean sem of fold change in cr versus al . total rna was extracted from muscle obtained for microarray and real - time quantitative pcr ( qpcr ) assays . frozen tissues were placed in a mortar in liquid nitrogen , and the tissue was pulverized into powder using a pestle on dry ice . total rna was isolated from the tissue powder using trizol reagent ( invitrogen , carlsbad , ca ) . after dnase i ( invitrogen ) digestion , rna was further purified with an rneasy mini kit ( qiagen , germantown , md ) . rna concentration and quality were measured by an rna 6000 nano labchip kit ( agilent technologies , santa clara , ca ) . the applied biosystems human genome survey microarray version 2.0 chip containing 32,878 oligonucleotide probes ( 60-mer ) representing 29,098 individual human genes and more than 1,000 control probes was used for microarray profiling for 10 animals , 5 randomly chosen from each group . based dna arrays can be used effectively to detect differential gene expression in a nonhuman primate ( 28 ) . digoxigenin - utp labeled crna was generated and linearly amplified from 1 g of total rna from each sample using an applied biosystems chemiluminescent rt - ivt labeling kit according to the manufacturer 's protocol . after crna was fragmented by heating at 60c for 30 min , 10 g of crna fragments were hybridized at 55c for 16 h. chemiluminescence detection , image acquisition , and analysis were performed according to the manufacturer 's protocol ( applied biosystems , foster , ca ) . signals were quantified and corrected for background , and final images and feature data were processed by applied biosystems 1700 chemiluminescent microarray analyzer software version 1.1 . the primer sequences of candidate genes were designed using primer express software version 3.0 ( applied biosystems ) . a 1-g aliquot of total rna for each sample was reverse transcribed in a 100-l reaction volume with a commercial high - capacity cdna archive kit ( applied biosystems ) according to the manufacturer 's protocol . the qpcr was conducted in 384-well microtiter plates on the abi prism sequence detector 7900 ( applied biosystems ) with bio - rad itaqtm sybr green supermix with rox kits . for each sample of each gene , the mrna content of each candidate gene was determined simultaneously in 10 paired ( cr and al ) muscle samples assessed by dna array analysis . 20s proteasome activity in muscle lysates obtained in the basal state was measured in duplicate using a 20s proteasome activity assay kit ( chemicon international , temecula , ca ) . 20s proteasome chymotrypsin activity was measured by incubating 20 g of each lysate with fluorophore 7-amino-4-methylcoumarin ( amc)-labeled peptide substrate llvy - amc at 37c for 60 min . the free amc released by proteasome activity was quantified using a 380/460-nm filter set in a fluorometer ( biotex , winooski , vt ) . proteasome activity was confirmed using purified 20s proteasome as the positive control and is reported as mol / l amc per mg protein per h. each sample / substrate combination was measured both in the presence and in the absence of mg132 ( 10 mol / l ) or epoxomicin ( 1 mol / l ) , a highly specific 20s proteasome inhibitor ( boston biochem , cambridge , ma ) ( 29 ) , to account for any nonproteasomal degradation of the substrate . the effects of cr on the trial evaluations measured at the specified intervals postrandomization were estimated using repeated - measures ancova . analysis of group differences was adjusted for the prerandomization levels of the outcome measure tested in order to reduce the variance explained by prerandomization predictors . whenever a baseline value was used as a covariate in an ancova model , an interaction term between the group and the covariate was initially included to check the parallelism assumption . if the interaction was not significant at the 0.10 level of significance , and it was always the case , the interaction term was omitted . for gene expression , within - array normalization was done with the scanning software from applied biosystems based on housekeeping genes on each array . differentially expressed genes between the cr and al groups were determined based on the following criteria : bonferroni - adjusted p < 0.05 and fold change in the cr over the al group of 1.5 . public databases including david / ease , genmapp , panther , gotm , and treeview version 1.6 were used to assess functional gene cluster analysis ( 31 ) . insulin sensitivity was assessed at 6-month intervals using a modified minimal model and at the end of the study using a hyperinsulinemic - euglycemic clamp as described ( 14 ) . at the baseline of the clamp , after the insulin infusion , repeat biopsies were obtained at 5 , 20 , and 40 min of the clamp . muscle samples ( 200 mg wet wt ) were immediately placed into liquid nitrogen and then stored at 80c . there was no difference between the steady state plasma glucose ( 5.38 0.10 vs. 5.42 0.10 mmol / l ) or plasma insulin levels observed during the clamp ( 1,472 145 vs. 1,535 155 muscle tissue lysates were prepared by dissection and homogenized in buffer a ( 25 mmol / l hepes , ph 7.4 , 1% nonidet p-40 , 137 mmol / l nacl , 1 mmol / l phenylmethylsulfonyl fluoride , 10 g / ml aprotinin , 1 g / ml pepstatin , and 5 g / ml leupeptin ) , using a pro 200 homogenizer ( pro scientific , oxford , ct ) . the samples were centrifuged at 14,000 g for 20 min at 4c , and protein content of the supernatant was determined ( bio - rad protein assay kit ; bio - rad laboratories , hercules , ca ) . supernatants ( 50 g ) were resolved by sds - page and subjected to immunoblotting using chemiluminescence reagent ( perkinelmer life science , boston , ma ) and quantified as described ( 22 ) . the 19s proteasome base anti - s5a / rpn10 antibodies were ordered from calbiochem ( gibbstown , nj ) . antibodies for phospho insulin receptor substrate ( irs)-1 ( tyr612 ) , phospho insulin receptor ( ir ) ( tyr1150/1151 ) , phosphoinositol ( pi ) 3-kinase protein 85 ( p85 of pi 3-kinase ) , phospho - akt ( ser473 ) , irs-1 and irs-2 , 20s proteasome subunit 2i , akt , serum- and glucocorticoid - inducible kinase 1 ( sgk1 ) , signal transducer and activator of transcription 3 ( stat3 ) , and sirt1 antibodies were obtained from upstate biotech ( lake placid , ny ) . anti-19s proteasome lid subunits s9/rpn6 and s14/rpn12 antibodies were ordered from biomol international ( plymouth meeting , pa ) . lipoprotein lipase ( lpl ) antibody was purchased from genetex ( san antonio , tx ) and -actin from affinity bioreagents ( golden , co ) . ir tyrosine kinase activity was assessed as described by le marchand - brustel et al . briefly , 500 g of muscle lysate at each time point was added to 50 l of agarose bound wheat germ agglutinin incubated at room temperature for 2 h. after washing with buffer a , bound receptor was eluted by 3 ir kinase activity was initialed by adding 40 l of reaction solution consisting of 1 mmol / l dtt , 10 mmol / l mgcl2 , 3 mmol / l mncl2 , and 5 mol / l [ r - p]atp . reactions were carried out for 30 min at 22c and terminated by adding 2 l of 0.5 mol / l edta and 10 l of 500 mol / l atp . then 2 g of ir -subunit antibody and 50 l of protein a agarose beads were added to the reaction mixture and incubated at room temperature for 30 min . after extensive washing with buffer a , 40 l of electrophoresis sample buffer was added and heated at 95c for 4 min . following sds - page , ir -subunit tyrosine phosphorylation and ir -subunit abundance were measured by western blot techniques ( 24,25 ) . briefly , 50 g of lysates prepared as described above was subjected to 8% sds - page and transferred to nitrocellulose membrane . ir -subunit phosphorylation was detected with anti phospho - ir ( tyr1150/1151 ) antibody . the membrane was striped with strip buffer and reprobed with anti ir -subunit and -actin antibodies , respectively . to assess irs-1 phosphorylation , muscle lysates were subjected to sds - page ( 24 ) . the bands were detected with anti phospho - irs-1 ( tyr612 ) antibody ( 26 ) . after measuring phospho - irs-1 abundance , the membrane was striped with strip buffer and reprobed with anti total irs-1 antibody to obtain abundance . irs-1associated pi 3-kinase activities of the muscle at each time point were assessed ( 2527 ) . we confirm that wortmannin treatment could completely inhibit irs-1associated pi 3-kinase activity of muscle tissues ( data not shown ) . total akt and pakt ( ser473 ) at each time point were similarly assessed with western blot techniques . protein content for irs-2 , glut4 , pi 3-kinase ( p85 ) , lpl , sgk1 , sirt1 , and stat3 in the lysates was measured also using western blot analysis . results of scanning for each gel were normalized by -actin , and the data are presented as mean sem of fold change in cr versus al . total rna was extracted from muscle obtained for microarray and real - time quantitative pcr ( qpcr ) assays . frozen tissues were placed in a mortar in liquid nitrogen , and the tissue was pulverized into powder using a pestle on dry ice . total rna was isolated from the tissue powder using trizol reagent ( invitrogen , carlsbad , ca ) . after dnase i ( invitrogen ) digestion , rna was further purified with an rneasy mini kit ( qiagen , germantown , md ) . rna concentration and quality were measured by an rna 6000 nano labchip kit ( agilent technologies , santa clara , ca ) . the applied biosystems human genome survey microarray version 2.0 chip containing 32,878 oligonucleotide probes ( 60-mer ) representing 29,098 individual human genes and more than 1,000 control probes was used for microarray profiling for 10 animals , 5 randomly chosen from each group . based dna arrays can be used effectively to detect differential gene expression in a nonhuman primate ( 28 ) . digoxigenin - utp labeled crna was generated and linearly amplified from 1 g of total rna from each sample using an applied biosystems chemiluminescent rt - ivt labeling kit according to the manufacturer 's protocol . after crna was fragmented by heating at 60c for 30 min , 10 g of crna fragments were hybridized at 55c for 16 h. chemiluminescence detection , image acquisition , and analysis were performed according to the manufacturer 's protocol ( applied biosystems , foster , ca ) . signals were quantified and corrected for background , and final images and feature data were processed by applied biosystems 1700 chemiluminescent microarray analyzer software version 1.1 . the primer sequences of candidate genes were designed using primer express software version 3.0 ( applied biosystems ) . a 1-g aliquot of total rna for each sample was reverse transcribed in a 100-l reaction volume with a commercial high - capacity cdna archive kit ( applied biosystems ) according to the manufacturer 's protocol . the qpcr was conducted in 384-well microtiter plates on the abi prism sequence detector 7900 ( applied biosystems ) with bio - rad itaqtm sybr green supermix with rox kits . for each sample of each gene , the mrna content of each candidate gene was determined simultaneously in 10 paired ( cr and al ) muscle samples assessed by dna array analysis . 20s proteasome activity in muscle lysates obtained in the basal state was measured in duplicate using a 20s proteasome activity assay kit ( chemicon international , temecula , ca ) . 20s proteasome chymotrypsin activity was measured by incubating 20 g of each lysate with fluorophore 7-amino-4-methylcoumarin ( amc)-labeled peptide substrate llvy - amc at 37c for 60 min . the free amc released by proteasome activity was quantified using a 380/460-nm filter set in a fluorometer ( biotex , winooski , vt ) . proteasome activity was confirmed using purified 20s proteasome as the positive control and is reported as mol / l amc per mg protein per h. each sample / substrate combination was measured both in the presence and in the absence of mg132 ( 10 mol / l ) or epoxomicin ( 1 mol / l ) , a highly specific 20s proteasome inhibitor ( boston biochem , cambridge , ma ) ( 29 ) , to account for any nonproteasomal degradation of the substrate . the effects of cr on the trial evaluations measured at the specified intervals postrandomization were estimated using repeated - measures ancova . analysis of group differences was adjusted for the prerandomization levels of the outcome measure tested in order to reduce the variance explained by prerandomization predictors . all tests of hypotheses and reported p values were two - sided . whenever a baseline value was used as a covariate in an ancova model , an interaction term between the group and the covariate was initially included to check the parallelism assumption . if the interaction was not significant at the 0.10 level of significance , and it was always the case , the interaction term was omitted . for gene expression , within - array normalization was done with the scanning software from applied biosystems based on housekeeping genes on each array . differentially expressed genes between the cr and al groups were determined based on the following criteria : bonferroni - adjusted p < 0.05 and fold change in the cr over the al group of 1.5 . public databases including david / ease , genmapp , panther , gotm , and treeview version 1.6 were used to assess functional gene cluster analysis ( 31 ) . the clinical , phenotypic , and metabolic effects of cr for the cohort of cynomolgus monkeys ( macaca fascicularis ) evaluated were previously reported in detail ( 14 ) . compared with the al group , animals randomized to cr were observed to have significantly reduced dietary intake , reduced body weight , increased insulin sensitivity , and reduced intra - abdominal fat with aging ( fig . the effect of cr was noted during the 1st year of observation and maintained over the 4 years of observation , as demonstrated not only from periodic assessment of insulin sensitivity with the minimal model ( fig . 2c ) , but also from the assessment with hyperinsulinemic - euglycemic clamps obtained at study end ( fig . 2d ) ( 14 ) . caloric intake ( a ) and body weight ( b ) for both treatment groups over 4 years of observations are demonstrated . c demonstrates results of insulin sensitivity assessed every 6 months over the course of study , using the minimal model technique . d demonstrates insulin sensitivity assessed by hyperinsulinemic - euglycemic clamps conducted at the end of the study . si units = 10 min u ml . * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 for cr versus al . ( all data presented in this figure have been previously published , and the figure was modified from cefalu et al . with permission . ) animals randomized to cr had significantly increased skeletal muscle protein abundance of irs-1 , irs-2 , ir -subunit , pi 3-kinase ( p85 ) , and glut4 compared with that in the al group ( fig . when compared with al animals , animals randomized to cr had enhanced insulin - stimulated skeletal muscle ir tyrosine kinase activity ( data not shown ) and increased ir -subunit phosphorylation ( fig . irs-1 protein levels measured at the 5- , 20- , and 40-min time points did not differ from the value assessed as the 0 time point . although there was no significant difference in basal pi 3-kinase activity between the cr and al groups , insulin - stimulated pi 3-kinase activities were significantly higher when assessed at all time points post insulin stimulation in the cr group compared with those in the al group ( fig . akt phosphorylation post insulin stimulation was also increased in skeletal muscle with cr when compared with al ( fig . 4d ) . content of insulin receptor signaling proteins in the monkey skeletal muscle obtained at basal state is demonstrated . irs-1 , irs-2 , ir -subunit , pi 3-kinase ( p85 ) , and glut4 protein abundance in the muscle were measured by western blot analysis . data are means sem ( n = 13 per group ) as fold change of al at baseline . * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 for cr versus al . ir -subunit phosphorylation ( a ) , irs-1 phosphorylation ( b ) , irs-1associated pi 3-kinase activities ( c ) , and total akt and pakt ( ser473 ) ( d ) in the muscles at baseline ( 0 time point ) and at 5 , 20 , and 40 min post insulin stimulation are demonstrated . data are means sem ( n = 6 per group ) . * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 for cr versus al . a total of 241 genes were identified as significantly differentially expressed with cr from 10,163 probes with a satisfactory quality of signals over all the array slides . among them , 179 genes were observed to be downregulated and 62 genes upregulated . using cluster analysis , gene expression differed in 11 categories of biological processes with 18% of genes involved in either carbohydrate and lipid metabolism or signaltransduction ( fig . tables 1 and 2 list genes of interest in the muscle that were either significantly upregulated or downregulated with cr for each biological process , respectively . percentage of 241 genes modulated by cr and sorted by biological process for which fold - change was 1.5 and p was < genes observed to be upregulated by cr genes downregulated by cr to confirm the microarray findings , real - time qpcr assays were conducted . altogether , 27 genes were selected from the microarray analysis to measure transcription levels using rt - qpcr for which 22 were confirmed to have significant changes in the cr compared with the al group ( table 3 ) . two genes , i.e. , stat3 and interleukin 6 signal transducer ( il6st ) , did not show significant changes between al and cr conditions as assessed with real - time qpcr assays as opposed to the microarray analysis , and expression of the type 3 iodothyronine deiodinase ( dio3 ) gene with rt - qpcr did not agree with the findings from the microarray . in addition , sirt1 transcriptional level in muscle from the cr monkeys was significantly increased compared with that from the al monkeys , as assessed by rt - qpcr assay . genes confirmed by rt - qpcr in skeletal muscle from the list of genes confirmed by rt - qpcr , lpl , sgk1 , sirt1 , and stat3 were assessed for protein abundance ( fig . the protein abundance of lpl , sgk1 , and sirt1 was significantly increased in the cr compared with the al group ( 97 , 57 , and 35% , respectively ) . in agreement with the pcr data , protein expression of stat3 in the cr monkeys because increased gene expression of insulin signaling proteins ( i.e. , irs-1 , irs-2 , and pi 3-kinase ) was not noted , yet increased protein content was observed , factors that regulate protein content ( i.e. , degradation ) were sought . the majority of intracellular proteins are degraded by the 26s proteasome ( 32 ) . given the inhibitory effect of insulin reported for proteasome - dependent protein degradation ( 19,20 ) , 20s proteasome activity in muscle lysates was significantly reduced in the cr group compared with the al group , although nonproteasomal degradation measured in the presence of two independent proteasome inhibitors , i.e. , mg132 or epoxomicin , was unchanged ( fig . 7a ) . decreased 20s proteasome activity with cr was associated with significantly decreased abundance of selected subunits of the 26s proteasome , including a subunit of the 20s catalytic core ( 2i ) and subunits of the 19s regulatory complex base ( s5a , which contains a ubiquitin binding site ) and lid ( rpn6 and rpn12 , two of eight non - atpase subunits ) ( fig . protein content for lpl , sgk1 , sirt1 , and stat3 in the muscle tissues obtained at the basal time point . data are means sem ( n = 13 per group ) . * p < 0.05 , * * p < 0.01 , * * * p < 0.001 for cr versus al . abundance of selected 26s proteasome subunits and 20s proteasome activity measurements in the muscle of cr and al monkeys at the basal time point . a : 20s proteasome activity measured in the absence or presence of the proteasome inhibitors mg132 ( 10 mol / l ) or epoxomicin ( 1 20s proteasome activity was measured as the hydrolysis of the fluorogenic peptidyl substrate suc - llvy - amc and is reported as mol / l amc per mg protein per h. b and c : levels of the 20s proteasome subunit 2i and 19s complex subunits s5a / rpn10 , s9/rpn6 , and s14/rpn12 in muscle lysate analyzed by western blot analysis . * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 for cr versus al . animals randomized to cr had significantly increased skeletal muscle protein abundance of irs-1 , irs-2 , ir -subunit , pi 3-kinase ( p85 ) , and glut4 compared with that in the al group ( fig . 3 ) . when compared with al animals , animals randomized to cr had enhanced insulin - stimulated skeletal muscle ir tyrosine kinase activity ( data not shown ) and increased ir -subunit phosphorylation ( fig . irs-1 protein levels measured at the 5- , 20- , and 40-min time points did not differ from the value assessed as the 0 time point . although there was no significant difference in basal pi 3-kinase activity between the cr and al groups , insulin - stimulated pi 3-kinase activities were significantly higher when assessed at all time points post insulin stimulation in the cr group compared with those in the al group ( fig . akt phosphorylation post insulin stimulation was also increased in skeletal muscle with cr when compared with al ( fig . 4d ) . content of insulin receptor signaling proteins in the monkey skeletal muscle obtained at basal state is demonstrated . irs-1 , irs-2 , ir -subunit , pi 3-kinase ( p85 ) , and glut4 protein abundance in the muscle were measured by western blot analysis . data are means sem ( n = 13 per group ) as fold change of al at baseline . * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 for cr versus al . ir -subunit phosphorylation ( a ) , irs-1 phosphorylation ( b ) , irs-1associated pi 3-kinase activities ( c ) , and total akt and pakt ( ser473 ) ( d ) in the muscles at baseline ( 0 time point ) and at 5 , 20 , and 40 min post insulin stimulation are demonstrated . * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 for cr versus al . a total of 241 genes were identified as significantly differentially expressed with cr from 10,163 probes with a satisfactory quality of signals over all the array slides . among them , 179 genes were observed to be downregulated and 62 genes upregulated . using cluster analysis , gene expression differed in 11 categories of biological processes with 18% of genes involved in either carbohydrate and lipid metabolism or signaltransduction ( fig . tables 1 and 2 list genes of interest in the muscle that were either significantly upregulated or downregulated with cr for each biological process , respectively . percentage of 241 genes modulated by cr and sorted by biological process for which fold - change was 1.5 and p was < genes observed to be upregulated by cr genes downregulated by cr to confirm the microarray findings , real - time qpcr assays were conducted . altogether , 27 genes were selected from the microarray analysis to measure transcription levels using rt - qpcr for which 22 were confirmed to have significant changes in the cr compared with the al group ( table 3 ) . two genes , i.e. , stat3 and interleukin 6 signal transducer ( il6st ) , did not show significant changes between al and cr conditions as assessed with real - time qpcr assays as opposed to the microarray analysis , and expression of the type 3 iodothyronine deiodinase ( dio3 ) gene with rt - qpcr did not agree with the findings from the microarray . in addition , sirt1 transcriptional level in muscle from the cr monkeys was significantly increased compared with that from the al monkeys , as assessed by rt - qpcr assay . genes confirmed by rt - qpcr in skeletal muscle from the list of genes confirmed by rt - qpcr , lpl , sgk1 , sirt1 , and stat3 were assessed for protein abundance ( fig . the protein abundance of lpl , sgk1 , and sirt1 was significantly increased in the cr compared with the al group ( 97 , 57 , and 35% , respectively ) . in agreement with the pcr data , protein expression of stat3 in the cr monkeys because increased gene expression of insulin signaling proteins ( i.e. , irs-1 , irs-2 , and pi 3-kinase ) was not noted , yet increased protein content was observed , factors that regulate protein content ( i.e. , degradation ) were sought . the majority of intracellular proteins are degraded by the 26s proteasome ( 32 ) . given the inhibitory effect of insulin reported for proteasome - dependent protein degradation ( 19,20 ) , 20s proteasome activity was assayed in the monkey muscle lysates . 20s proteasome activity in muscle lysates was significantly reduced in the cr group compared with the al group , although nonproteasomal degradation measured in the presence of two independent proteasome inhibitors , i.e. , mg132 or epoxomicin , was unchanged ( fig . 7a ) . decreased 20s proteasome activity with cr was associated with significantly decreased abundance of selected subunits of the 26s proteasome , including a subunit of the 20s catalytic core ( 2i ) and subunits of the 19s regulatory complex base ( s5a , which contains a ubiquitin binding site ) and lid ( rpn6 and rpn12 , two of eight non - atpase subunits ) ( fig . protein content for lpl , sgk1 , sirt1 , and stat3 in the muscle tissues obtained at the basal time point . data are means sem ( n = 13 per group ) . * p < 0.05 , * * p < 0.01 , * * * p < 0.001 for cr versus al . abundance of selected 26s proteasome subunits and 20s proteasome activity measurements in the muscle of cr and al monkeys at the basal time point . a : 20s proteasome activity measured in the absence or presence of the proteasome inhibitors mg132 ( 10 mol / l ) or epoxomicin ( 1 mol / l ) and performed in duplicate . 20s proteasome activity was measured as the hydrolysis of the fluorogenic peptidyl substrate suc - llvy - amc and is reported as mol / l amc per mg protein per h. b and c : levels of the 20s proteasome subunit 2i and 19s complex subunits s5a / rpn10 , s9/rpn6 , and s14/rpn12 in muscle lysate analyzed by western blot analysis . * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 for cr versus al . one of the most consistent physiological changes observed with cr is the favorable effect on glucoregulation , i.e. , increased insulin sensitivity , as reported for both rhesus monkeys and rodents ( 13,1517 ) . our data confirm that cr markedly improved insulin sensitivity in cynomolgus monkeys when evaluated over a 4-year period ( 14 ) . in addition , we provide novel data regarding the cellular mechanism of action of cr because the enhanced insulin action observed in animals randomized to cr , as opposed to al , was associated with increased content of proteins of the ir signal transduction pathway . enhanced insulin signaling was demonstrated in our study with the finding of increases in ir kinase activities , ir -subunit phosphorylation , and irs-1associated pi 3-kinase activities following insulin stimulation in the muscle tissues in the cr group . the mechanism by which insulin signaling is enhanced with cr has been suggested to be secondary to an effect on transcriptional regulation . for example , it has been reported that liver ir , igf-1r , and irs-1 mrna were greater in older rats subjected to 25 months of 40% cr than in al - fed rats ( 33 ) and that cr also had greater effect on the cardiac gene expression of ir , irs-1 , igf-1 , igf-1r , and glut4 compared with that in age - matched controls ( 34 ) . in contrast , studies reported for primates subjected to cr do not support the observations in rodents as related to transcription . ( 35 ) evaluated skeletal muscle from rhesus monkeys subjected to cr and reported increases in gene expression of glut4 , which agree with our data . there were clear differences in our study compared with the other reported studies in that we evaluated a different primate species ( i.e. , cynomolgus vs. rhesus monkeys ) , a different dietary content ( i.e. , atherogenic diet vs. chow ) , and a later start date for initiation of cr ( 1418,35 ) . given our observations , the major question would be the mechanism by which insulin signaling protein content in muscle is increased without a significant effect on transcriptional regulation . ambient protein levels are determined by the coordinated interplay of metabolic processes that involve transcription , mrna translation , and degradation ( 36 ) . transcription and degradation mechanisms have received significant attention , and regulation at the level of mrna translation as an independent mechanism is suggested when mrna and protein levels do not correlate ( 36 ) . it is also reported that the content of irs-1 and irs-2 is influenced by many factors , including growth factors and cytokines , and conditions associated with insulin resistance have been reported to exhibit increases in whole - body protein degradation ( 37,38 ) . studies using cell lines or isolated primary cells chronically exposed to insulin have revealed that the reduced level of irs-1 protein is due to enhanced degradation ( 39 ) . other studies have demonstrated that a major effect of insulin is regulation of protein degradation mediated by the proteasome ( 19,20 ) . based on these reports and from our microarray findings , the next logical step was to evaluate whether the proteins involved in proteasomal degradation are modulated by cr . ubiquitin - dependent proteolysis plays an important role in regulating fundamental biological functions , including cell division and cellular differentiation . the proteasome degradation system is composed of two distinct and successive steps : ubiquitin conjugation and proteasome degradation ( 40 ) . following activation , one of several e2 enzymes ( ubiquitin - conjugating proteins ) transfers ubiquitin from e1 to a member of the ubiquitin - protein ligase family , e3 , to which the substrate protein is specifically bound . e3 catalyzes the last step in the conjugation process , covalent attachment of ubiquitin to the substrate ( 41 ) . ubiquitin - tagged proteins are then recognized and degraded by the 26s proteasome complex , which consists of the 20s catalytic core complex capped on one or both ends by 19s regulatory complexes ( 32 ) . in support of the hypothesis that cr may modulate the ubiquitin - proteasome system , transcriptional levels of ubiquitin - specific peptidase 2 and 18 ( usp2 and usp18 ) were reduced . moreover , skeletal muscle from animals randomized to cr had decreased 20s proteasome activity and greatly reduced protein abundance of selected proteasome subunits . collectively , our data support the hypothesis that cr enhances the insulin signaling cascade , and one of the contributing mechanisms may be secondary to modulating the ubiquitin - proteasome system in skeletal muscle . there were other genes of interest that were modulated with cr including those involved in lipid metabolism , e.g. , lpl and related enzyme transcriptional levels ( tables 13 ) . in addition , we observed significantly increased sirt1 protein abundance and gene expression with cr in primates . this result appears to be consistent with data found in rodent studies ( 11,42 ) , but other investigators suggest that the regulation of sirt1 activity during cr may be tissue specific ( 43 ) . thus , the relationship between sirt1 and insulin signaling in cr animals needs to be further studied . our data demonstrating that insulin signaling proteins are increased in muscle with cr appear to be in contrast to many published studies . for example , gazdag et al . ( 18 ) evaluated cr in rhesus monkeys and reported only that the content of irs-1 approached significance ( p = 0.051 ) . friedman et al . ( 44 ) reported no change in glut4 protein in skeletal muscle but observed a significant improvement in insulin sensitivity in obese human subjects after an average weight loss of 36% . ( 45 ) reported improvement in insulin sensitivity with enhanced insulin - stimulated receptor and irs-1 tyrosine phosphorylation without change in protein content in muscle . previously , we reported that there was no change in glut4 levels for heart or diaphragm muscle between al or cr animals despite increase in insulin sensitivity ( 22 ) . thus , it is important to note that an increase in insulin signaling proteins or glut4 abundance does not appear to be essential for increased insulin sensitivity with reduction in caloric intake . taken together , the mechanism by which cr has its effects on insulin action in cynomolgus monkeys may be different from those reported in other species that demonstrate improved insulin action with cr ( i.e. , humans , rhesus monkeys , rats , and mice ) ( 13,1318,42 ) . it is not known at this time whether the differences are related to the species , composition of diet , age at cr initiation , length of cr intervention , or other factors . in summary , our data demonstrate that cr enhances insulin sensitivity and skeletal muscle content of insulin signaling proteins in cynomolgus monkeys . because ambient protein levels are determined by transcription , mrna translation , and degradation , any of these processes could have contributed to the observations . however , the data do support the finding that a contributing cellular mechanism by which cr enhances insulin action in vivo may be secondary to modulation of protein degradation via the ubiquitin - proteasome system . the mechanism for the specificity for the sparing of degradation of insulin signaling proteins , but not most proteins , remains unexplained and will need to be confirmed with more precise mechanistic studies .

objectivecaloric restriction ( cr ) has been shown to retard aging processes , extend maximal life span , and consistently increase insulin action in experimental animals . the mechanism by which cr enhances insulin action , specifically in higher species , is not precisely known . we sought to examine insulin receptor signaling and transcriptional alterations in skeletal muscle of nonhuman primates subjected to cr over a 4-year period.research design and methodsat baseline , 32 male adult cynomolgus monkeys ( macaca fascicularis ) were randomized to an ad libitum ( al ) diet or to 30% cr . dietary intake , body weight , and insulin sensitivity were obtained at routine intervals over 4 years . at the end of the study , hyperinsulinemic - euglycemic clamps were performed and skeletal muscle ( vastus lateralis ) was obtained in the basal and insulin - stimulated states for insulin receptor signaling and gene expression profiling.resultscr significantly increased whole - body insulin mediated glucose disposal compared with al diet and increased insulin receptor signaling , i.e. , insulin receptor substrate ( irs)-1 , insulin receptor phosphorylation , and irs associated pi 3-kinase activity in skeletal muscle ( p < 0.01 , p < 0.01 , and p < 0.01 , respectively ) . gene expression for insulin signaling proteins , i.e. , irs-1 and irs-2 , were not increased with cr , although a significant increase in protein abundance was noted . components of the ubiquitin - proteasome system , i.e. , 20s and 19s proteasome subunit abundance and 20s proteasome activity , were significantly decreased by cr.conclusionscr increases insulin sensitivity on a whole - body level and enhances insulin receptor signaling in this higher species . cr in cynomolgus monkeys may alter insulin signaling in vivo by modulating protein content of insulin receptor signaling proteins .

RESEARCH DESIGN AND METHODS Insulin sensitivity. Tissue preparation. IR tyrosine kinase activity. IR -subunit tyrosine phosphorylation. Insulin-stimulated tyrosine phosphorylation of IRS-1, PI 3-kinase activity, and Akt phosphorylation. RNA extraction. Gene expression. Real-time qPCR. 20S proteasome activity assay. Statistical analysis. RESULTS Insulin signaling. Genomic analysis. DISCUSSION

the effect of chronic cr to modulate cellular insulin signaling and transcriptional regulation was assessed in skeletal muscle obtained from nonhuman primates subjected to a 4-year period of 30% cr compared with ad libitum ( al ) feeding conditions . specifically , 32 feral adult male cynomolgus monkeys ( macaca fascicularis ) ( mean sem age 8.2 1.2 years ) were part of a randomized trial in which the independent effect of cr and its interaction with insulin resistance and atherosclerotic lesion extent and composition were evaluated ( fig . insulin sensitivity was assessed at 6-month intervals using a modified minimal model and at the end of the study using a hyperinsulinemic - euglycemic clamp as described ( 14 ) . at the baseline of the clamp , after the insulin infusion , repeat biopsies were obtained at 5 , 20 , and 40 min of the clamp . the samples were centrifuged at 14,000 g for 20 min at 4c , and protein content of the supernatant was determined ( bio - rad protein assay kit ; bio - rad laboratories , hercules , ca ) . antibodies for phospho insulin receptor substrate ( irs)-1 ( tyr612 ) , phospho insulin receptor ( ir ) ( tyr1150/1151 ) , phosphoinositol ( pi ) 3-kinase protein 85 ( p85 of pi 3-kinase ) , phospho - akt ( ser473 ) , irs-1 and irs-2 , 20s proteasome subunit 2i , akt , serum- and glucocorticoid - inducible kinase 1 ( sgk1 ) , signal transducer and activator of transcription 3 ( stat3 ) , and sirt1 antibodies were obtained from upstate biotech ( lake placid , ny ) . protein content for irs-2 , glut4 , pi 3-kinase ( p85 ) , lpl , sgk1 , sirt1 , and stat3 in the lysates was measured also using western blot analysis . 20s proteasome activity in muscle lysates obtained in the basal state was measured in duplicate using a 20s proteasome activity assay kit ( chemicon international , temecula , ca ) . proteasome activity was confirmed using purified 20s proteasome as the positive control and is reported as mol / l amc per mg protein per h. each sample / substrate combination was measured both in the presence and in the absence of mg132 ( 10 mol / l ) or epoxomicin ( 1 mol / l ) , a highly specific 20s proteasome inhibitor ( boston biochem , cambridge , ma ) ( 29 ) , to account for any nonproteasomal degradation of the substrate . differentially expressed genes between the cr and al groups were determined based on the following criteria : bonferroni - adjusted p < 0.05 and fold change in the cr over the al group of 1.5 . insulin sensitivity was assessed at 6-month intervals using a modified minimal model and at the end of the study using a hyperinsulinemic - euglycemic clamp as described ( 14 ) . at the baseline of the clamp , after the insulin infusion , repeat biopsies were obtained at 5 , 20 , and 40 min of the clamp . the samples were centrifuged at 14,000 g for 20 min at 4c , and protein content of the supernatant was determined ( bio - rad protein assay kit ; bio - rad laboratories , hercules , ca ) . antibodies for phospho insulin receptor substrate ( irs)-1 ( tyr612 ) , phospho insulin receptor ( ir ) ( tyr1150/1151 ) , phosphoinositol ( pi ) 3-kinase protein 85 ( p85 of pi 3-kinase ) , phospho - akt ( ser473 ) , irs-1 and irs-2 , 20s proteasome subunit 2i , akt , serum- and glucocorticoid - inducible kinase 1 ( sgk1 ) , signal transducer and activator of transcription 3 ( stat3 ) , and sirt1 antibodies were obtained from upstate biotech ( lake placid , ny ) . protein content for irs-2 , glut4 , pi 3-kinase ( p85 ) , lpl , sgk1 , sirt1 , and stat3 in the lysates was measured also using western blot analysis . 20s proteasome activity in muscle lysates obtained in the basal state was measured in duplicate using a 20s proteasome activity assay kit ( chemicon international , temecula , ca ) . proteasome activity was confirmed using purified 20s proteasome as the positive control and is reported as mol / l amc per mg protein per h. each sample / substrate combination was measured both in the presence and in the absence of mg132 ( 10 mol / l ) or epoxomicin ( 1 mol / l ) , a highly specific 20s proteasome inhibitor ( boston biochem , cambridge , ma ) ( 29 ) , to account for any nonproteasomal degradation of the substrate . differentially expressed genes between the cr and al groups were determined based on the following criteria : bonferroni - adjusted p < 0.05 and fold change in the cr over the al group of 1.5 . the clinical , phenotypic , and metabolic effects of cr for the cohort of cynomolgus monkeys ( macaca fascicularis ) evaluated were previously reported in detail ( 14 ) . compared with the al group , animals randomized to cr were observed to have significantly reduced dietary intake , reduced body weight , increased insulin sensitivity , and reduced intra - abdominal fat with aging ( fig . the effect of cr was noted during the 1st year of observation and maintained over the 4 years of observation , as demonstrated not only from periodic assessment of insulin sensitivity with the minimal model ( fig . 2c ) , but also from the assessment with hyperinsulinemic - euglycemic clamps obtained at study end ( fig . caloric intake ( a ) and body weight ( b ) for both treatment groups over 4 years of observations are demonstrated . d demonstrates insulin sensitivity assessed by hyperinsulinemic - euglycemic clamps conducted at the end of the study . * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 for cr versus al . animals randomized to cr had significantly increased skeletal muscle protein abundance of irs-1 , irs-2 , ir -subunit , pi 3-kinase ( p85 ) , and glut4 compared with that in the al group ( fig . when compared with al animals , animals randomized to cr had enhanced insulin - stimulated skeletal muscle ir tyrosine kinase activity ( data not shown ) and increased ir -subunit phosphorylation ( fig . although there was no significant difference in basal pi 3-kinase activity between the cr and al groups , insulin - stimulated pi 3-kinase activities were significantly higher when assessed at all time points post insulin stimulation in the cr group compared with those in the al group ( fig . akt phosphorylation post insulin stimulation was also increased in skeletal muscle with cr when compared with al ( fig . content of insulin receptor signaling proteins in the monkey skeletal muscle obtained at basal state is demonstrated . irs-1 , irs-2 , ir -subunit , pi 3-kinase ( p85 ) , and glut4 protein abundance in the muscle were measured by western blot analysis . * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 for cr versus al . ir -subunit phosphorylation ( a ) , irs-1 phosphorylation ( b ) , irs-1associated pi 3-kinase activities ( c ) , and total akt and pakt ( ser473 ) ( d ) in the muscles at baseline ( 0 time point ) and at 5 , 20 , and 40 min post insulin stimulation are demonstrated . * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 for cr versus al . genes confirmed by rt - qpcr in skeletal muscle from the list of genes confirmed by rt - qpcr , lpl , sgk1 , sirt1 , and stat3 were assessed for protein abundance ( fig . the protein abundance of lpl , sgk1 , and sirt1 was significantly increased in the cr compared with the al group ( 97 , 57 , and 35% , respectively ) . in agreement with the pcr data , protein expression of stat3 in the cr monkeys because increased gene expression of insulin signaling proteins ( i.e. , irs-1 , irs-2 , and pi 3-kinase ) was not noted , yet increased protein content was observed , factors that regulate protein content ( i.e. given the inhibitory effect of insulin reported for proteasome - dependent protein degradation ( 19,20 ) , 20s proteasome activity in muscle lysates was significantly reduced in the cr group compared with the al group , although nonproteasomal degradation measured in the presence of two independent proteasome inhibitors , i.e. decreased 20s proteasome activity with cr was associated with significantly decreased abundance of selected subunits of the 26s proteasome , including a subunit of the 20s catalytic core ( 2i ) and subunits of the 19s regulatory complex base ( s5a , which contains a ubiquitin binding site ) and lid ( rpn6 and rpn12 , two of eight non - atpase subunits ) ( fig . protein content for lpl , sgk1 , sirt1 , and stat3 in the muscle tissues obtained at the basal time point . * p < 0.05 , * * p < 0.01 , * * * p < 0.001 for cr versus al . abundance of selected 26s proteasome subunits and 20s proteasome activity measurements in the muscle of cr and al monkeys at the basal time point . a : 20s proteasome activity measured in the absence or presence of the proteasome inhibitors mg132 ( 10 mol / l ) or epoxomicin ( 1 20s proteasome activity was measured as the hydrolysis of the fluorogenic peptidyl substrate suc - llvy - amc and is reported as mol / l amc per mg protein per h. b and c : levels of the 20s proteasome subunit 2i and 19s complex subunits s5a / rpn10 , s9/rpn6 , and s14/rpn12 in muscle lysate analyzed by western blot analysis . * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 for cr versus al . animals randomized to cr had significantly increased skeletal muscle protein abundance of irs-1 , irs-2 , ir -subunit , pi 3-kinase ( p85 ) , and glut4 compared with that in the al group ( fig . when compared with al animals , animals randomized to cr had enhanced insulin - stimulated skeletal muscle ir tyrosine kinase activity ( data not shown ) and increased ir -subunit phosphorylation ( fig . although there was no significant difference in basal pi 3-kinase activity between the cr and al groups , insulin - stimulated pi 3-kinase activities were significantly higher when assessed at all time points post insulin stimulation in the cr group compared with those in the al group ( fig . akt phosphorylation post insulin stimulation was also increased in skeletal muscle with cr when compared with al ( fig . content of insulin receptor signaling proteins in the monkey skeletal muscle obtained at basal state is demonstrated . irs-1 , irs-2 , ir -subunit , pi 3-kinase ( p85 ) , and glut4 protein abundance in the muscle were measured by western blot analysis . * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 for cr versus al . ir -subunit phosphorylation ( a ) , irs-1 phosphorylation ( b ) , irs-1associated pi 3-kinase activities ( c ) , and total akt and pakt ( ser473 ) ( d ) in the muscles at baseline ( 0 time point ) and at 5 , 20 , and 40 min post insulin stimulation are demonstrated . * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 for cr versus al . tables 1 and 2 list genes of interest in the muscle that were either significantly upregulated or downregulated with cr for each biological process , respectively . genes confirmed by rt - qpcr in skeletal muscle from the list of genes confirmed by rt - qpcr , lpl , sgk1 , sirt1 , and stat3 were assessed for protein abundance ( fig . the protein abundance of lpl , sgk1 , and sirt1 was significantly increased in the cr compared with the al group ( 97 , 57 , and 35% , respectively ) . in agreement with the pcr data , protein expression of stat3 in the cr monkeys because increased gene expression of insulin signaling proteins ( i.e. , irs-1 , irs-2 , and pi 3-kinase ) was not noted , yet increased protein content was observed , factors that regulate protein content ( i.e. given the inhibitory effect of insulin reported for proteasome - dependent protein degradation ( 19,20 ) , 20s proteasome activity was assayed in the monkey muscle lysates . 20s proteasome activity in muscle lysates was significantly reduced in the cr group compared with the al group , although nonproteasomal degradation measured in the presence of two independent proteasome inhibitors , i.e. decreased 20s proteasome activity with cr was associated with significantly decreased abundance of selected subunits of the 26s proteasome , including a subunit of the 20s catalytic core ( 2i ) and subunits of the 19s regulatory complex base ( s5a , which contains a ubiquitin binding site ) and lid ( rpn6 and rpn12 , two of eight non - atpase subunits ) ( fig . protein content for lpl , sgk1 , sirt1 , and stat3 in the muscle tissues obtained at the basal time point . * p < 0.05 , * * p < 0.01 , * * * p < 0.001 for cr versus al . abundance of selected 26s proteasome subunits and 20s proteasome activity measurements in the muscle of cr and al monkeys at the basal time point . a : 20s proteasome activity measured in the absence or presence of the proteasome inhibitors mg132 ( 10 mol / l ) or epoxomicin ( 1 mol / l ) and performed in duplicate . 20s proteasome activity was measured as the hydrolysis of the fluorogenic peptidyl substrate suc - llvy - amc and is reported as mol / l amc per mg protein per h. b and c : levels of the 20s proteasome subunit 2i and 19s complex subunits s5a / rpn10 , s9/rpn6 , and s14/rpn12 in muscle lysate analyzed by western blot analysis . * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 for cr versus al . one of the most consistent physiological changes observed with cr is the favorable effect on glucoregulation , i.e. our data confirm that cr markedly improved insulin sensitivity in cynomolgus monkeys when evaluated over a 4-year period ( 14 ) . in addition , we provide novel data regarding the cellular mechanism of action of cr because the enhanced insulin action observed in animals randomized to cr , as opposed to al , was associated with increased content of proteins of the ir signal transduction pathway . enhanced insulin signaling was demonstrated in our study with the finding of increases in ir kinase activities , ir -subunit phosphorylation , and irs-1associated pi 3-kinase activities following insulin stimulation in the muscle tissues in the cr group . the mechanism by which insulin signaling is enhanced with cr has been suggested to be secondary to an effect on transcriptional regulation . for example , it has been reported that liver ir , igf-1r , and irs-1 mrna were greater in older rats subjected to 25 months of 40% cr than in al - fed rats ( 33 ) and that cr also had greater effect on the cardiac gene expression of ir , irs-1 , igf-1 , igf-1r , and glut4 compared with that in age - matched controls ( 34 ) . ( 35 ) evaluated skeletal muscle from rhesus monkeys subjected to cr and reported increases in gene expression of glut4 , which agree with our data . given our observations , the major question would be the mechanism by which insulin signaling protein content in muscle is increased without a significant effect on transcriptional regulation . it is also reported that the content of irs-1 and irs-2 is influenced by many factors , including growth factors and cytokines , and conditions associated with insulin resistance have been reported to exhibit increases in whole - body protein degradation ( 37,38 ) . in support of the hypothesis that cr may modulate the ubiquitin - proteasome system , transcriptional levels of ubiquitin - specific peptidase 2 and 18 ( usp2 and usp18 ) were reduced . moreover , skeletal muscle from animals randomized to cr had decreased 20s proteasome activity and greatly reduced protein abundance of selected proteasome subunits . collectively , our data support the hypothesis that cr enhances the insulin signaling cascade , and one of the contributing mechanisms may be secondary to modulating the ubiquitin - proteasome system in skeletal muscle . in addition , we observed significantly increased sirt1 protein abundance and gene expression with cr in primates . ( 18 ) evaluated cr in rhesus monkeys and reported only that the content of irs-1 approached significance ( p = 0.051 ) . ( 44 ) reported no change in glut4 protein in skeletal muscle but observed a significant improvement in insulin sensitivity in obese human subjects after an average weight loss of 36% . ( 45 ) reported improvement in insulin sensitivity with enhanced insulin - stimulated receptor and irs-1 tyrosine phosphorylation without change in protein content in muscle . thus , it is important to note that an increase in insulin signaling proteins or glut4 abundance does not appear to be essential for increased insulin sensitivity with reduction in caloric intake . taken together , the mechanism by which cr has its effects on insulin action in cynomolgus monkeys may be different from those reported in other species that demonstrate improved insulin action with cr ( i.e. in summary , our data demonstrate that cr enhances insulin sensitivity and skeletal muscle content of insulin signaling proteins in cynomolgus monkeys . however , the data do support the finding that a contributing cellular mechanism by which cr enhances insulin action in vivo may be secondary to modulation of protein degradation via the ubiquitin - proteasome system . the mechanism for the specificity for the sparing of degradation of insulin signaling proteins , but not most proteins , remains unexplained and will need to be confirmed with more precise mechanistic studies .

chronic lymphocytic leukemia ( cll ) is a lymphoid malignancy characterized by the accumulation and proliferation of nonfunctional and monoclonal small cd5/cd19/cd-20/cd23-positive lymphocytes in the blood , bone marrow , and lymphoid tissues [ 1 , 2 ] . it is the most common adult leukemia in the united states , with 15,680 new cases and estimated 4,850 deaths reported by the american cancer society in 2013 . cll is primarily a disease of old age with the median age at diagnosis being 72 years ; its incidence in the male population is reported to be twice that of the female population . diagnosis of cll requires the presence of at least 5,000 monoclonal mature appearing b - lymphocytes per microliter in the peripheral blood . cll is a slowly progressive disease , with an 82% five - year survival rate . the treatment strategies of cll are highly individualized with patients in the early and stable stages of cll not requiring treatment . however , those with progressive or clinically advanced disease will require treatment . cytotoxic drugs , such as the alkylating agents ( chlorambucil , cyclophosphamide , and bendamustine ) , have been the mainstay of chemotherapeutic treatment in cll . however , their lack of specificity for cll cells and toxicity to normal cells , particularly hematopoietic and immune cells , have limited their efficacy . other treatment modalities include purine nucleoside analogs ( pna ) such as fludarabine and immunotherapeutic agents such as anti - cd20 monoclonal antibodies ( rituximab , ofatumumab , and alemtuzumab ) [ 1 , 4 , 6 ] . several regimens using the combination of immunotherapy with chemotherapeutics drugs are also currently being used in the treatment of cll . a treatment regimen combining fludarabine , cyclophosphamide , and rituximab ( fcr ) is currently the gold standard of initial treatment for cll and has also shown response in relapsed / refractory cases [ 1 , 6 ] . unfortunately , however , despite the availability of various therapeutic agents for cll , the disease is currently considered incurable with most patients eventually relapsing after initial treatment . the poor outcomes of the current treatment strategies , especially in patients with high - risk features ( del 17p , del 11q , igvh mutations , zap-70 , and cd38 expression ) , and the lack of tolerability of cytotoxic drugs by the older patients have prompted research into the development of novel drug therapies [ 4 , 7 ] . the standard fcr regimen can not be tolerated by the majority of cll patients who begin treatment after the age of 70 and suffer from other comorbid diseases . the advancement in our understanding of the signal transduction pathways involved in cll has shifted focus towards targeted therapy involving inhibitors of signal transducers in cll . some of the drugs being tested in various stages of preclinical and clinical trials include inhibitors of lyn ( dasatinib ) , syk ( fostamatinib ) , pi3k ( idelalisib , rigosertib ) , btk ( ibrutinib , avl-292 ) , mtor ( everolimus , temsirolimus ) , cereblon ( lenalidomide ) , cxcr4/cxcl12 ( nox - a12 , plerixafor ) , and bcl2 ( navitoclax ) . in this review , we particularly focus on the phosphatidylinositol 3 kinase ( pi3k ) inhibitor idelalisib ( gs-1101 , cal 101 ) , the clinical rationale behind its use in the treatment of cll , and the outcomes of various preclinical and clinical trials studying the use of idelalisib alone or in combination with other therapeutic agents for treatment of both initial and relapse / refractory cases of cll . prior to reviewing the role of pi3k inhibitor as a therapeutic agent for cll , it is essential to present a brief overview of the cll microenvironment and bcr - signaling pathway in b lymphocytes . the intricate interactions between the b cells and their microenvironment are central to the pathogenesis of cll . cll cells residing in the body constantly recirculate between the peripheral blood , bone marrow , and the lymphoid organs . while cll cells residing in the peripheral blood are in a resting state , those located within the bone marrow and secondary lymphatic organs actively proliferate in anatomic tissue sites labelled proliferation centers or pseudofollicles . within these proliferation centers , the malignant b cells interact with components of the tissue microenvironment , including bone marrow stromal cells , t cells , and monocyte derived nurse cells [ 7 , 10 , 11 ] . additionally , there is a complex interplay between b - cell antigen receptor ( bcr ) , chemokines , chemokine receptors , and adhesion molecules , which is responsible for homing , expansion , and survival of the malignant b cells [ 7 , 10 ] . the bcr is transmembrane receptor protein composed of two parts : an antigen - specific membrane bound immunoglobulin ( ig ) and an intracellular signal transduction moiety ig ( ig , cd79a)/ig ( ig , cd79b ) chains [ 7 , 12 ] . bcr activation leading to intracellular signaling cascades can either be antigen - induced or may result from an antigen - independent autonomous signaling resulting from interaction between hcdr3 region of the bcr and the fr2 epitope of the same or adjacent bcrs on the same cell [ 1214 ] . bcr stimulation results in the activation of several intracellular signaling pathways , including the plcy / calcium / nfat pathway , the pi3k / akt / mtor pathway , the nf-b pathway , and erk pathway , leading to b - cell maturation and differentiation [ 10 , 14 ] . pi3k are lipid kinases that play a central role in normal cellular functions such as cell growth , differentiation , proliferation , and survival . constitutive activation and overexpression of the pi3k pathway have been implicated in the pathogenesis of several human cancers . the pi3k family is subdivided into three classes containing eight different isoforms . the class i kinases , which comprise isoforms pi3k , , , and , act by phosphorylation of several membrane inositol phospholipids . each of the class i pi3k isoforms is heterodimer molecules comprising a catalytic subunit p110 ( p110 , p110 , p110 , or p110 ) and a regulatory subunit p85 ( p85 , p85 , p55 , p55 , p50 , and p101 ) [ 16 , 17 ] . binding of an antigen to the antigen - specific extracellular component of the bcr leads to phosphorylation of itams ( immunoreceptor tyrosine - based activation motifs ) by tyrosine kinases lyn and spleen tyrosine kinase ( syk ) . itams are intracytoplasmic specific sequences of the cd79a / b part of bcr which are responsible for further downstream signal cascades in b cells . upon antigen binding to the bcr , lyn phosphorylates syk , which in turn amplifies the initial bcr signal by autophosphorylation and phosphorylation of itams . moreover , lyn also controls inhibition of the bcr signal transduction pathway by activating phosphatases , which inhibit signal transduction through the bcr . lyn and syk lead to pi3k activation by phosphorylating cd19 and b - cell adaptor protein ( bcap ) [ 18 , 19 ] . this results in recruitment of the p85/p110 complex to the cell membrane . following activation of pi3k , downstream signaling mediators including akt , bruton 's tyrosine kinase ( btk ) , and phospholipase c- ( plc2 ) are recruited . plc2 catalyzes the hydrolysis of pip2 ( phosphatidylinositol-4,5-bisphosphate ) to release the second messengers dag ( 1,2-diacylglycerol ) and pip3 ( phosphatidylinositol-3,4 , 5-trisphosphate ) . pip3 is responsible for the reorganization of the cell cytoskeleton and regulates cellular functions such as cell adhesion via calcium flux into the cells , whereas dag activates several isoforms of protein kinase c and other downstream signal mediators . the p110 pi3k signaling pathway can also be activated by receptors other than the bcr such as by cytokines il-4 , il-6 , and baff [ 20 , 21 ] , by the chemokine cxcl13 , and by costimulatory receptors cd40 and toll - like receptors ( tlrs ) [ 2326 ] . pi3k ( p110 ) is mainly expressed in circulating leukocytes and lymphoid tissues along with the ubiquitously expressed pi3k . it has been demonstrated through various studies in p110 knock - out ( ko ) and p110 knock - in ( ki ) mice that p110 is essential for b - cell survival , migration , and proliferation . mice deficient in a functional p110 protein produced fewer mature b cells and exhibited impaired function in b cells that do manage to develop . additionally , in cd19/bcap double knock - out mice , the disruption of pi3k activation arrested generation of immature and mature b cells within the bone marrow and spleen . the importance of p110 is further highlighted by the finding that complete deficiency of pi3k in cells makes them prone to apoptosis , whereas , when a constitutively active form of pi3k isoform is introduced into cells lacking bcr expression , they are rescued from apoptosis . furthermore , it has been demonstrated that overexpression of the pi3k isoform as a wild - type protein induces transformation and constitutive activation of the akt signaling pathway in cultured cells . the above findings are a clear indication of the importance that the pi3k pathway has not only in normal b - cell development but for the proliferation , survival , and migration of cll cells . in cll tumor cells , there is increased activity of pi3k in contrast with normal b - cells [ 17 , 18 ] . the tumor microenvironment , via activation of pi3k , provides prosurvival signals to cll cells and also leads to their homing and retention in the bone marrow and lymphoid tissue . it has been reported that in vitro idelalisib , via inactivation of pi3k , leads to a decrease in akt and erk phosphorylation thus interrupting downstream bcr signaling . it induces apoptosis in cll cells in a time- and dose - dependent manner via a caspase dependent pathway [ 17 , 28 ] . this cytotoxicity is independent of genomic features such as chromosomal deletions del(11q22.3 ) and del(17p13.1 ) and igvh gene mutational status demonstrating that idelalisib is equally effective in treating patients with adverse prognostic factors such as p53 deletions and igvh mutations . data from preclinical and clinical studies has shown that idelalisib shows selective cytotoxicity to cll cells compared with normal b cells . furthermore , unlike pan - pi3k inhibitors , idelalisib is not significantly cytotoxic to normal nk and t cells . however , it does alter the function of nk cells and t cells by decreasing production of cytokines il-6 , il-4 , il-10 , and interferon- by these immune effector cells resulting in impaired cll cell proliferation [ 4 , 29 ] . moreover , idelalisib antagonizes the microenvironmental triggers tnf- , cd40l , baff , and fibronectin , all of which act by increasing akt phosphorylation in cll cells and protecting them from spontaneous apoptosis [ 4 , 17 , 29 ] . these findings demonstrate that idelalisib targets multiple mechanisms of cll cell survival by not only direct inhibition of the pi3k signaling pathway but also by inhibition of extrinsic activation of this pathway by microenvironmental stimuli from cd40l , baff , tnf- , and fibronectin . this provides a strong rationale for the use of idelalisib as a therapeutic agent in cll patients . multiple preclinical and clinical studies are being performed to investigate idelalisib as a therapeutic agent for cll as monotherapy or in combination with rituximab and/or bendamustine , fludarabine , ofatumumab , chlorambucil , chlorambucil + rituximab , and lenalidomide + rituximab . a study investigating the synergistic effect of idelalisib in combination with histone deacetylase inhibitor ( hdi ) , panobinostat ( lbh589 ) , and suberoylanilide hydroxamic acid ( saha ) in vitro has also been performed in cll and primary non - hodgkin lymphoma ( nhl ) . the study indicated that pi3k inhibitor synergistically potentiates histone deacetylase inhibitor - induced proliferation inhibition and apoptosis in malignant hematopoietic cells through the inactivation of pi3k and extracellular signal - regulated kinase pathways . the first phase i trial on idelalisib studied the clinical pharmacokinetics of the drug in healthy volunteers and in those with lymphoid malignancies . following administration of single doses up to a maximum of 400 mg and multiple doses of up to 200 mg twice a day for a week in healthy subjects , it was found that idelalisib is well tolerated with no apparent maximum tolerated dose . subsequently , idelalisib was tested in patients with lymphoid malignancies in dose levels up to a maximum of 350 mg / kg over several months . the drug was also well tolerated in these patients with reversible transaminase elevations in only some patients with lymphoid malignancies . the clinical data from this study supports dosing at 150 mg twice a day as this achieves steady - state plasma concentrations . idelalisib can be orally administered with or without food and is metabolized to a single metabolite in the plasma with hepatic metabolism . it is interesting to note that , although idelalisib is metabolized by the cyp450 3a4 enzyme , it does not appear to be a sensitive substrate for the enzyme and can be coadministered with inhibitors of cyp450 3a4 . preliminary reports from a phase i trial studying the clinical activity of idelalisib in relapsed or refractory b - cell malignancies such as chronic lymphocytic leukemia ( n = 55 ) , indolent non - hodgkin lymphoma ( n = 63 ) , mantle cell lymphoma ( n = 40 ) , diffuse large b - cell lymphoma ( n = 9 ) as well as in acute myelogenous leukemia ( n = 12 ) , and multiple myeloma ( n = 12 ) has shown that responses were obtained in patients with cll ( 4/17 ) , indolent lymphoma ( 9/15 ) , and mantle cell lymphoma ( 6/7 ) at all dose levels . however , no response was visible in patients with acute myeloid leukemia or diffuse large b - cell lymphoma . the level of pakt expression was measured in some cll patients and was found to be markedly decreased following idelalisib dosing on day 1 ( 1.5 h and 4 h after dosing ) and on day 8 prior to dosing . in the final results reported from a phase i study investigating the clinical activity of idelalisib in fifty - four patients with relapsed / refractory cll , being given single - agent oral idelalisib 50350 mg / dose , overall response rate ( orr ) was 56% including two complete remissions ( cr ) and twenty - eight partial remissions ( pr ) . of the twenty - eight pr , twenty - two met the hallek criteria ( 2008 ) and six met the pr with lymphocytosis cheson criteria ( 2012 ) . the median time to first response was 1.9 months while the median progression free survival ( pfs ) was 17 months . furthermore , with treatment 81% patients showed a lymph node response that is a 50% reduction in the nodal spd ( sum of the product of the longest perpendicular dimensions ) ; there was a resolution of splenomegaly ( 70% patients ) and normalization of cytopenias : anemia ( 68% ) , thrombocytopenia ( 79% ) , and neutropenia ( 100% ) . there were no dose - limiting toxicities reported and the most common adverse effects included fatigue , diarrhea , pyrexia , rash , upper respiratory tract infection , and pneumonia . of the 15% patients that discontinued due to adverse effects , 7% were potentially treatment - related . as with other novel agents targeting the bcr pathway in cll , an initial increase in the absolute lymphocyte count ( alc ) is seen with idelalisib therapy . according to the iwcll response criteria , lymphocytosis is a sign of disease progression ; however , cheson et al . have recommended a modification of the response criteria and cautioned against labelling the peripheral lymphocytosis initially seen with bcr - targeting drugs as a progressive disease . this lymphocytosis may be attributed to reduced cll cell adhesion to stromal cells and reduced responsiveness to cxcl12 and cxcl13 , which may interfere with cll cell tissue homing . consequently , in the peripheral blood the lack of prosurvival signals from the tissue microenvironment makes the cll cells prone to programmed cell death or apoptosis . one of the approaches to eliminate the increase in alc to keep with the current response criteria has been to study idelalisib in combination with other therapeutic agents . preliminary results from a phase i study indicate that combinations of idelalisib with fludarabine , bendamustine , and/or rituximab resolve the peripheral lymphocytosis seen with idelalisib monotherapy . in a recent study investigating combinations of idelalisib with rituximab and/or bendamustine , coutre et al . report that when idelalisib ( 150 mg bid continuously ) was given with rituximab at a dose of 375 mg / m weekly for 8 doses and/or 90 mg / m bendamustine on days 1 and 2 of each cycle for 6 cycles , overall response rates ( orr ) for the idelalisib + rituximab , idelalisib + bendamustine , and idelalisib + rituximab + bendamustine regimens were 78% , 82% , and 87% , respectively , and the one - year progression - free survival ( pfs ) rates were 74% , 88% , and 87% , respectively . moreover , almost all studied patients showed a rapid decrease in lymphadenopathy . some of the grade 3 adverse events were anemia , neutropenia , febrile neutropenia , thrombocytopenia , infections , pneumonia / pneumonitis , rash , diarrhea , and hepatic transaminase elevation studied idelalisib in combination with rituximab and bendamustine in patients with relapsed or refractory cll . when idelalisib was given with rituximab and/or bendamustine in the identical dosing regimens specified above , overall response rates ( orr ) the median time to response was 1.9 months ; the 2-year progression - free survival ( pfs ) was 62% and os ( overall survival ) was 85% . the aes ( any grade/grade 3 ) were pyrexia , diarrhea , cough , fatigue , nausea , pneumonia , rash , and alt / ast elevation . in another phase i study evaluating the combination of idelalisib with bendamustine / rituximab or chlorambucil / rituximab in relapsed / refractory cll patients , wagner - johnston et al . reported an orr of 89.7% , demonstrating good efficacy and tolerability of idelalisib in these patients when used in combination regimens . vos et al . studied the combination of idelalisib with bendamustine , fludarabine , or chlorambucil in patients with relapsed or refractory cll in a phase i study and reported an orr of 78% and estimated dor and pfs of 28.5 months . the results from these studies indicate that double or triple combination treatment regimens of idelalisib with rituximab , bendamustine , chlorambucil , or fludarabine are highly active with an excellent safety profile and tolerability and necessitate further phase iii studies evaluating their efficacy and safety . a phase iii trial evaluating the efficacy and safety of idelalisib in combination with bendamustine and rituximab for previously treated cll was initiated in june , 2012 , and is currently underway . a phase i study evaluating idelalisib in combination with rituximab or ofatumumab was conducted in patients with relapsed / refractory cll . they were given idelalisib continuously at 150 mg bid in combination with a total of 8 infusions of rituximab ( r , 375 mg / m2 weekly 8) or a total of 12 infusions of ofatumumab ( o , 300 mg initial dose either on day 1 or day 2 relative to the first dose of idelalisib , then 1,000 mg weekly for 7 cycles , and then 1,000 mg every 4 weeks for cycles ) . those patients who were on treatment after 48 weeks were eligible to continue idelalisib on an extension study . the orr was 83% with 3 crs ( 8% ) and a median time to response of 1.9 months . the pfs for all patients and duration of response ( dor ) were 20 and 19 months , respectively . among the 11 patients with del(17p ) and/or tp53 mutations , the response rate was 73% and the median pfs and dor were 19 months . this study demonstrates that the combination of idelalisib with anti - cd20 antibodies such as rituximab or ofatumumab has good clinical activity and tumor control along with an acceptable safety profile in patients with relapsed / refractory cll . phase iii trials evaluating the efficacy and safety of idelalisib in combination with these two anti - cd20 antibodies , rituximab or ofatumumab , are currently underway ( nct01539512 , nct01659021 ) . in a phase ii study , o'brien at al . investigated the combination of idelalisib with rituximab in treatment - naive patients 65 years of age , with cll or sll . they were treated with rituximab 375 mg / m weekly 8 doses and idelalisib 150 mg bid continuously for 48 weeks ; those who completed 48 weeks without progression were enrolled in an extension study and continued to receive idelalisib . the orr was 96% , and the median time to response was 1.9 months with no on - study relapses . at 24 months , most notably , all of the six patients with 17p deletion ( del(17p ) ) responded with 1 cr and 5 pr . the aes reported were diarrhea , pyrexia , chills , fatigue , rash , pneumonia , and elevated alt / ast . there are several genetic mutations that serve as markers of poor prognosis in cll including del(17p)/tp53 mutation , del(11q ) , ighv mutation , and notch1 mutation [ 4446 ] . coutre et al . reported the effect of these mutations on the clinical activity of idelalisib in both treatment - nave and relapsed / refractory cll patients . all subjects were enrolled into one of three trials , the first two of which were for relapsed / refractory patients ; a phase i study with monotherapy , escalation dosing from 50350 mg bid ; phase i combination with either rituximab , ofatumumab , bendamustine rituximab , fludarabine , or chlorambucil rituximab ; phase ii combination with rituximab in previously untreated patients aged 65 years . the results from this study indicate that idelalisib shows good activity in cll regardless of these high - risk prognostic markers . a combination of idelalisib with cx-4945 , an orally bioavailable selective inhibitor of casein kinase 2 ( ck2 ) , has also been tested and has shown promising activity in cll . another preclinical study exploring the synergism between idelalisib and a highly selective spleen tyrosine kinase ( syk ) inhibitor , gs-9973 , has shown significant activity of this combination in samples collected from cll patients . lenalidomide - mediated disease - specific toxicity of tumor flare and cytokine release is attenuated by inhibition of pi3k pathway by idelalisib . furman et al . enrolled 220 patients with relapsed cll and coexistent medical conditions such as decreased renal function , chemotherapy related bone marrow suppression , and other major comorbidities . the patients were divided into two groups : rituximab with idelalisib and rituximab with placebo . it was demonstrated that combination with rituximab and idelalisib led to greater disease free survival , treatment response rate , and survival among patients with relapsed cll . to date , the results from trials evaluating the clinical efficacy and safety of idelalisib , both as monotherapy and as part of combination regimens , have been encouraging . however , there are still questions to be answered about the long - term application of idelalisib , particularly about its safety , tolerability , drug interactions , and most importantly the duration and durability of response . there is a concern that , with specific inhibition of such a complex pathway as the pi3k pathway , there exists a potential for escape by upregulation or activation of other pi3k isoforms , resulting in drug resistance . future research into idelalisib therapy for cll will have to focus on all these aspects including determination of the optimal combination regimens and long - term outcomes in both previously untreated and refractory / relapsed cll patients . the extraordinarily promising results shown by targeted therapy have revolutionized the current treatment strategies for cll . the preclinical and clinical data thus far demonstrate that idelalisib produces a dramatic and durable response in cll patients without causing significant toxicity , which is a drawback of other nonspecific therapies . moving forward , the ongoing clinical trials will help address the various questions currently being raised regarding the long - term application and safety of idelalisib . with greater clinical experience following more widespread use of idelalisib , we will be able to determine the optimal combination therapies in treatment - nave and relapsed / refractory patients , resulting in more individualized therapeutic strategies for cll patients .

chronic lymphocytic leukemia is the most common leukemia in the united states . it is a slowly progressive disease , with an 82% five - year survival rate . the treatment strategies are highly individualized with patients in the early and stable stages typically not requiring treatment . however , those with progressive or clinically advanced disease will require treatment . cytotoxic drugs , such as the alkylating agents , purine nucleoside antagonists , and immunotherapeutic agents , have been the mainstay of chemotherapeutic treatment in cll . however , given the lack of therapeutic specificity , these medications ( especially older ones ) have limited tolerability due to side effects . in this paper , we will discuss the data on the use of phosphatidylinositol 3 kinase inhibitor idelalisib in the management of patients with chronic lymphocytic leukemia . the preclinical and clinical data thus far demonstrate that idelalisib produces a dramatic and durable response in patients with chronic lymphocytic leukemia and without causing significant toxicity . moving forward , the ongoing clinical trials will help address the various questions currently being raised regarding the long - term application and safety of idelalisib . with greater clinical experience following more widespread use of idelalisib , we will be able to determine the optimal combination therapies in treatment - nave and relapsed / refractory patients , resulting in more individualized therapeutic strategies for patients with chronic lymphocytic leukemia .

1. Introduction 2. Biology of PIK3 in CLL 3. Conclusion

chronic lymphocytic leukemia ( cll ) is a lymphoid malignancy characterized by the accumulation and proliferation of nonfunctional and monoclonal small cd5/cd19/cd-20/cd23-positive lymphocytes in the blood , bone marrow , and lymphoid tissues [ 1 , 2 ] . it is the most common adult leukemia in the united states , with 15,680 new cases and estimated 4,850 deaths reported by the american cancer society in 2013 . cll is a slowly progressive disease , with an 82% five - year survival rate . the treatment strategies of cll are highly individualized with patients in the early and stable stages of cll not requiring treatment . however , those with progressive or clinically advanced disease will require treatment . cytotoxic drugs , such as the alkylating agents ( chlorambucil , cyclophosphamide , and bendamustine ) , have been the mainstay of chemotherapeutic treatment in cll . however , their lack of specificity for cll cells and toxicity to normal cells , particularly hematopoietic and immune cells , have limited their efficacy . other treatment modalities include purine nucleoside analogs ( pna ) such as fludarabine and immunotherapeutic agents such as anti - cd20 monoclonal antibodies ( rituximab , ofatumumab , and alemtuzumab ) [ 1 , 4 , 6 ] . several regimens using the combination of immunotherapy with chemotherapeutics drugs are also currently being used in the treatment of cll . a treatment regimen combining fludarabine , cyclophosphamide , and rituximab ( fcr ) is currently the gold standard of initial treatment for cll and has also shown response in relapsed / refractory cases [ 1 , 6 ] . unfortunately , however , despite the availability of various therapeutic agents for cll , the disease is currently considered incurable with most patients eventually relapsing after initial treatment . the poor outcomes of the current treatment strategies , especially in patients with high - risk features ( del 17p , del 11q , igvh mutations , zap-70 , and cd38 expression ) , and the lack of tolerability of cytotoxic drugs by the older patients have prompted research into the development of novel drug therapies [ 4 , 7 ] . the advancement in our understanding of the signal transduction pathways involved in cll has shifted focus towards targeted therapy involving inhibitors of signal transducers in cll . some of the drugs being tested in various stages of preclinical and clinical trials include inhibitors of lyn ( dasatinib ) , syk ( fostamatinib ) , pi3k ( idelalisib , rigosertib ) , btk ( ibrutinib , avl-292 ) , mtor ( everolimus , temsirolimus ) , cereblon ( lenalidomide ) , cxcr4/cxcl12 ( nox - a12 , plerixafor ) , and bcl2 ( navitoclax ) . in this review , we particularly focus on the phosphatidylinositol 3 kinase ( pi3k ) inhibitor idelalisib ( gs-1101 , cal 101 ) , the clinical rationale behind its use in the treatment of cll , and the outcomes of various preclinical and clinical trials studying the use of idelalisib alone or in combination with other therapeutic agents for treatment of both initial and relapse / refractory cases of cll . prior to reviewing the role of pi3k inhibitor as a therapeutic agent for cll , it is essential to present a brief overview of the cll microenvironment and bcr - signaling pathway in b lymphocytes . cll cells residing in the body constantly recirculate between the peripheral blood , bone marrow , and the lymphoid organs . while cll cells residing in the peripheral blood are in a resting state , those located within the bone marrow and secondary lymphatic organs actively proliferate in anatomic tissue sites labelled proliferation centers or pseudofollicles . within these proliferation centers , the malignant b cells interact with components of the tissue microenvironment , including bone marrow stromal cells , t cells , and monocyte derived nurse cells [ 7 , 10 , 11 ] . additionally , there is a complex interplay between b - cell antigen receptor ( bcr ) , chemokines , chemokine receptors , and adhesion molecules , which is responsible for homing , expansion , and survival of the malignant b cells [ 7 , 10 ] . bcr stimulation results in the activation of several intracellular signaling pathways , including the plcy / calcium / nfat pathway , the pi3k / akt / mtor pathway , the nf-b pathway , and erk pathway , leading to b - cell maturation and differentiation [ 10 , 14 ] . pi3k are lipid kinases that play a central role in normal cellular functions such as cell growth , differentiation , proliferation , and survival . constitutive activation and overexpression of the pi3k pathway have been implicated in the pathogenesis of several human cancers . each of the class i pi3k isoforms is heterodimer molecules comprising a catalytic subunit p110 ( p110 , p110 , p110 , or p110 ) and a regulatory subunit p85 ( p85 , p85 , p55 , p55 , p50 , and p101 ) [ 16 , 17 ] . following activation of pi3k , downstream signaling mediators including akt , bruton 's tyrosine kinase ( btk ) , and phospholipase c- ( plc2 ) are recruited . the p110 pi3k signaling pathway can also be activated by receptors other than the bcr such as by cytokines il-4 , il-6 , and baff [ 20 , 21 ] , by the chemokine cxcl13 , and by costimulatory receptors cd40 and toll - like receptors ( tlrs ) [ 2326 ] . in cll tumor cells , there is increased activity of pi3k in contrast with normal b - cells [ 17 , 18 ] . the tumor microenvironment , via activation of pi3k , provides prosurvival signals to cll cells and also leads to their homing and retention in the bone marrow and lymphoid tissue . it induces apoptosis in cll cells in a time- and dose - dependent manner via a caspase dependent pathway [ 17 , 28 ] . this cytotoxicity is independent of genomic features such as chromosomal deletions del(11q22.3 ) and del(17p13.1 ) and igvh gene mutational status demonstrating that idelalisib is equally effective in treating patients with adverse prognostic factors such as p53 deletions and igvh mutations . data from preclinical and clinical studies has shown that idelalisib shows selective cytotoxicity to cll cells compared with normal b cells . however , it does alter the function of nk cells and t cells by decreasing production of cytokines il-6 , il-4 , il-10 , and interferon- by these immune effector cells resulting in impaired cll cell proliferation [ 4 , 29 ] . moreover , idelalisib antagonizes the microenvironmental triggers tnf- , cd40l , baff , and fibronectin , all of which act by increasing akt phosphorylation in cll cells and protecting them from spontaneous apoptosis [ 4 , 17 , 29 ] . these findings demonstrate that idelalisib targets multiple mechanisms of cll cell survival by not only direct inhibition of the pi3k signaling pathway but also by inhibition of extrinsic activation of this pathway by microenvironmental stimuli from cd40l , baff , tnf- , and fibronectin . this provides a strong rationale for the use of idelalisib as a therapeutic agent in cll patients . multiple preclinical and clinical studies are being performed to investigate idelalisib as a therapeutic agent for cll as monotherapy or in combination with rituximab and/or bendamustine , fludarabine , ofatumumab , chlorambucil , chlorambucil + rituximab , and lenalidomide + rituximab . a study investigating the synergistic effect of idelalisib in combination with histone deacetylase inhibitor ( hdi ) , panobinostat ( lbh589 ) , and suberoylanilide hydroxamic acid ( saha ) in vitro has also been performed in cll and primary non - hodgkin lymphoma ( nhl ) . following administration of single doses up to a maximum of 400 mg and multiple doses of up to 200 mg twice a day for a week in healthy subjects , it was found that idelalisib is well tolerated with no apparent maximum tolerated dose . subsequently , idelalisib was tested in patients with lymphoid malignancies in dose levels up to a maximum of 350 mg / kg over several months . preliminary reports from a phase i trial studying the clinical activity of idelalisib in relapsed or refractory b - cell malignancies such as chronic lymphocytic leukemia ( n = 55 ) , indolent non - hodgkin lymphoma ( n = 63 ) , mantle cell lymphoma ( n = 40 ) , diffuse large b - cell lymphoma ( n = 9 ) as well as in acute myelogenous leukemia ( n = 12 ) , and multiple myeloma ( n = 12 ) has shown that responses were obtained in patients with cll ( 4/17 ) , indolent lymphoma ( 9/15 ) , and mantle cell lymphoma ( 6/7 ) at all dose levels . however , no response was visible in patients with acute myeloid leukemia or diffuse large b - cell lymphoma . in the final results reported from a phase i study investigating the clinical activity of idelalisib in fifty - four patients with relapsed / refractory cll , being given single - agent oral idelalisib 50350 mg / dose , overall response rate ( orr ) was 56% including two complete remissions ( cr ) and twenty - eight partial remissions ( pr ) . furthermore , with treatment 81% patients showed a lymph node response that is a 50% reduction in the nodal spd ( sum of the product of the longest perpendicular dimensions ) ; there was a resolution of splenomegaly ( 70% patients ) and normalization of cytopenias : anemia ( 68% ) , thrombocytopenia ( 79% ) , and neutropenia ( 100% ) . there were no dose - limiting toxicities reported and the most common adverse effects included fatigue , diarrhea , pyrexia , rash , upper respiratory tract infection , and pneumonia . of the 15% patients that discontinued due to adverse effects , 7% were potentially treatment - related . as with other novel agents targeting the bcr pathway in cll , an initial increase in the absolute lymphocyte count ( alc ) is seen with idelalisib therapy . according to the iwcll response criteria , lymphocytosis is a sign of disease progression ; however , cheson et al . have recommended a modification of the response criteria and cautioned against labelling the peripheral lymphocytosis initially seen with bcr - targeting drugs as a progressive disease . consequently , in the peripheral blood the lack of prosurvival signals from the tissue microenvironment makes the cll cells prone to programmed cell death or apoptosis . one of the approaches to eliminate the increase in alc to keep with the current response criteria has been to study idelalisib in combination with other therapeutic agents . preliminary results from a phase i study indicate that combinations of idelalisib with fludarabine , bendamustine , and/or rituximab resolve the peripheral lymphocytosis seen with idelalisib monotherapy . in a recent study investigating combinations of idelalisib with rituximab and/or bendamustine , coutre et al . report that when idelalisib ( 150 mg bid continuously ) was given with rituximab at a dose of 375 mg / m weekly for 8 doses and/or 90 mg / m bendamustine on days 1 and 2 of each cycle for 6 cycles , overall response rates ( orr ) for the idelalisib + rituximab , idelalisib + bendamustine , and idelalisib + rituximab + bendamustine regimens were 78% , 82% , and 87% , respectively , and the one - year progression - free survival ( pfs ) rates were 74% , 88% , and 87% , respectively . some of the grade 3 adverse events were anemia , neutropenia , febrile neutropenia , thrombocytopenia , infections , pneumonia / pneumonitis , rash , diarrhea , and hepatic transaminase elevation studied idelalisib in combination with rituximab and bendamustine in patients with relapsed or refractory cll . the aes ( any grade/grade 3 ) were pyrexia , diarrhea , cough , fatigue , nausea , pneumonia , rash , and alt / ast elevation . in another phase i study evaluating the combination of idelalisib with bendamustine / rituximab or chlorambucil / rituximab in relapsed / refractory cll patients , wagner - johnston et al . reported an orr of 89.7% , demonstrating good efficacy and tolerability of idelalisib in these patients when used in combination regimens . studied the combination of idelalisib with bendamustine , fludarabine , or chlorambucil in patients with relapsed or refractory cll in a phase i study and reported an orr of 78% and estimated dor and pfs of 28.5 months . the results from these studies indicate that double or triple combination treatment regimens of idelalisib with rituximab , bendamustine , chlorambucil , or fludarabine are highly active with an excellent safety profile and tolerability and necessitate further phase iii studies evaluating their efficacy and safety . a phase iii trial evaluating the efficacy and safety of idelalisib in combination with bendamustine and rituximab for previously treated cll was initiated in june , 2012 , and is currently underway . a phase i study evaluating idelalisib in combination with rituximab or ofatumumab was conducted in patients with relapsed / refractory cll . they were given idelalisib continuously at 150 mg bid in combination with a total of 8 infusions of rituximab ( r , 375 mg / m2 weekly 8) or a total of 12 infusions of ofatumumab ( o , 300 mg initial dose either on day 1 or day 2 relative to the first dose of idelalisib , then 1,000 mg weekly for 7 cycles , and then 1,000 mg every 4 weeks for cycles ) . among the 11 patients with del(17p ) and/or tp53 mutations , the response rate was 73% and the median pfs and dor were 19 months . this study demonstrates that the combination of idelalisib with anti - cd20 antibodies such as rituximab or ofatumumab has good clinical activity and tumor control along with an acceptable safety profile in patients with relapsed / refractory cll . phase iii trials evaluating the efficacy and safety of idelalisib in combination with these two anti - cd20 antibodies , rituximab or ofatumumab , are currently underway ( nct01539512 , nct01659021 ) . investigated the combination of idelalisib with rituximab in treatment - naive patients 65 years of age , with cll or sll . the orr was 96% , and the median time to response was 1.9 months with no on - study relapses . at 24 months , most notably , all of the six patients with 17p deletion ( del(17p ) ) responded with 1 cr and 5 pr . the aes reported were diarrhea , pyrexia , chills , fatigue , rash , pneumonia , and elevated alt / ast . there are several genetic mutations that serve as markers of poor prognosis in cll including del(17p)/tp53 mutation , del(11q ) , ighv mutation , and notch1 mutation [ 4446 ] . reported the effect of these mutations on the clinical activity of idelalisib in both treatment - nave and relapsed / refractory cll patients . all subjects were enrolled into one of three trials , the first two of which were for relapsed / refractory patients ; a phase i study with monotherapy , escalation dosing from 50350 mg bid ; phase i combination with either rituximab , ofatumumab , bendamustine rituximab , fludarabine , or chlorambucil rituximab ; phase ii combination with rituximab in previously untreated patients aged 65 years . the results from this study indicate that idelalisib shows good activity in cll regardless of these high - risk prognostic markers . a combination of idelalisib with cx-4945 , an orally bioavailable selective inhibitor of casein kinase 2 ( ck2 ) , has also been tested and has shown promising activity in cll . enrolled 220 patients with relapsed cll and coexistent medical conditions such as decreased renal function , chemotherapy related bone marrow suppression , and other major comorbidities . it was demonstrated that combination with rituximab and idelalisib led to greater disease free survival , treatment response rate , and survival among patients with relapsed cll . to date , the results from trials evaluating the clinical efficacy and safety of idelalisib , both as monotherapy and as part of combination regimens , have been encouraging . however , there are still questions to be answered about the long - term application of idelalisib , particularly about its safety , tolerability , drug interactions , and most importantly the duration and durability of response . there is a concern that , with specific inhibition of such a complex pathway as the pi3k pathway , there exists a potential for escape by upregulation or activation of other pi3k isoforms , resulting in drug resistance . future research into idelalisib therapy for cll will have to focus on all these aspects including determination of the optimal combination regimens and long - term outcomes in both previously untreated and refractory / relapsed cll patients . the extraordinarily promising results shown by targeted therapy have revolutionized the current treatment strategies for cll . the preclinical and clinical data thus far demonstrate that idelalisib produces a dramatic and durable response in cll patients without causing significant toxicity , which is a drawback of other nonspecific therapies . moving forward , the ongoing clinical trials will help address the various questions currently being raised regarding the long - term application and safety of idelalisib . with greater clinical experience following more widespread use of idelalisib , we will be able to determine the optimal combination therapies in treatment - nave and relapsed / refractory patients , resulting in more individualized therapeutic strategies for cll patients .

translational efficiency of eukaryotic mrnas depends on the structural features of the 5-untranslated region ( 5-utr , or leader sequence ) and the nucleotide sequence flanking the translation start codon ( start codon context ) . both experimental ( 15 ) and statistical ( 617 ) approaches have been used to reveal the characteristics of the 5-utrs that influence translation . in particular , 5-utr length ( 18 ) , secondary structure ( 19 ) and the presence of aug triplets upstream of the true translation start in mrna , known as upstream augs ( 20 ) ( hereinafter uaugs ; for the sake of uniformity , we designate start codons aug disregarding the fact that it should be rendered as atg when dna sequences are considered ) , have been shown to affect the efficiency of translation . although , in most cases , translation of eukaryotic mrnas is initiated at the first aug of the 5-utr , according to the scanning model of kozak , many exceptions to this rule have been described ( 2,3,1925 ) . the context of the start codon is another important regulatory factor . a consensus sequence , ( gcc)gccrccaugg ( where r = g or a ) , has been derived for the start codon context of mammalian mrnas ( 6,8 ) . the most critical sites near the start aug codon ( saug ) are 3 ( the a of the saug is position + 1 ) , usually occupied by a purine , and + 4 , typically occupied by a guanine ( 19,26 ) . other sites near the start codon seem to be less important , although the influence of nucleotides in different positions may vary from species to species ( 2730 ) . generally , uaugs decrease mrna translation efficiency and may be considered strong negative translational regulatory signals ( 3,31,32 ) , since the major mechanism of translation initiation in eukaryotes appears to be the scanning model , according to which eukaryotic ribosomes initiate translation at the 5-proximal aug codon ( 1,6 ) . this model is supported by the observation that mammalian 5-utrs contain significantly fewer uaugs than expected by chance , suggesting that purifying selection caused elimination of uaugs in many 5-utrs ( 13,17 ) . weak start codon context ( low information content ) and conversely , high information content of the start codon context is typical of short 5-utrs containing no uaugs ( 13 ) . this finding seems to be compatible with the possibility that uaugs function as attenuators of translation initiation by engaging ribosomes in futile initiation and precluding them from reaching the saug . however , the alternative possibility remains that cdnas with aug - containing 5-utrs might represent mainly non - functional sequences , such as incorrectly processed transcripts and transcripts of pseudogenes , artifacts of cloning and coding region annotation , such that the purported uaugs actually belong to misannotated 5-portions of coding regions , or mrnas of genes with multiple transcription start sites ( 2,13,19,31,32 ) . indeed , follow - up studies have shown that some cdna sequences containing many augs in the 5-utr do not correspond to functional mrnas ( 31,32,36 ) . a natural approach to address the issue of the functional significance of uaugs is to examine the evolutionary conservation of these triplets on genome scale . by comparing sequences of human , mouse and rat orthologous genes , we show here that in 5-utrs of mammalian cdnas but not in 3-utrs or coding sequences , aug is conserved to a significantly greater extent than any of the other 63 nt triplets . this observation can be hardly explained by annotation errors because pronounced excess of conserved uaugs was detected in 5-utrs containing an in - frame stop codon and because many of the conserved augs are found in different frames , which is consistent with the location in non - coding sequences . these observations strongly suggest that at least some of the conserved uaugs are functional elements of translation initiation regulation . mammalian cdna sequences were extracted from the refseq database ( 16 773 human cdnas , 19 777 mouse cdnas and 21 178 rat cdnas ) . all 5-utrs that showed statistically significant blastx ( 37 ) hits ( e - value < 10 ) to the non - redundant protein database ( national center for biotechnology information , nih , bethesda ) and , accordingly , were probable to represent that undetected coding sequences were removed from the datasets . in addition , the identity between the first 50 amino acids of the refseq proteins encoded in the analyzed genes and the corresponding swissprot entries were checked . two 5-utrs were considered redundant if their masked sequences ( repeatview , ) ( 38 ) produced a bidirectional blast hit with an e - value < 10 . of all redundant 5-utrs , only the shortest versions were retained to exclude cloning artifacts and unspliced introns . probable one - to - one orthologs were identified by comparing symmetrical best hits ( 39 ) between proteins from the respective genomes using the blastp program ( 40 ) . the nucleotide sequence alignments for identified orthologous pairs of cdnas were produced using the blastn program ( 37 ) . only long pairwise alignments ( high - scoring segment pairs ) with length 50 bases and with a low expectation value ( e - value < 10 ) were retained for analysis . this ensures the reliability of nucleotide sequence alignments since blastn is known to produce conservative alignments , at least when applied with restrictive cut - off values as done here ( 41,42 ) . aligned sequences of orthologous genes from four yeast species , saccharomyces cerevisiae , saccharomyces paradoxus , saccharomyces mikatae and saccharomyces bayanus were extracted from the sgd database ( ) ( 43 ) . the alignment of start and stop codons was fixed in order to ensure the correct partitioning of 5-utr , the cds ( protein - coding sequence ) and the 3-utr . saccharomyces cdna sequences were extracted from genbank , and the reliability of annotation of the 5-utrs and cds was assessed as described above . the evolutionary conservation was calculated as the fraction of triplets that are conserved in pairwise ( for comparisons of mammalian sequences ) or multiple ( for comparisons of yeast sequences ) alignments of 5-utrs . we analyzed evolutionary conservation of uaugs in pairwise alignments of human mouse and mouse rat orthologs . conservative criteria were adopted to select reliable alignments for further analysis ( materials and methods ) . these fractions of uaug - containing 5-utrs are consistent with previous observations ( 13,3335 ) . examination of the evolutionary conservation of each of the 64 nt triplets in the aligned 5-utrs showed that aug was the most conserved triplet in both human mouse and mouse rat comparisons ( figure 1a and b ) . tables 1 and 2 compare the frequencies of conserved uaugs with those of the five triplets that are permutations of aug , in order to exclude any possible effect of nucleotide composition ; in both cases , the fraction of conserved augs was markedly greater than those of the other triplets ( p < 10 by the fisher 's exact test ) . in contrast , no excessive conservation of aug compared with the other 63 triplets was detected in 3-utrs and cdss ( excluding the start aug ) of mammalian mrnas ( figure 1c f ) , indicating that aug conservation was specific for 5-utrs . the conservation of uaugs seemed to be spread throughout the 5-utrs : a significant excess of conserved uaugs was observed both in the proximal ( region 100 : 1 nt ) and the distal ( region 200 : 101 nt ) parts of 5-utr alignments , without a significant enrichment in either ( supplementary tables s1s4 ) . an issue of concern with this observation is that the excess of conserved uaugs could be an artifact of erroneous annotation of coding regions , with the sequences annotated as 5-utr actually containing the upstream portion of the cds , including the typically conserved authentic start aug and , possibly , conserved methionine codons as well ( 36 ) . this did not seem to be a major factor because the observed excess of conserved uaug did not depend on whether we used refseq annotations ( tables 1 and 2 ) or only those refseq annotations that were consistent with the corresponding swissprot annotations ( supplementary tables s5 and s6 ) . nevertheless , to control such an artifact in a more direct fashion , we examined 5-utrs containing an upstream conserved stop codon(s ) in the same frame with the saug ( supplementary figure s1 ) ; 5-utrs containing in - phase stop codons are unlikely to represent undetected parts of the cds . figure 2 shows an example of a conserved in - frame stop codon in a human the main open reading frame ( orf ) encoding the 1 type iii collagen proprotein can not be extended in the 5 direction because , in both human and mouse 5-utrs , there is an in - frame stop codon uga . thus , the start codon of this gene apparently is annotated correctly , and the conserved uaugs are , indeed , located in the 5-utr ( figure 2 ) . analysis of the portions of 5-utrs located upstream of conserved in - frame stop codons ( 5-utr < stop ) yielded results that were fully compatible with those for complete 5-utrs . specifically , 19 and 22% of the human mouse and mouse rat alignments , respectively , contained conserved uaugs , and there was a highly significant excess of conserved uaugs compared with the five shuffled triplets ( tables 3 and 4 ) . further evidence of bona fide evolutionary conservation of uaugs was obtained by analysis of the phase distribution of the conserved uaugs ( phase 0 is the frame of the start codon , and phases 1 and 2 are frames shifted by 1 or 2 nt , respectively ) ( supplementary figure s2 ) . we found that human mouse and mouse rat alignments have a significant excess of conserved uaugs in the same phase ( 00 , 11 or 22 in table 5 ) . however , the fraction of conserved uaugs in different phases was also substantial ( 01 , 12 and so on ; table 5 ) which is not the expectation for protein - coding regions where frameshift mutations are , generally , not tolerated . it should be noted that blastn alignments are extremely conservative with respect to deletions / insertions , so the actual fraction of conserved uaugs in different phases might be even greater . the phase distribution of the conserved uaugs showed a significant excess of phase 11 and phase 22 uaugs compared with phase 00 uaugs ( table 5 ) . this may reflect the greater chance for uorfs in phase 00 to merge with the main orfs , thereby extending the main orfs in the 5 direction , which could be a mechanism of evolution of protein - coding regions yielding alternative translation starts ( 44,45 ) . however , the possibility can not be ruled out that , at least in part , the deficit of phase 00 is caused by the erroneous annotation of the 5-terminal aug as the start codon in a subset of mrnas , resulting in depletion of phase 0 uaugs . indeed , analysis of the 5-utr < stop regions which are , obviously , refractory to this artifact , showed a nearly uniform phase distribution ( table 6 ) . the lower excess of same - phase uaugs in the 5-utr < stop data set compared to the entire set of uaugs ( compare the data in tables 5 and 6 ) is probably owing to the lower sequence conservation and greater prevalence of indels in the distal parts of the 5-utrs ( 16 ) . thus , the phase distribution of the conserved uaugs is best compatible with their localization in non - coding regions . taken together , these results show that the observed exceptional conservation of uaugs in the 5-utrs of mammalian genes is not an artifact and strongly suggests that the uaugs are subject to purifying selection and hence have a function(s ) , most probably , related to the regulation of translation initiation . it has been reported recently that uorfs are , on average , slightly but significantly shorter than random orfs ( 17 ) . figure 3 compares the length distributions of uorfs starting from conserved uaugs , non - conserved uaugs and ugaus ( gau triplet , a permutation of aug , located in the 5-utrs ) . a noticeable and statistically highly significant excess of very short uorfs was observed for conserved uaugs : the length distribution of uorfs starting with conserved uaugs differed from the other two distributions with p < 10 ( test ) , and the excess of the shortest uorfs ( 020 codons ) was significant at p < 10 ( fisher 's exact test ) . the length distributions of uorf starting with conserved uaug were very similar for all three phases ( supplementary figure s3 ) . in contrast , the distributions of uorf lengths starting from non - conserved uaugs and gaus were statistically indistinguishable ( similar results were obtained with other permutations of aug ; data not shown ) . conceivably , the uorfs that start from conserved uaugs and may be engaged in the regulation of translation initiation were selected for short length optimization of the level of attenuation and prevent complete shutdown of initiation from the saug . there was no significant difference between the nucleotide contexts of the conserved and non - conserved uaugs ( data not shown ) ; in both cases , the information content of the uaug context was lower than that of the saug context ( 13,17 ) . having shown that uaugs are exceptionally conserved in 5-utrs of mammalian mrnas , we sought to investigate how general this pattern might be . to this end , we examined the conservation of uaugs in orthologous gene sets from four species of yeasts . since precise mrna mapping is unavailable for most yeast genes , we had to analyze , as a proxy for 5-utrs , genomic sequences ( 50 nt ) located upstream of saugs codons of yeast genes . this analysis revealed a highly significant excess of conserved uaugs compared with the permuted triplets ( table 7 ) . this result was confirmed also with genomic alignments of 5-utr < stop sequences ( see above ) and with 5-utrs of the available yeast cdna sequences ( table 7 ) . furthermore , 11% of the 253 genomic alignments of 5-utrs for known yeast cdna sequences contained uaugs conserved in all four yeast species for which genome sequences are available ( data not shown ) . the comparative genomic analysis reported here shows that the 5-utrs of at least 2030% of mammalian mrnas contain conserved uaugs ; qualitatively similar results were obtained with yeast 5-utrs . strikingly , in both mammals and yeasts , aug is by far the most conserved nucleotide triplet in 5-utr but not in 3-utrs or coding regions . these findings suggest that at least a fraction of the conserved uaugs perform a function related to the regulation of translation initiation . compatible with this conclusion , we found that uorfs starting from conserved ( and , potentially , functional ) uaugs are , on average , significantly shorter than those that start from non - conserved ( probably , non - functional ) augs . there was no single dominant functional trend among the genes containing multiple conserved uaugs but many of these genes encode transcription factors , receptors and other proteins involved in various forms of signal transduction ( supplementary table s7 and data not shown ) . these genes are subject to complex regulation which seems to be compatible with the proposed role of uaugs in modulation of translation initiation . the conclusion of the present work on the probably functional importance of conserved uaugs has to be considered in conjunction with the more or less orthogonal previous observation that mammalian 5-utrs are significantly depleted of augs , a trend that was confirmed in this study and is even stronger in yeast 5-utrs ( table 8) . apparently , purifying selection acts on the uaugs in two opposite directions : the uaugs that have no specific function tend to be deleterious and are eliminated during evolution ; in contrast , those uaugs that do serve a function are maintained . more specifically , what could be the regulatory functions of uaugs ? probably , the leading hypothesis is that the uaugs attenuate translation by diverting scanning ribosomes from the authentic saug . kozak ( 2,19 ) noticed that mrnas coding for regulatory proteins often contain multiple uaugs in the 5-utrs and proposed that this is an adaptation to prevent excessive and deleterious production of proto - oncogenes , transcription and growth factors , and other proteins that require tight regulation of expression . double suppression of mrna translational activity by uaugs and weak start codon context also might be employed for this purpose ( 12,13,46 ) . apparently , selection also operates at the level of the regulatory uorfs by shortening their length , possibly , to optimize the level of translation attenuation . an important role for down - regulation signals affecting translation initiation ( uaugs in 5-utr and weak saug context ) is supported by experimental data : deletion of aug - containing fragments of 5-utr has been shown to greatly increase the mrna translation rate and protein expression levels ( 47,48 ) . an attractive hypothesis adding another level of complexity to the regulation of translation initiation is that , in eukaryotic cells , the aug - containing portions of 5-utrs are similarly removed by alternative splicing , thus converting poorly translatable ( in some cases , virtually inactive ) mrnas into active ones . ( 49 ) found that the majority of alternative splicing events in human genes occurred in 5-utr regions . this observation suggests that aug - containing regions are removed by alternative splicing in many human mrnas and that the structures of cdnas , which reflect the hypothetical major forms of mrnas , and the corresponding functional mrnas ( hypothetical minor forms ) for certain genes ( especially those expressed at low levels ) are substantially different . if , indeed , alternative splicing is a common mechanism for the activation of aug - containing 5-utrs , then such 5-utrs are expected to be typical of weakly expressed proteins . in accord with this hypothesis , the density of uaugs is notably lower in yeast genes ( table 8) where alternative splicing does not seem to be a possibility , given the scarcity of introns and partial degradation of the splicing machinery ( 50,51 ) . conceivably , yeast genes are subject to stronger purifying selection against uaugs compared with mammalian genes , owing to the lack of the alternative splicing option in the former . however , the pronounced conservation of uaugs in yeast genes ( table 7 ) suggests the existence of a different regulatory mechanism , which is common to a broad variety if not all eukaryotes , possibly , sequestration of ribosomes by the uaugs . the conventional scanning mechanism ( 6 ) is likely to be completely or at least partially suppressed by uaugs . apart from the possibility of alternative splicing , several molecular mechanisms might provide for efficient translation of mrnas containing uaugs including leaky scanning , reinitiation ( 1 ) or internal initiation of translation ( 5255 ) . the relative contributions of these mechanisms remain uncertain ( 31,56,57 ) but several recent studies suggest that the impact of at least some of them might be substantial ( 3,5,17,23,24,32,5866 ) . additional functional signals , such as specific secondary structure elements within the 5-utr , might compensate for the negative effects of uaugs on translation initiation . such hypothetical signals , which might interact with still unknown regulatory proteins , could be used for regulation of gene expression in response to various stimuli . under this hypothesis , the uaugs may be regarded as regulatory elements that maintain the low constitutive level of expression of proteins that need to be sharply up - regulated in response to specific cues . it has been shown that the efficiency of translation of certain mrnas depends on unknown 5-utr elements other than the 3 or + 4 positions of the start codon context ( 30 ) although there is no clear evidence of a wide distribution of such regulatory signals ( 2,4,19,31,56 ) . a recent genome - wide analysis of uaugs and uorfs in a curated set of human and rodent cdnas has suggested that the ribosome - shunt mechanism , in which the small ribosome subunit binds to the mrna in a cap - dependent manner but then jumps over a large region of the 5-utr containing secondary structures , uorfs and uaugs to land near the saug , might be a widespread mode of translation regulation ( 17 ) . generally , the results of our analysis are compatible with ribosome scanning being the major mechanism of translation initiation in eukaryotes but they also indicate that , in a substantial fraction of eukaryotic mrnas , uaugs and uorfs might confer an additional level of translation regulation . the comparative - genomic results presented here suggest that there are thousands of genes in mammalian genomes that might be subject to translation initiation regulation involving uaugs . this finding is expected to stimulate experimental study of the repertoire and impact of such regulatory mechanisms . plots of nucleotide triplet conservation in mammalian cdnas . ( a ) human mouse 5-utrs . rat 3-utr . a mammalian 5-utr with conserved uaugs and an in - frame stop codon . the alignment of the 5utrs of human and mouse 1 type iii collagen proprotein ( gi numbers : 15 149 480 and 33 859 525 , respectively ) . uaugs are colored yellow , the collagen starting codon is colored green , and the open reading frames are colored grey . the protein - coding region can not be extended in the 5 direction because of the presence of a conserved in - frame uga stop codon ( dark gray ) . length distributions of human uorfs starting with conserved uaugs , non - conserved uaugs and pseudo - orfs starting with ugaus . the orf length is represented by bins , each including 10 codons ( i.e. bin 1 includes orfs from 0 to 10 codons , bin 2 orfs from 11 to 20 codons and so on ) . preferential conservation of uaugs in orthologous human and mouse 5-utrs fisher 's exact test : 1266 conserved uaugs , 218 non - conserved uaugs , 5690 conserved agu / gua / gau preferential conservation of uaugs in mouse and rat orthologous 5-utrs fishers 's exact test : 1539 conserved uaugs , 359 non - conserved uaugs , 11 218 conserved agu / gua / gau / uag / ugas , 5703 non - conserved agu / gua / gau / uag / ugas and p = 2.9 10 . mouse stop - codon - bounded 5-utr alignments ( 5-utr < stop set ) fishers 's exact test : 255 conserved uaugs , 49 non - conserved uaugs , 1124 conserved agu / gua / gau / uag / ugas , 491 non - conserved agu / gua / gau / uag / ugas and p = 1.52 10 . rat stop - codon - bounded 5-utr alignments ( 5-utr < stop set ) fishers 's exact test : 499 conserved uaugs , 124 non - conserved uaugs , 3262 conserved agu / gua / gau / uag / ugas , 1675 non - conserved agu / gua / gau / uag / ugas and p = 3.53 10 . phase distribution of conserved uaugs in alignments of mammalian 5-utrs phase distribution of conserved uaugs in stop - codon - bounded alignments of mammalian 5-utrs ( 5-utr < stop set ) preferential conservation of uaugs in yeast orthologous 5-utrs triplets were considered when present in the aligned sequences from all four yeast species . the aug data set consisted of alignments of genomic sequences located upstream ( 50 nt ) of saugs . the 5-utr < stop dataset consisted of the respective sequences containing in - frame stop codons . the aug(5-utr < stop + cdna ) dataset consisted of genomic alignments of 5-utr < stop sets that matched 5-utrs of the available of yeast cdna sequences . fisher 's exact test for the fraction of conserved aug triplets versus the fraction of conserved agu / gua / gau / uag / uga triplets produced highly significant results ( p < 10 ) for each of the three datasets . the shuffled triplet frequencies are shown for genomic sequences located upstream of saugs codons of yeast genes , this corresponds to the first row of the table ( the aug dataset ) . expected and observed numbers of uaug triplets and shuffled triplets per 1000 nt in mammalian and yeast 5-utrs the statistical significance of the differences between expected and observed frequencies of uaug and shuffled triplets were estimated using the test ( 2 2 tables ) . in all cases , the difference for uaugs was significantly greater than that for the combined shuffled triplets ( p < 0.001 ) .

by comparing sequences of human , mouse and rat orthologous genes , we show that in 5-untranslated regions ( 5-utrs ) of mammalian cdnas but not in 3-utrs or coding sequences , aug is conserved to a significantly greater extent than any of the other 63 nt triplets . this effect is likely to reflect , primarily , bona fide evolutionary conservation , rather than cdna annotation artifacts , because the excess of conserved upstream augs ( uaugs ) is seen in 5-utrs containing stop codons in - frame with the start aug and many of the conserved augs are found in different frames , consistent with the location in authentic non - coding sequences . altogether , conserved uaugs are present in at least 2030% of mammalian genes . qualitatively similar results were obtained by comparison of orthologous genes from different species of the yeast genus saccharomyces . together with the observation that mammalian and yeast 5-utrs are significantly depleted in overall aug content , these findings suggest that aug triplets in 5-utrs are subject to the pressure of purifying selection in two opposite directions : the uaugs that have no specific function tend to be deleterious and get eliminated during evolution , whereas those uaugs that do serve a function are conserved . most probably , the principal role of the conserved uaugs is attenuation of translation at the initiation stage , which is often additionally regulated by alternative splicing in the mammalian 5-utrs . consistent with this hypothesis , we found that open reading frames starting from conserved uaugs are significantly shorter than those starting from non - conserved uaugs , possibly , owing to selection for optimization of the level of attenuation .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION AND CONCLUSIONS SUPPLEMENTARY DATA Figures and Tables

in particular , 5-utr length ( 18 ) , secondary structure ( 19 ) and the presence of aug triplets upstream of the true translation start in mrna , known as upstream augs ( 20 ) ( hereinafter uaugs ; for the sake of uniformity , we designate start codons aug disregarding the fact that it should be rendered as atg when dna sequences are considered ) , have been shown to affect the efficiency of translation . although , in most cases , translation of eukaryotic mrnas is initiated at the first aug of the 5-utr , according to the scanning model of kozak , many exceptions to this rule have been described ( 2,3,1925 ) . the most critical sites near the start aug codon ( saug ) are 3 ( the a of the saug is position + 1 ) , usually occupied by a purine , and + 4 , typically occupied by a guanine ( 19,26 ) . other sites near the start codon seem to be less important , although the influence of nucleotides in different positions may vary from species to species ( 2730 ) . generally , uaugs decrease mrna translation efficiency and may be considered strong negative translational regulatory signals ( 3,31,32 ) , since the major mechanism of translation initiation in eukaryotes appears to be the scanning model , according to which eukaryotic ribosomes initiate translation at the 5-proximal aug codon ( 1,6 ) . this model is supported by the observation that mammalian 5-utrs contain significantly fewer uaugs than expected by chance , suggesting that purifying selection caused elimination of uaugs in many 5-utrs ( 13,17 ) . weak start codon context ( low information content ) and conversely , high information content of the start codon context is typical of short 5-utrs containing no uaugs ( 13 ) . this finding seems to be compatible with the possibility that uaugs function as attenuators of translation initiation by engaging ribosomes in futile initiation and precluding them from reaching the saug . however , the alternative possibility remains that cdnas with aug - containing 5-utrs might represent mainly non - functional sequences , such as incorrectly processed transcripts and transcripts of pseudogenes , artifacts of cloning and coding region annotation , such that the purported uaugs actually belong to misannotated 5-portions of coding regions , or mrnas of genes with multiple transcription start sites ( 2,13,19,31,32 ) . a natural approach to address the issue of the functional significance of uaugs is to examine the evolutionary conservation of these triplets on genome scale . by comparing sequences of human , mouse and rat orthologous genes , we show here that in 5-utrs of mammalian cdnas but not in 3-utrs or coding sequences , aug is conserved to a significantly greater extent than any of the other 63 nt triplets . this observation can be hardly explained by annotation errors because pronounced excess of conserved uaugs was detected in 5-utrs containing an in - frame stop codon and because many of the conserved augs are found in different frames , which is consistent with the location in non - coding sequences . these observations strongly suggest that at least some of the conserved uaugs are functional elements of translation initiation regulation . all 5-utrs that showed statistically significant blastx ( 37 ) hits ( e - value < 10 ) to the non - redundant protein database ( national center for biotechnology information , nih , bethesda ) and , accordingly , were probable to represent that undetected coding sequences were removed from the datasets . in addition , the identity between the first 50 amino acids of the refseq proteins encoded in the analyzed genes and the corresponding swissprot entries were checked . aligned sequences of orthologous genes from four yeast species , saccharomyces cerevisiae , saccharomyces paradoxus , saccharomyces mikatae and saccharomyces bayanus were extracted from the sgd database ( ) ( 43 ) . the alignment of start and stop codons was fixed in order to ensure the correct partitioning of 5-utr , the cds ( protein - coding sequence ) and the 3-utr . the evolutionary conservation was calculated as the fraction of triplets that are conserved in pairwise ( for comparisons of mammalian sequences ) or multiple ( for comparisons of yeast sequences ) alignments of 5-utrs . we analyzed evolutionary conservation of uaugs in pairwise alignments of human mouse and mouse rat orthologs . examination of the evolutionary conservation of each of the 64 nt triplets in the aligned 5-utrs showed that aug was the most conserved triplet in both human mouse and mouse rat comparisons ( figure 1a and b ) . tables 1 and 2 compare the frequencies of conserved uaugs with those of the five triplets that are permutations of aug , in order to exclude any possible effect of nucleotide composition ; in both cases , the fraction of conserved augs was markedly greater than those of the other triplets ( p < 10 by the fisher 's exact test ) . in contrast , no excessive conservation of aug compared with the other 63 triplets was detected in 3-utrs and cdss ( excluding the start aug ) of mammalian mrnas ( figure 1c f ) , indicating that aug conservation was specific for 5-utrs . the conservation of uaugs seemed to be spread throughout the 5-utrs : a significant excess of conserved uaugs was observed both in the proximal ( region 100 : 1 nt ) and the distal ( region 200 : 101 nt ) parts of 5-utr alignments , without a significant enrichment in either ( supplementary tables s1s4 ) . an issue of concern with this observation is that the excess of conserved uaugs could be an artifact of erroneous annotation of coding regions , with the sequences annotated as 5-utr actually containing the upstream portion of the cds , including the typically conserved authentic start aug and , possibly , conserved methionine codons as well ( 36 ) . this did not seem to be a major factor because the observed excess of conserved uaug did not depend on whether we used refseq annotations ( tables 1 and 2 ) or only those refseq annotations that were consistent with the corresponding swissprot annotations ( supplementary tables s5 and s6 ) . nevertheless , to control such an artifact in a more direct fashion , we examined 5-utrs containing an upstream conserved stop codon(s ) in the same frame with the saug ( supplementary figure s1 ) ; 5-utrs containing in - phase stop codons are unlikely to represent undetected parts of the cds . figure 2 shows an example of a conserved in - frame stop codon in a human the main open reading frame ( orf ) encoding the 1 type iii collagen proprotein can not be extended in the 5 direction because , in both human and mouse 5-utrs , there is an in - frame stop codon uga . thus , the start codon of this gene apparently is annotated correctly , and the conserved uaugs are , indeed , located in the 5-utr ( figure 2 ) . analysis of the portions of 5-utrs located upstream of conserved in - frame stop codons ( 5-utr < stop ) yielded results that were fully compatible with those for complete 5-utrs . specifically , 19 and 22% of the human mouse and mouse rat alignments , respectively , contained conserved uaugs , and there was a highly significant excess of conserved uaugs compared with the five shuffled triplets ( tables 3 and 4 ) . further evidence of bona fide evolutionary conservation of uaugs was obtained by analysis of the phase distribution of the conserved uaugs ( phase 0 is the frame of the start codon , and phases 1 and 2 are frames shifted by 1 or 2 nt , respectively ) ( supplementary figure s2 ) . we found that human mouse and mouse rat alignments have a significant excess of conserved uaugs in the same phase ( 00 , 11 or 22 in table 5 ) . however , the fraction of conserved uaugs in different phases was also substantial ( 01 , 12 and so on ; table 5 ) which is not the expectation for protein - coding regions where frameshift mutations are , generally , not tolerated . it should be noted that blastn alignments are extremely conservative with respect to deletions / insertions , so the actual fraction of conserved uaugs in different phases might be even greater . the phase distribution of the conserved uaugs showed a significant excess of phase 11 and phase 22 uaugs compared with phase 00 uaugs ( table 5 ) . this may reflect the greater chance for uorfs in phase 00 to merge with the main orfs , thereby extending the main orfs in the 5 direction , which could be a mechanism of evolution of protein - coding regions yielding alternative translation starts ( 44,45 ) . however , the possibility can not be ruled out that , at least in part , the deficit of phase 00 is caused by the erroneous annotation of the 5-terminal aug as the start codon in a subset of mrnas , resulting in depletion of phase 0 uaugs . the lower excess of same - phase uaugs in the 5-utr < stop data set compared to the entire set of uaugs ( compare the data in tables 5 and 6 ) is probably owing to the lower sequence conservation and greater prevalence of indels in the distal parts of the 5-utrs ( 16 ) . thus , the phase distribution of the conserved uaugs is best compatible with their localization in non - coding regions . taken together , these results show that the observed exceptional conservation of uaugs in the 5-utrs of mammalian genes is not an artifact and strongly suggests that the uaugs are subject to purifying selection and hence have a function(s ) , most probably , related to the regulation of translation initiation . figure 3 compares the length distributions of uorfs starting from conserved uaugs , non - conserved uaugs and ugaus ( gau triplet , a permutation of aug , located in the 5-utrs ) . a noticeable and statistically highly significant excess of very short uorfs was observed for conserved uaugs : the length distribution of uorfs starting with conserved uaugs differed from the other two distributions with p < 10 ( test ) , and the excess of the shortest uorfs ( 020 codons ) was significant at p < 10 ( fisher 's exact test ) . in contrast , the distributions of uorf lengths starting from non - conserved uaugs and gaus were statistically indistinguishable ( similar results were obtained with other permutations of aug ; data not shown ) . conceivably , the uorfs that start from conserved uaugs and may be engaged in the regulation of translation initiation were selected for short length optimization of the level of attenuation and prevent complete shutdown of initiation from the saug . there was no significant difference between the nucleotide contexts of the conserved and non - conserved uaugs ( data not shown ) ; in both cases , the information content of the uaug context was lower than that of the saug context ( 13,17 ) . having shown that uaugs are exceptionally conserved in 5-utrs of mammalian mrnas , we sought to investigate how general this pattern might be . this analysis revealed a highly significant excess of conserved uaugs compared with the permuted triplets ( table 7 ) . the comparative genomic analysis reported here shows that the 5-utrs of at least 2030% of mammalian mrnas contain conserved uaugs ; qualitatively similar results were obtained with yeast 5-utrs . strikingly , in both mammals and yeasts , aug is by far the most conserved nucleotide triplet in 5-utr but not in 3-utrs or coding regions . these findings suggest that at least a fraction of the conserved uaugs perform a function related to the regulation of translation initiation . compatible with this conclusion , we found that uorfs starting from conserved ( and , potentially , functional ) uaugs are , on average , significantly shorter than those that start from non - conserved ( probably , non - functional ) augs . these genes are subject to complex regulation which seems to be compatible with the proposed role of uaugs in modulation of translation initiation . the conclusion of the present work on the probably functional importance of conserved uaugs has to be considered in conjunction with the more or less orthogonal previous observation that mammalian 5-utrs are significantly depleted of augs , a trend that was confirmed in this study and is even stronger in yeast 5-utrs ( table 8) . apparently , purifying selection acts on the uaugs in two opposite directions : the uaugs that have no specific function tend to be deleterious and are eliminated during evolution ; in contrast , those uaugs that do serve a function are maintained . probably , the leading hypothesis is that the uaugs attenuate translation by diverting scanning ribosomes from the authentic saug . apparently , selection also operates at the level of the regulatory uorfs by shortening their length , possibly , to optimize the level of translation attenuation . an attractive hypothesis adding another level of complexity to the regulation of translation initiation is that , in eukaryotic cells , the aug - containing portions of 5-utrs are similarly removed by alternative splicing , thus converting poorly translatable ( in some cases , virtually inactive ) mrnas into active ones . this observation suggests that aug - containing regions are removed by alternative splicing in many human mrnas and that the structures of cdnas , which reflect the hypothetical major forms of mrnas , and the corresponding functional mrnas ( hypothetical minor forms ) for certain genes ( especially those expressed at low levels ) are substantially different . if , indeed , alternative splicing is a common mechanism for the activation of aug - containing 5-utrs , then such 5-utrs are expected to be typical of weakly expressed proteins . in accord with this hypothesis , the density of uaugs is notably lower in yeast genes ( table 8) where alternative splicing does not seem to be a possibility , given the scarcity of introns and partial degradation of the splicing machinery ( 50,51 ) . conceivably , yeast genes are subject to stronger purifying selection against uaugs compared with mammalian genes , owing to the lack of the alternative splicing option in the former . however , the pronounced conservation of uaugs in yeast genes ( table 7 ) suggests the existence of a different regulatory mechanism , which is common to a broad variety if not all eukaryotes , possibly , sequestration of ribosomes by the uaugs . the conventional scanning mechanism ( 6 ) is likely to be completely or at least partially suppressed by uaugs . under this hypothesis , the uaugs may be regarded as regulatory elements that maintain the low constitutive level of expression of proteins that need to be sharply up - regulated in response to specific cues . it has been shown that the efficiency of translation of certain mrnas depends on unknown 5-utr elements other than the 3 or + 4 positions of the start codon context ( 30 ) although there is no clear evidence of a wide distribution of such regulatory signals ( 2,4,19,31,56 ) . a recent genome - wide analysis of uaugs and uorfs in a curated set of human and rodent cdnas has suggested that the ribosome - shunt mechanism , in which the small ribosome subunit binds to the mrna in a cap - dependent manner but then jumps over a large region of the 5-utr containing secondary structures , uorfs and uaugs to land near the saug , might be a widespread mode of translation regulation ( 17 ) . generally , the results of our analysis are compatible with ribosome scanning being the major mechanism of translation initiation in eukaryotes but they also indicate that , in a substantial fraction of eukaryotic mrnas , uaugs and uorfs might confer an additional level of translation regulation . a mammalian 5-utr with conserved uaugs and an in - frame stop codon . uaugs are colored yellow , the collagen starting codon is colored green , and the open reading frames are colored grey . the protein - coding region can not be extended in the 5 direction because of the presence of a conserved in - frame uga stop codon ( dark gray ) . length distributions of human uorfs starting with conserved uaugs , non - conserved uaugs and pseudo - orfs starting with ugaus . preferential conservation of uaugs in orthologous human and mouse 5-utrs fisher 's exact test : 1266 conserved uaugs , 218 non - conserved uaugs , 5690 conserved agu / gua / gau preferential conservation of uaugs in mouse and rat orthologous 5-utrs fishers 's exact test : 1539 conserved uaugs , 359 non - conserved uaugs , 11 218 conserved agu / gua / gau / uag / ugas , 5703 non - conserved agu / gua / gau / uag / ugas and p = 2.9 10 . mouse stop - codon - bounded 5-utr alignments ( 5-utr < stop set ) fishers 's exact test : 255 conserved uaugs , 49 non - conserved uaugs , 1124 conserved agu / gua / gau / uag / ugas , 491 non - conserved agu / gua / gau / uag / ugas and p = 1.52 10 . rat stop - codon - bounded 5-utr alignments ( 5-utr < stop set ) fishers 's exact test : 499 conserved uaugs , 124 non - conserved uaugs , 3262 conserved agu / gua / gau / uag / ugas , 1675 non - conserved agu / gua / gau / uag / ugas and p = 3.53 10 . phase distribution of conserved uaugs in alignments of mammalian 5-utrs phase distribution of conserved uaugs in stop - codon - bounded alignments of mammalian 5-utrs ( 5-utr < stop set ) preferential conservation of uaugs in yeast orthologous 5-utrs triplets were considered when present in the aligned sequences from all four yeast species . the 5-utr < stop dataset consisted of the respective sequences containing in - frame stop codons . fisher 's exact test for the fraction of conserved aug triplets versus the fraction of conserved agu / gua / gau / uag / uga triplets produced highly significant results ( p < 10 ) for each of the three datasets . the shuffled triplet frequencies are shown for genomic sequences located upstream of saugs codons of yeast genes , this corresponds to the first row of the table ( the aug dataset ) . expected and observed numbers of uaug triplets and shuffled triplets per 1000 nt in mammalian and yeast 5-utrs the statistical significance of the differences between expected and observed frequencies of uaug and shuffled triplets were estimated using the test ( 2 2 tables ) .

patients with copd who are hospitalized for an acute exacerbation often receive oxygen therapy as a part of their treatment . despite current recommendations,1 oxygen therapy is often suboptimally adjusted during acute exacerbation of copd , with episodes of both hypoxemia and hyperoxia reported.2 hyperoxia may be particularly problematic in copd because of its association with hypercapnia,37 especially in the acute phase of exacerbations,3,4,8 where it may be associated with adverse clinical outcomes , including respiratory acidosis and even increased mortality.4,8,9 continuous adjustment of oxygen flows is time consuming , and clinicians are more concerned with oxygen desaturation than with hyperoxia , potentially leading to the utilization of higher than required oxygen flows for longer than necessary . closed - loop adjustment of oxygen administration based on pulsed oxygen saturation ( spo2 ) may optimize oxygen therapy and improve patients safety.10 we recently developed a closed - loop device , freeo2 , that automatically adjusts the oxygen flows administered to spontaneously breathing patients to maintain spo2 in a predefined target set by physicians and to progressively and automatically wean patients from oxygen.11 this pilot trial was intended to test the acceptability of freeo2 by nurses and attending physicians and the potential clinical impact of using such a device in patients hospitalized in the respiratory ward for acute exacerbation of copd . our hypotheses were that 1 ) nurses and physicians would conclude that freeo2 is an acceptable oxygen administration device , 2 ) time spent in the target spo2 zone would be increased by automatic o2 flow adjustments in comparison to manual o2 titration , and 3 ) weaning time from oxygen would be shortened by the automatic o2 adjustment device ( freeo2 ) . we conducted a randomized trial ( clinical trial registration : nct01393015 ) comparing manual oxygen titration and automated oxygen adjustment with freeo2 in patients hospitalized for an acute exacerbation of copd in whom oxygen therapy was prescribed by the attending physician based on the documentation of resting hypoxemia ( spo2 < 90% ) . we included patients aged 40 years with a past or current smoking history of at least 10 pack - years . patients had to maintain an spo2 of 92% with supplemental oxygen at a maximum flow rate of 8 l / min . we excluded patients admitted for > 24 hours , patients infected with multidrug - resistant bacteria , patients on intermittent noninvasive ventilation ( including cpap for obstructive sleep apnea ) , and patients with cognitive impairment precluding informed consent or collaboration . the study was conducted at the institut universitaire de cardiologie et de pneumologie de qubec . the study was approved by the institutional ethics committee ( # 20694 ) , and a signed consent was obtained from each participant . the freeo2 system automatically adjusts oxygen flow rates administered via nasal cannula or nonocclusive mask , using a closed - loop algorithm based on physiological data.11 it also provides continuous monitoring of respiratory parameters in spontaneously breathing patients.11 the main parameter that is monitored within this closed - loop system is spo2 that continuously feeds the algorithm at a rate of 1 value / s . a proportional integral controller adjusts the oxygen flow delivered by a mass - flow controller from 0 l / min to 20 l / min ( flow accuracy 0.1 l / min ) , with the aim of maintaining spo2 at a predefined target . the system was developed by the authors ( fl and elh ) in collaboration with the department of electronic and informatics engineering , laval university , quebec ( figure 1 ) . patients were allocated to either 1 ) automated oxygen titration by freeo2 with continuous monitoring of physiological parameters , including spo2 and oxygen flow rates ( freeo2 arm ) , at bedside and remotely at the nursing station or 2 ) manual oxygen administration with bedside titration from usual oxygen flow - meter ( rotameter ) and usual oxygen monitoring with bedside pulsed oximetry ( control arm ) . in the control group , the nurses manually adjusted the oxygen flows guided by local protocols and by spo2 target provided by the attending physician before randomization . spo2 monitoring was conducted as per the local practice ( ie , approximately three times per day ) . however , there were no specific training to the oxygen management guidelines and no recording of the compliance to these guidelines . in the freeo2 arm , oxygen was titrated automatically every second to maintain spo2 in the target zone set by the attending physician before randomization . the principal investigators of the study did not manage the patients included in the study and were not involved in the treatment and discharge decisions . at baseline , demographic data , arterial or capillary blood gases , oxygen flow rate , and respiratory rate were recorded . pulmonary function tests ( forced expiratory volume in 1 second and forced vital capacity ) were conducted using standardized postbronchodilator spirometry . these pulmonary parameters were recorded during a stable period of copd within 1 year before or after exacerbation and during hospitalization . in order to evaluate whether freeo2 could be used in daily practice and to quantify its acceptance and ease of use by caregivers ( primary outcomes ) , the nurses and physicians perception of the appropriateness of the oxygen management was monitored by direct interview when available on a daily basis using a 10-point likert scale ( from 0 : not appropriate at all to 10 : perfectly appropriate ) . the secondary outcomes were 1 ) time spent within the spo2 target ( 2% ) , 2 ) time with severe desaturation ( spo2 < 85% ) , 3 ) time with hyperoxia ( spo2 > 5% above the target ) , 4 ) total duration of oxygen therapy , and 5 ) hospital length of stay . patients in both groups had continuous monitoring of spo2 , respiratory rate , heart rate , and end - tidal co2 with the freeo2 system set in the freeo2 mode ( automated closed - loop adjustment of oxygen and continuous recording ) or in the recording mode ( continuous recording of physiological data without administration of oxygen by the device for those in the control group ) . capillary blood gases were collected every day during the first week or up to discharge . we could not calculate a priori a sample size based on the primary outcome as we did not have any data to estimate how the system would be accepted by the caregivers . nevertheless , we chose to include 50 patients to obtain sufficient exposure and experience with freeo2 . data were expressed using mean with standard deviation or median with interquartile ranges for continuous variables or proportions for categorical data , respectively . categorical variables were compared between groups using the chi - square or fisher s exact tests , and continuous variables were compared using one - way analyses of variance . wilcoxon rank sum tests ( nonparametric tests ) were performed for data that did not fulfill the normality or variance assumptions after transformation . when different conclusions between analyses from one - way analysis of variance and nonparametric tests occurred , the latest approach was retained . all analyses were conducted using the statistical package sas , version 9.4 ( sas institute inc . , cary , nc , usa ) . we conducted a randomized trial ( clinical trial registration : nct01393015 ) comparing manual oxygen titration and automated oxygen adjustment with freeo2 in patients hospitalized for an acute exacerbation of copd in whom oxygen therapy was prescribed by the attending physician based on the documentation of resting hypoxemia ( spo2 < 90% ) . we included patients aged 40 years with a past or current smoking history of at least 10 pack - years . patients had to maintain an spo2 of 92% with supplemental oxygen at a maximum flow rate of 8 l / min . we excluded patients admitted for > 24 hours , patients infected with multidrug - resistant bacteria , patients on intermittent noninvasive ventilation ( including cpap for obstructive sleep apnea ) , and patients with cognitive impairment precluding informed consent or collaboration . the study was conducted at the institut universitaire de cardiologie et de pneumologie de qubec . the study was approved by the institutional ethics committee ( # 20694 ) , and a signed consent was obtained from each participant . the freeo2 system automatically adjusts oxygen flow rates administered via nasal cannula or nonocclusive mask , using a closed - loop algorithm based on physiological data.11 it also provides continuous monitoring of respiratory parameters in spontaneously breathing patients.11 the main parameter that is monitored within this closed - loop system is spo2 that continuously feeds the algorithm at a rate of 1 value / s . a proportional integral controller adjusts the oxygen flow delivered by a mass - flow controller from 0 l / min to 20 l / min ( flow accuracy 0.1 l / min ) , with the aim of maintaining spo2 at a predefined target . the system was developed by the authors ( fl and elh ) in collaboration with the department of electronic and informatics engineering , laval university , quebec ( figure 1 ) . patients were allocated to either 1 ) automated oxygen titration by freeo2 with continuous monitoring of physiological parameters , including spo2 and oxygen flow rates ( freeo2 arm ) , at bedside and remotely at the nursing station or 2 ) manual oxygen administration with bedside titration from usual oxygen flow - meter ( rotameter ) and usual oxygen monitoring with bedside pulsed oximetry ( control arm ) . in the control group , the nurses manually adjusted the oxygen flows guided by local protocols and by spo2 target provided by the attending physician before randomization . spo2 monitoring was conducted as per the local practice ( ie , approximately three times per day ) . however , there were no specific training to the oxygen management guidelines and no recording of the compliance to these guidelines . in the freeo2 arm , oxygen was titrated automatically every second to maintain spo2 in the target zone set by the attending physician before randomization . the principal investigators of the study did not manage the patients included in the study and were not involved in the treatment and discharge decisions . at baseline , demographic data , arterial or capillary blood gases , oxygen flow rate , and respiratory rate were recorded . pulmonary function tests ( forced expiratory volume in 1 second and forced vital capacity ) were conducted using standardized postbronchodilator spirometry . these pulmonary parameters were recorded during a stable period of copd within 1 year before or after exacerbation and during hospitalization . in order to evaluate whether freeo2 could be used in daily practice and to quantify its acceptance and ease of use by caregivers ( primary outcomes ) , the nurses and physicians perception of the appropriateness of the oxygen management was monitored by direct interview when available on a daily basis using a 10-point likert scale ( from 0 : not appropriate at all to 10 : perfectly appropriate ) . the secondary outcomes were 1 ) time spent within the spo2 target ( 2% ) , 2 ) time with severe desaturation ( spo2 < 85% ) , 3 ) time with hyperoxia ( spo2 > 5% above the target ) , 4 ) total duration of oxygen therapy , and 5 ) hospital length of stay . patients in both groups had continuous monitoring of spo2 , respiratory rate , heart rate , and end - tidal co2 with the freeo2 system set in the freeo2 mode ( automated closed - loop adjustment of oxygen and continuous recording ) or in the recording mode ( continuous recording of physiological data without administration of oxygen by the device for those in the control group ) . capillary blood gases were collected every day during the first week or up to discharge . we could not calculate a priori a sample size based on the primary outcome as we did not have any data to estimate how the system would be accepted by the caregivers . nevertheless , we chose to include 50 patients to obtain sufficient exposure and experience with freeo2 . data were expressed using mean with standard deviation or median with interquartile ranges for continuous variables or proportions for categorical data , respectively . categorical variables were compared between groups using the chi - square or fisher s exact tests , and continuous variables were compared using one - way analyses of variance . wilcoxon rank sum tests ( nonparametric tests ) were performed for data that did not fulfill the normality or variance assumptions after transformation . when different conclusions between analyses from one - way analysis of variance and nonparametric tests occurred , the latest approach was retained . all analyses were conducted using the statistical package sas , version 9.4 ( sas institute inc . , cary , nc , usa ) . we randomized 25 patients with copd in each group ( total of 50 patients ) from august 2011 to february 2015 . the mean age was 729 years , and the mean forced expiratory volume in 1 second was 1.000.49 l , representing 37%22% predicted . there were 924 evaluations of the oxygen adjustment and monitoring by nurses and 96 evaluations by physicians . nurses and physicians considered freeo2 adjustments and monitoring to be at least as appropriate and as acceptable as manual oxygen management . only monitoring was deemed slightly better with freeo2 by the physicians , but the difference was not statistically significant ( table 2 ) . we did not directly evaluate patient s tolerance of the system , but 80% of the patients completed the study in both groups . the main reason to prematurely stop the study was related to the difficulty in continuously wearing a pulse oximeter and the reduced mobility ( 70% of the reasons to stop ) ; this occurred similarly in both groups . spo2 targets chosen by physicians were similar in both groups , averaging 90.0% and 90.1% for freeo2 and manual o2 adjustment , respectively . the mean spo2 during the study was 90.91.2 in the freeo2 group and 91.91.2 in the manual adjustment group ( p=0.009 ) . the proportion of time within spo2 target was 81.2%19.9% with freeo2 vs 51.3%19.7% with manual o2 adjustments ( p<0.001 ) . the percentage of time with severe desaturation ( spo2 < 85% ) and with hyperoxia ( spo2 > 5% above the target ) was significantly lower with freeo2 in comparison with manual oxygen adjustment ( figure 3 and table 3 ) . there was no significant difference in blood gases measured on day 3 and day 7 between the two groups . duration of oxygen administration was reduced by 1.8 days with freeo2 , but this difference did not reach statistical significance ( table 4 ) . time from randomization to hospital discharge was reduced by 2.6 days with freeo2 ( p=0.051 ) . there was no difference between the two groups in the requirement for noninvasive ventilation during hospitalization , need of transfer to the intensive care unit , or death . the readmission rates at 30 , 60 , and 180 days were also similar in the two groups . there was no safety issue as we did not record any oxygen delivery interruption with freeo2 . few technical issues occurred with end - tidal co2 monitoring , but none concerned the oxygen delivery valve , and this was not associated with safety issues . there were 924 evaluations of the oxygen adjustment and monitoring by nurses and 96 evaluations by physicians . nurses and physicians considered freeo2 adjustments and monitoring to be at least as appropriate and as acceptable as manual oxygen management . only monitoring was deemed slightly better with freeo2 by the physicians , but the difference was not statistically significant ( table 2 ) . we did not directly evaluate patient s tolerance of the system , but 80% of the patients completed the study in both groups . the main reason to prematurely stop the study was related to the difficulty in continuously wearing a pulse oximeter and the reduced mobility ( 70% of the reasons to stop ) ; this occurred similarly in both groups . spo2 targets chosen by physicians were similar in both groups , averaging 90.0% and 90.1% for freeo2 and manual o2 adjustment , respectively . the mean spo2 during the study was 90.91.2 in the freeo2 group and 91.91.2 in the manual adjustment group ( p=0.009 ) . the proportion of time within spo2 target was 81.2%19.9% with freeo2 vs 51.3%19.7% with manual o2 adjustments ( p<0.001 ) . ( spo2 > 5% above the target ) was significantly lower with freeo2 in comparison with manual oxygen adjustment ( figure 3 and table 3 ) . there was no significant difference in blood gases measured on day 3 and day 7 between the two groups . duration of oxygen administration was reduced by 1.8 days with freeo2 , but this difference did not reach statistical significance ( table 4 ) . time from randomization to hospital discharge was reduced by 2.6 days with freeo2 ( p=0.051 ) . there was no difference between the two groups in the requirement for noninvasive ventilation during hospitalization , need of transfer to the intensive care unit , or death . the readmission rates at 30 , 60 , and 180 days were also similar in the two groups . there was no safety issue as we did not record any oxygen delivery interruption with freeo2 . few technical issues occurred with end - tidal co2 monitoring , but none concerned the oxygen delivery valve , and this was not associated with safety issues . spo2 targets chosen by physicians were similar in both groups , averaging 90.0% and 90.1% for freeo2 and manual o2 adjustment , respectively . the mean spo2 during the study was 90.91.2 in the freeo2 group and 91.91.2 in the manual adjustment group ( p=0.009 ) . the proportion of time within spo2 target was 81.2%19.9% with freeo2 vs 51.3%19.7% with manual o2 adjustments ( p<0.001 ) . ( spo2 > 5% above the target ) was significantly lower with freeo2 in comparison with manual oxygen adjustment ( figure 3 and table 3 ) . there was no significant difference in blood gases measured on day 3 and day 7 between the two groups . duration of oxygen administration was reduced by 1.8 days with freeo2 , but this difference did not reach statistical significance ( table 4 ) . time from randomization to hospital discharge was reduced by 2.6 days with freeo2 ( p=0.051 ) . there was no difference between the two groups in the requirement for noninvasive ventilation during hospitalization , need of transfer to the intensive care unit , or death . the readmission rates at 30 , 60 , and 180 days were also similar in the two groups . there was no safety issue as we did not record any oxygen delivery interruption with freeo2 . few technical issues occurred with end - tidal co2 monitoring , but none concerned the oxygen delivery valve , and this was not associated with safety issues . our pilot trial demonstrates the feasibility of providing oxygen therapy with freeo2 to patients hospitalized for an acute exacerbation of copd , even during prolonged administration of oxygen ( up to 8 consecutive days ) . in addition , our results suggest that freeo2 may have the potential to reduce hospital length of stay , although this pilot observation requires formal testing in a proper clinical trial . our study is the first to investigate the potential effects of a continuous and an automated oxygen titration and weaning system on physiological and clinical outcomes in patients hospitalized for an acute exacerbation of copd . several studies evaluated similar systems in patients with copd in other settings , such as home care12 or during rehabilitation,13,14 and in other populations , including neonates15,16 and in patients with acute respiratory failure in the emergency department.17 automated therapy has always proved effective in safely providing oxygen in these settings and populations , and our results are consistent with this notion . the introduction of new devices in clinical practice may not be well accepted , and barriers to technology acceptance should not be overlooked.18 the primary study outcome was selected accordingly . freeo2 was deemed at least as adequate for oxygen administration and monitoring as usual oxygen administration . also , most patients tolerated the instrument , as 80% completed the study in both groups . few patients did not complete the study due to reduced mobility related to the continuous oximetry recording . implementation of an oximeter with wireless communication features may further improve the acceptance of o2 automated systems in the future . we were successful in continuously recording respiratory parameters with identical oximeters in both groups with second - by - second data available 90.4% and 82.4% of the time in the freeo2 and control groups , respectively . as expected and in line with other evaluations of automated oxygen titration,10,14,17 oxygenation parameters were improved in the freeo2 group , with more time spent in the targeted range of spo2 and less time with oxygen desaturation or hyperoxia . this may be clinically relevant when considering that hyperoxia may induce hypercapnia37 with potentially severe consequences.4,8,9 hyperoxia is also of concern in patients with copd who frequently have coronary artery disease,19 as it may increase coronary artery resistance.20,21 on the other hand , periods of oxygen desaturations may be responsible for arythmias and myocardial ischemia,22,23 and they should also be avoided . in the control group , patients were in the spo2 target 50% of the time , which may not be compared with other data , as these are the first data with continuous recording of spo2 in this situation to our knowledge . in the recent review of cousin et al,24 the mean rate of accurate prescription of oxygen therapy in eleven studies was 17.4% before and 51.2% after implementation of specific interventions . one strength of our study is that we could evaluate an automated oxygen therapy during prolonged duration ( ie , up to 8 days ) , compared with previous studies where automated o2 titration was provided for a duration of < 1 hour11,13,14 and up to 3 hours.17 an important result of our trial is the trend toward a reduction in duration of oxygen therapy and hospital length of stay with automated oxygen titration , although we can not ascertain whether this effect was related to closer o2 monitoring and surveillance as a part of the freeo2 protocol or whether this was due to the freeo2 device itself . although the trial was not primarily designed to demonstrate an impact on these outcomes , this finding is in agreement with the hypothesis that unnecessarily high flows and prolonged oxygen therapy are common25 and that automated weaning may be useful to abbreviate oxygen therapy in hospitalized patients with copd . length of stay must be interpreted in comparison to local statistics obtained off - protocol . in 2010 , the mean length of stay for acute exacerbation of copd in our institution was 9.88.1 days ( unpublished data ) , which is in - line with the length of stay in the control group of the present study . reducing the length of hospital stay by 31% as found in this study would be expected to have major logistic and financial impacts on our health care setting . this study was underpowered to conclude on the hospital length of stay , and larger studies will be required to confirm this potential benefit of automated oxygen titration . first , this was a pilot study with a small sample size . a more complete evaluation of relevant clinical outcomes , such as length of stay and cost effectiveness , second , the recruitment rate was low as we recruited only 50 patients in 3 years . the main reason was the limited resources during prolonged periods that did not allow continuous screening of potential study participants . there were > 500 copd hospitalized every year during the study period , and several of these would have been eligible to the trial . nurses had to be aware of the patients allocation as they were in charge of the manual titration in the control group , and remote monitoring of cardiorespiratory parameters at the nursing station was available only in the freeo2 arm . to limit the impact of this potential source of bias this study demonstrates the feasibility of using automated oxygen titration and weaning as well as remote monitoring with the freeo2 system in patients hospitalized for acute exacerbation of copd . automated oxygen titration provided benefits in terms of safety ( ie , reduction in the time with severe desaturation and hyperoxia ) and may have contributed to a trend toward the reduced hospital length of stay , a potentially important finding , if these benefits are confirmed in subsequent studies specifically designed to demonstrate these effects .

introductionwe developed a device ( freeo2 ) that automatically adjusts the oxygen flow rates based on patients needs , in order to limit hyperoxia and hypoxemia and to automatically wean them from oxygen.objectivethe aim of this study was to evaluate the feasibility of using freeo2 in patients hospitalized in the respiratory ward for an acute exacerbation of copd.methodswe conducted a randomized controlled trial comparing freeo2 vs manual oxygen titration in the respiratory ward of a university hospital . we measured the perception of appropriateness of oxygen titration and monitoring in both groups by nurses and attending physicians using a likert scale . we evaluated the time in the target range of oxygen saturation ( spo2 ) as defined for each patient by the attending physician , the time with severe desaturation ( spo2 < 85% ) , and the time with hyperoxia ( spo2 > 5% above the target ) . we also recorded length of stay , intensive care unit admissions , and readmission rate . fifty patients were randomized ( 25 patients in both groups ; mean age : 728 years ; mean forced expiratory volume in 1 second : 1.000.49 l ; and mean initial o2 flow 2.01.0 l / min).resultsnurses and attending physicians felt that oxygen titration and monitoring were equally appropriate with both o2 administration systems . the percentage of time within the spo2 target was significantly higher with freeo2 , and the time with severe desaturation and hyperoxia was significantly reduced with freeo2 . time from study inclusion to hospital discharge was 5.84.4 days with freeo2 and 8.46.0 days with usual oxygen administration ( p=0.051).conclusionfreeo2 was deemed as an appropriate oxygen administration system by nurses and physicians of a respiratory unit . this system maintained spo2 at the target level better than did manual titration and reduced periods of desaturation and hyperoxia . our results also suggest that freeo2 has the potential to reduce the hospital length of stay .

Introduction Methods Participants FreeO Protocol Measurements Statistical analyses Results Primary outcomes Secondary outcomes Oxygenation and blood gases Clinical outcomes Safety Discussion Conclusion

patients with copd who are hospitalized for an acute exacerbation often receive oxygen therapy as a part of their treatment . despite current recommendations,1 oxygen therapy is often suboptimally adjusted during acute exacerbation of copd , with episodes of both hypoxemia and hyperoxia reported.2 hyperoxia may be particularly problematic in copd because of its association with hypercapnia,37 especially in the acute phase of exacerbations,3,4,8 where it may be associated with adverse clinical outcomes , including respiratory acidosis and even increased mortality.4,8,9 continuous adjustment of oxygen flows is time consuming , and clinicians are more concerned with oxygen desaturation than with hyperoxia , potentially leading to the utilization of higher than required oxygen flows for longer than necessary . closed - loop adjustment of oxygen administration based on pulsed oxygen saturation ( spo2 ) may optimize oxygen therapy and improve patients safety.10 we recently developed a closed - loop device , freeo2 , that automatically adjusts the oxygen flows administered to spontaneously breathing patients to maintain spo2 in a predefined target set by physicians and to progressively and automatically wean patients from oxygen.11 this pilot trial was intended to test the acceptability of freeo2 by nurses and attending physicians and the potential clinical impact of using such a device in patients hospitalized in the respiratory ward for acute exacerbation of copd . our hypotheses were that 1 ) nurses and physicians would conclude that freeo2 is an acceptable oxygen administration device , 2 ) time spent in the target spo2 zone would be increased by automatic o2 flow adjustments in comparison to manual o2 titration , and 3 ) weaning time from oxygen would be shortened by the automatic o2 adjustment device ( freeo2 ) . we conducted a randomized trial ( clinical trial registration : nct01393015 ) comparing manual oxygen titration and automated oxygen adjustment with freeo2 in patients hospitalized for an acute exacerbation of copd in whom oxygen therapy was prescribed by the attending physician based on the documentation of resting hypoxemia ( spo2 < 90% ) . we excluded patients admitted for > 24 hours , patients infected with multidrug - resistant bacteria , patients on intermittent noninvasive ventilation ( including cpap for obstructive sleep apnea ) , and patients with cognitive impairment precluding informed consent or collaboration . the study was conducted at the institut universitaire de cardiologie et de pneumologie de qubec . the study was approved by the institutional ethics committee ( # 20694 ) , and a signed consent was obtained from each participant . the freeo2 system automatically adjusts oxygen flow rates administered via nasal cannula or nonocclusive mask , using a closed - loop algorithm based on physiological data.11 it also provides continuous monitoring of respiratory parameters in spontaneously breathing patients.11 the main parameter that is monitored within this closed - loop system is spo2 that continuously feeds the algorithm at a rate of 1 value / s . a proportional integral controller adjusts the oxygen flow delivered by a mass - flow controller from 0 l / min to 20 l / min ( flow accuracy 0.1 l / min ) , with the aim of maintaining spo2 at a predefined target . patients were allocated to either 1 ) automated oxygen titration by freeo2 with continuous monitoring of physiological parameters , including spo2 and oxygen flow rates ( freeo2 arm ) , at bedside and remotely at the nursing station or 2 ) manual oxygen administration with bedside titration from usual oxygen flow - meter ( rotameter ) and usual oxygen monitoring with bedside pulsed oximetry ( control arm ) . in the control group , the nurses manually adjusted the oxygen flows guided by local protocols and by spo2 target provided by the attending physician before randomization . in the freeo2 arm , oxygen was titrated automatically every second to maintain spo2 in the target zone set by the attending physician before randomization . at baseline , demographic data , arterial or capillary blood gases , oxygen flow rate , and respiratory rate were recorded . pulmonary function tests ( forced expiratory volume in 1 second and forced vital capacity ) were conducted using standardized postbronchodilator spirometry . in order to evaluate whether freeo2 could be used in daily practice and to quantify its acceptance and ease of use by caregivers ( primary outcomes ) , the nurses and physicians perception of the appropriateness of the oxygen management was monitored by direct interview when available on a daily basis using a 10-point likert scale ( from 0 : not appropriate at all to 10 : perfectly appropriate ) . the secondary outcomes were 1 ) time spent within the spo2 target ( 2% ) , 2 ) time with severe desaturation ( spo2 < 85% ) , 3 ) time with hyperoxia ( spo2 > 5% above the target ) , 4 ) total duration of oxygen therapy , and 5 ) hospital length of stay . patients in both groups had continuous monitoring of spo2 , respiratory rate , heart rate , and end - tidal co2 with the freeo2 system set in the freeo2 mode ( automated closed - loop adjustment of oxygen and continuous recording ) or in the recording mode ( continuous recording of physiological data without administration of oxygen by the device for those in the control group ) . we could not calculate a priori a sample size based on the primary outcome as we did not have any data to estimate how the system would be accepted by the caregivers . we conducted a randomized trial ( clinical trial registration : nct01393015 ) comparing manual oxygen titration and automated oxygen adjustment with freeo2 in patients hospitalized for an acute exacerbation of copd in whom oxygen therapy was prescribed by the attending physician based on the documentation of resting hypoxemia ( spo2 < 90% ) . we excluded patients admitted for > 24 hours , patients infected with multidrug - resistant bacteria , patients on intermittent noninvasive ventilation ( including cpap for obstructive sleep apnea ) , and patients with cognitive impairment precluding informed consent or collaboration . the study was conducted at the institut universitaire de cardiologie et de pneumologie de qubec . the study was approved by the institutional ethics committee ( # 20694 ) , and a signed consent was obtained from each participant . the freeo2 system automatically adjusts oxygen flow rates administered via nasal cannula or nonocclusive mask , using a closed - loop algorithm based on physiological data.11 it also provides continuous monitoring of respiratory parameters in spontaneously breathing patients.11 the main parameter that is monitored within this closed - loop system is spo2 that continuously feeds the algorithm at a rate of 1 value / s . a proportional integral controller adjusts the oxygen flow delivered by a mass - flow controller from 0 l / min to 20 l / min ( flow accuracy 0.1 l / min ) , with the aim of maintaining spo2 at a predefined target . patients were allocated to either 1 ) automated oxygen titration by freeo2 with continuous monitoring of physiological parameters , including spo2 and oxygen flow rates ( freeo2 arm ) , at bedside and remotely at the nursing station or 2 ) manual oxygen administration with bedside titration from usual oxygen flow - meter ( rotameter ) and usual oxygen monitoring with bedside pulsed oximetry ( control arm ) . in the control group , the nurses manually adjusted the oxygen flows guided by local protocols and by spo2 target provided by the attending physician before randomization . in the freeo2 arm , oxygen was titrated automatically every second to maintain spo2 in the target zone set by the attending physician before randomization . at baseline , demographic data , arterial or capillary blood gases , oxygen flow rate , and respiratory rate were recorded . pulmonary function tests ( forced expiratory volume in 1 second and forced vital capacity ) were conducted using standardized postbronchodilator spirometry . in order to evaluate whether freeo2 could be used in daily practice and to quantify its acceptance and ease of use by caregivers ( primary outcomes ) , the nurses and physicians perception of the appropriateness of the oxygen management was monitored by direct interview when available on a daily basis using a 10-point likert scale ( from 0 : not appropriate at all to 10 : perfectly appropriate ) . the secondary outcomes were 1 ) time spent within the spo2 target ( 2% ) , 2 ) time with severe desaturation ( spo2 < 85% ) , 3 ) time with hyperoxia ( spo2 > 5% above the target ) , 4 ) total duration of oxygen therapy , and 5 ) hospital length of stay . patients in both groups had continuous monitoring of spo2 , respiratory rate , heart rate , and end - tidal co2 with the freeo2 system set in the freeo2 mode ( automated closed - loop adjustment of oxygen and continuous recording ) or in the recording mode ( continuous recording of physiological data without administration of oxygen by the device for those in the control group ) . we could not calculate a priori a sample size based on the primary outcome as we did not have any data to estimate how the system would be accepted by the caregivers . nevertheless , we chose to include 50 patients to obtain sufficient exposure and experience with freeo2 . categorical variables were compared between groups using the chi - square or fisher s exact tests , and continuous variables were compared using one - way analyses of variance . the mean age was 729 years , and the mean forced expiratory volume in 1 second was 1.000.49 l , representing 37%22% predicted . there were 924 evaluations of the oxygen adjustment and monitoring by nurses and 96 evaluations by physicians . nurses and physicians considered freeo2 adjustments and monitoring to be at least as appropriate and as acceptable as manual oxygen management . only monitoring was deemed slightly better with freeo2 by the physicians , but the difference was not statistically significant ( table 2 ) . we did not directly evaluate patient s tolerance of the system , but 80% of the patients completed the study in both groups . the main reason to prematurely stop the study was related to the difficulty in continuously wearing a pulse oximeter and the reduced mobility ( 70% of the reasons to stop ) ; this occurred similarly in both groups . spo2 targets chosen by physicians were similar in both groups , averaging 90.0% and 90.1% for freeo2 and manual o2 adjustment , respectively . the mean spo2 during the study was 90.91.2 in the freeo2 group and 91.91.2 in the manual adjustment group ( p=0.009 ) . the proportion of time within spo2 target was 81.2%19.9% with freeo2 vs 51.3%19.7% with manual o2 adjustments ( p<0.001 ) . the percentage of time with severe desaturation ( spo2 < 85% ) and with hyperoxia ( spo2 > 5% above the target ) was significantly lower with freeo2 in comparison with manual oxygen adjustment ( figure 3 and table 3 ) . duration of oxygen administration was reduced by 1.8 days with freeo2 , but this difference did not reach statistical significance ( table 4 ) . time from randomization to hospital discharge was reduced by 2.6 days with freeo2 ( p=0.051 ) . there was no difference between the two groups in the requirement for noninvasive ventilation during hospitalization , need of transfer to the intensive care unit , or death . the readmission rates at 30 , 60 , and 180 days were also similar in the two groups . there was no safety issue as we did not record any oxygen delivery interruption with freeo2 . few technical issues occurred with end - tidal co2 monitoring , but none concerned the oxygen delivery valve , and this was not associated with safety issues . there were 924 evaluations of the oxygen adjustment and monitoring by nurses and 96 evaluations by physicians . nurses and physicians considered freeo2 adjustments and monitoring to be at least as appropriate and as acceptable as manual oxygen management . only monitoring was deemed slightly better with freeo2 by the physicians , but the difference was not statistically significant ( table 2 ) . we did not directly evaluate patient s tolerance of the system , but 80% of the patients completed the study in both groups . the main reason to prematurely stop the study was related to the difficulty in continuously wearing a pulse oximeter and the reduced mobility ( 70% of the reasons to stop ) ; this occurred similarly in both groups . spo2 targets chosen by physicians were similar in both groups , averaging 90.0% and 90.1% for freeo2 and manual o2 adjustment , respectively . the mean spo2 during the study was 90.91.2 in the freeo2 group and 91.91.2 in the manual adjustment group ( p=0.009 ) . the proportion of time within spo2 target was 81.2%19.9% with freeo2 vs 51.3%19.7% with manual o2 adjustments ( p<0.001 ) . ( spo2 > 5% above the target ) was significantly lower with freeo2 in comparison with manual oxygen adjustment ( figure 3 and table 3 ) . duration of oxygen administration was reduced by 1.8 days with freeo2 , but this difference did not reach statistical significance ( table 4 ) . time from randomization to hospital discharge was reduced by 2.6 days with freeo2 ( p=0.051 ) . there was no difference between the two groups in the requirement for noninvasive ventilation during hospitalization , need of transfer to the intensive care unit , or death . the readmission rates at 30 , 60 , and 180 days were also similar in the two groups . few technical issues occurred with end - tidal co2 monitoring , but none concerned the oxygen delivery valve , and this was not associated with safety issues . spo2 targets chosen by physicians were similar in both groups , averaging 90.0% and 90.1% for freeo2 and manual o2 adjustment , respectively . the mean spo2 during the study was 90.91.2 in the freeo2 group and 91.91.2 in the manual adjustment group ( p=0.009 ) . the proportion of time within spo2 target was 81.2%19.9% with freeo2 vs 51.3%19.7% with manual o2 adjustments ( p<0.001 ) . ( spo2 > 5% above the target ) was significantly lower with freeo2 in comparison with manual oxygen adjustment ( figure 3 and table 3 ) . duration of oxygen administration was reduced by 1.8 days with freeo2 , but this difference did not reach statistical significance ( table 4 ) . time from randomization to hospital discharge was reduced by 2.6 days with freeo2 ( p=0.051 ) . there was no difference between the two groups in the requirement for noninvasive ventilation during hospitalization , need of transfer to the intensive care unit , or death . the readmission rates at 30 , 60 , and 180 days were also similar in the two groups . there was no safety issue as we did not record any oxygen delivery interruption with freeo2 . few technical issues occurred with end - tidal co2 monitoring , but none concerned the oxygen delivery valve , and this was not associated with safety issues . our pilot trial demonstrates the feasibility of providing oxygen therapy with freeo2 to patients hospitalized for an acute exacerbation of copd , even during prolonged administration of oxygen ( up to 8 consecutive days ) . in addition , our results suggest that freeo2 may have the potential to reduce hospital length of stay , although this pilot observation requires formal testing in a proper clinical trial . our study is the first to investigate the potential effects of a continuous and an automated oxygen titration and weaning system on physiological and clinical outcomes in patients hospitalized for an acute exacerbation of copd . several studies evaluated similar systems in patients with copd in other settings , such as home care12 or during rehabilitation,13,14 and in other populations , including neonates15,16 and in patients with acute respiratory failure in the emergency department.17 automated therapy has always proved effective in safely providing oxygen in these settings and populations , and our results are consistent with this notion . the introduction of new devices in clinical practice may not be well accepted , and barriers to technology acceptance should not be overlooked.18 the primary study outcome was selected accordingly . freeo2 was deemed at least as adequate for oxygen administration and monitoring as usual oxygen administration . also , most patients tolerated the instrument , as 80% completed the study in both groups . we were successful in continuously recording respiratory parameters with identical oximeters in both groups with second - by - second data available 90.4% and 82.4% of the time in the freeo2 and control groups , respectively . as expected and in line with other evaluations of automated oxygen titration,10,14,17 oxygenation parameters were improved in the freeo2 group , with more time spent in the targeted range of spo2 and less time with oxygen desaturation or hyperoxia . this may be clinically relevant when considering that hyperoxia may induce hypercapnia37 with potentially severe consequences.4,8,9 hyperoxia is also of concern in patients with copd who frequently have coronary artery disease,19 as it may increase coronary artery resistance.20,21 on the other hand , periods of oxygen desaturations may be responsible for arythmias and myocardial ischemia,22,23 and they should also be avoided . in the control group , patients were in the spo2 target 50% of the time , which may not be compared with other data , as these are the first data with continuous recording of spo2 in this situation to our knowledge . in the recent review of cousin et al,24 the mean rate of accurate prescription of oxygen therapy in eleven studies was 17.4% before and 51.2% after implementation of specific interventions . one strength of our study is that we could evaluate an automated oxygen therapy during prolonged duration ( ie , up to 8 days ) , compared with previous studies where automated o2 titration was provided for a duration of < 1 hour11,13,14 and up to 3 hours.17 an important result of our trial is the trend toward a reduction in duration of oxygen therapy and hospital length of stay with automated oxygen titration , although we can not ascertain whether this effect was related to closer o2 monitoring and surveillance as a part of the freeo2 protocol or whether this was due to the freeo2 device itself . length of stay must be interpreted in comparison to local statistics obtained off - protocol . in 2010 , the mean length of stay for acute exacerbation of copd in our institution was 9.88.1 days ( unpublished data ) , which is in - line with the length of stay in the control group of the present study . reducing the length of hospital stay by 31% as found in this study would be expected to have major logistic and financial impacts on our health care setting . this study was underpowered to conclude on the hospital length of stay , and larger studies will be required to confirm this potential benefit of automated oxygen titration . a more complete evaluation of relevant clinical outcomes , such as length of stay and cost effectiveness , second , the recruitment rate was low as we recruited only 50 patients in 3 years . nurses had to be aware of the patients allocation as they were in charge of the manual titration in the control group , and remote monitoring of cardiorespiratory parameters at the nursing station was available only in the freeo2 arm . to limit the impact of this potential source of bias this study demonstrates the feasibility of using automated oxygen titration and weaning as well as remote monitoring with the freeo2 system in patients hospitalized for acute exacerbation of copd . automated oxygen titration provided benefits in terms of safety ( ie , reduction in the time with severe desaturation and hyperoxia ) and may have contributed to a trend toward the reduced hospital length of stay , a potentially important finding , if these benefits are confirmed in subsequent studies specifically designed to demonstrate these effects .

forty patients ( 20 men and 20 women ) with type 1 diabetes and 40 patients ( 20 men and 20 women ) with type 2 diabetes were recruited among the patients followed in our department . patients with arterial hypertension , renal insufficiency , or cvd or taking medications interfering with vascular reactivity ( including any type of antihypertensive agents ) were excluded from the study . all patients with type 1 diabetes received intensified insulin therapy with multiple daily insulin injections ( n = 36 ) or continuous subcutaneous insulin infusion via a portable pump ( n = 4 ) . patients with type 2 diabetes received various types of oral glucose - lowering therapies ( metformin alone , sulfonylurea alone , or metformin - sulfonylurea combination ) ( n = 25 ) or insulin alone ( n = 5 ) or combined with metformin ( n = 10 ) . two groups of healthy subjects were used as control subjects and matched for bmi with either type 1 diabetic patients or type 2 diabetic patients ( table 1 ) . characteristics of middle - aged diabetic patients with type 1 diabetes , patients with type 2 diabetes , nondiabetic lean control subjects , and nondiabetic overweight / obese control subjects and average values recorded during the whole 3-min squatting test data are means sd . lc , nondiabetic lean control subjects ; na , not applicable ; oc , nondiabetic overweight / obese control subjects ; t1 dm , patients with type 1 diabetes , t2 dm , patients with type 2 diabetes . the squatting test ( successively 1 min standing , 1 min squatting , and 1 min standing ) is an original active orthostatic maneuver that leads to the most important and fast variations of the hydrostatic level with posture ( 9 ) . squatting produces a prompt increase in cardiac output and arterial blood pressure , essentially attributed to augmented venous return from compression of leg veins . these changes result in a significant increase in mean arterial blood pressure and pulse pressure ( 7,8 ) , which is accompanied by an immediate decrease in heart rate and forearm vascular resistance , probably due to activation of cardiopulmonary and arterial baroreflexes , implicating the autonomic nervous system . later on , the active transition from squatting to standing results in a profound initial blood pressure decrease inducing a reflex tachycardia , which can be used to detect diabetic cardiac autonomic neuropathy ( can ) ( 10,11 ) and assess baroreflex sensitivity ( 12 ) . changes in systolic blood pressure ( sbp ) , diastolic blood pressure ( dbp ) , and heart rate were measured continuously with a finapres instrument ( ohmeda ) that allows careful study of cardiovascular reflexes , especially during an orthostatic maneuver ( 13 ) . the finapres is based on servoplethysmomanometry , using the volume clamp technique at the finger level . a good concordance was reported between finapres blood pressure measurements and direct intra - arterial measurements ( 13 ) . pulse pressure , i.e. , sbp minus dbp , was automatically calculated throughout the test . mean arterial blood pressure ( mbp ) was calculated by the formula ( sbp + 2 dbp)/3 . to quantify the relative magnitude of the pulsatile to mean artery pressure ( pulsatility index ) , we normalized the pulse pressure to the mbp and referred to this value as fractional pulse pressure ( 14 ) . pulsatile stress was defined as the double product of pulse pressure and heart rate ( pphr ) ) ; it has been shown to be largely regulated by arterial stiffness and by sympathetic nerve activity and to be associated with a higher risk of ( micro)albuminuria ( 15 ) . we also calculated the sbphr double product , an index of cardiac load that has been shown to be associated with an increased cvd risk ( 16 ) . for each variable or parameter , mean levels were calculated for each subject during the whole period of the test , during the initial standing position , and during the squatting position , after exclusion of the initial transition phase , as described previously ( 7,8 ) . during the transition from squatting to standing , there is an abrupt drop in blood pressure associated with a reflex tachycardia , which is followed by a rapid return to baseline values of both parameters ( blood pressure increase and heart rate decrease ) . the mirror changes in heart rate and sbp allow the calculation of a baroreflex gain by plotting the pulse intervals ( r - r ) against sbp values , and the slope of this relation represents the baroreflex sensitivity ( 17 ) . we also calculated both a vagal index ( ratio between the baseline cardiac r - r interval and the longest r - r interval in the first 15 s of squatting [ sqtv ] ) and a sympathetic index ( ratio between the baseline cardiac r - r interval and the shortest r - r interval in the first 1020 s of standing after squatting [ sqts ] ) , as described previously ( 10,11 ) . these indexes , based on heart rate reduction during squatting and reflex tachycardia during standing , were considered as markers of can : a higher sqtv value indicates a parasympathetic neuropathy , whereas a lower sqts is an indicator of sympathetic neuropathy ( 1012 ) . concomitant a1c levels ( normal values 46% ) were measured to assess recent blood glucose control in diabetic patients ; for each patient , the corresponding a1c mean level corresponded to the average of one to three measurements . lipid profiles were also collected in diabetic patients and the prevalence of the metabolic syndrome ( national cholesterol education program adult treatment panel iii criteria ) was calculated in patients with type 1 diabetes and in patients with type 2 diabetes . the required sample size to have an 80% chance of detecting as significant ( at the two - sided 5% level ) 10 mmhg difference in pulse pressure between two different subgroups , with an assumed sd of pulse pressure of 14 mmhg , was 32 individuals . a difference of 10 mmhg was chosen as clinically significant because it has been shown to be associated with increased cardiovascular mortality in type 2 diabetes ( 1 ) and total mortality in the large eurodiab cohort of patients with type 1 diabetes ( 5 ) . the relationship between two variables , i.e. , between pulsatile stress and baroreflex gain as a marker of can , was assessed with the spearman correlation coefficient . the squatting test ( successively 1 min standing , 1 min squatting , and 1 min standing ) is an original active orthostatic maneuver that leads to the most important and fast variations of the hydrostatic level with posture ( 9 ) . squatting produces a prompt increase in cardiac output and arterial blood pressure , essentially attributed to augmented venous return from compression of leg veins . these changes result in a significant increase in mean arterial blood pressure and pulse pressure ( 7,8 ) , which is accompanied by an immediate decrease in heart rate and forearm vascular resistance , probably due to activation of cardiopulmonary and arterial baroreflexes , implicating the autonomic nervous system . later on , the active transition from squatting to standing results in a profound initial blood pressure decrease inducing a reflex tachycardia , which can be used to detect diabetic cardiac autonomic neuropathy ( can ) ( 10,11 ) and assess baroreflex sensitivity ( 12 ) . changes in systolic blood pressure ( sbp ) , diastolic blood pressure ( dbp ) , and heart rate were measured continuously with a finapres instrument ( ohmeda ) that allows careful study of cardiovascular reflexes , especially during an orthostatic maneuver ( 13 ) . the finapres is based on servoplethysmomanometry , using the volume clamp technique at the finger level . a good concordance was reported between finapres blood pressure measurements and direct intra - arterial measurements ( 13 ) . pulse pressure , i.e. , sbp minus dbp , was automatically calculated throughout the test . mean arterial blood pressure ( mbp ) was calculated by the formula ( sbp + 2 dbp)/3 . to quantify the relative magnitude of the pulsatile to mean artery pressure ( pulsatility index ) , we normalized the pulse pressure to the mbp and referred to this value as fractional pulse pressure ( 14 ) . pulsatile stress was defined as the double product of pulse pressure and heart rate ( pphr ) ) ; it has been shown to be largely regulated by arterial stiffness and by sympathetic nerve activity and to be associated with a higher risk of ( micro)albuminuria ( 15 ) . we also calculated the sbphr double product , an index of cardiac load that has been shown to be associated with an increased cvd risk ( 16 ) . for each variable or parameter , mean levels were calculated for each subject during the whole period of the test , during the initial standing position , and during the squatting position , after exclusion of the initial transition phase , as described previously ( 7,8 ) . during the transition from squatting to standing , there is an abrupt drop in blood pressure associated with a reflex tachycardia , which is followed by a rapid return to baseline values of both parameters ( blood pressure increase and heart rate decrease ) . the mirror changes in heart rate and sbp allow the calculation of a baroreflex gain by plotting the pulse intervals ( r - r ) against sbp values , and the slope of this relation represents the baroreflex sensitivity ( 17 ) . we also calculated both a vagal index ( ratio between the baseline cardiac r - r interval and the longest r - r interval in the first 15 s of squatting [ sqtv ] ) and a sympathetic index ( ratio between the baseline cardiac r - r interval and the shortest r - r interval in the first 1020 s of standing after squatting [ sqts ] ) , as described previously ( 10,11 ) . these indexes , based on heart rate reduction during squatting and reflex tachycardia during standing , were considered as markers of can : a higher sqtv value indicates a parasympathetic neuropathy , whereas a lower sqts is an indicator of sympathetic neuropathy ( 1012 ) . concomitant a1c levels ( normal values 46% ) were measured to assess recent blood glucose control in diabetic patients ; for each patient , the corresponding a1c mean level corresponded to the average of one to three measurements . lipid profiles were also collected in diabetic patients and the prevalence of the metabolic syndrome ( national cholesterol education program adult treatment panel iii criteria ) was calculated in patients with type 1 diabetes and in patients with type 2 diabetes . the required sample size to have an 80% chance of detecting as significant ( at the two - sided 5% level ) 10 mmhg difference in pulse pressure between two different subgroups , with an assumed sd of pulse pressure of 14 mmhg , was 32 individuals . a difference of 10 mmhg was chosen as clinically significant because it has been shown to be associated with increased cardiovascular mortality in type 2 diabetes ( 1 ) and total mortality in the large eurodiab cohort of patients with type 1 diabetes ( 5 ) . the relationship between two variables , i.e. , between pulsatile stress and baroreflex gain as a marker of can , was assessed with the spearman correlation coefficient . compared with control subjects , patients with type 1 diabetes had similar mbp but were characterized throughout the test by significantly higher pulse pressure , heart rate , pulse pressure / mbp , pphr , and sbphr levels ( fig . when squatting was compared with the initial standing position , a trend for higher increases in pulse pressure , pp / mbp , and pphr was observed in patients with type 1 diabetes than in control subjects , with a significantly higher increase in sbphr ( table 2 ) . the baroreflex gain calculated during the transition from squatting to standing was markedly decreased in patients with type 1 diabetes compared with that in control subjects . sqtv and sqts indexes were also significantly different in patients with type 1 diabetes compared with those in lean control subjects ( table 2 ) . there was a significant inverse correlation between pulsatile stress ( pphr ) and baroreflex gain in patients with type 1 diabetes ( r = 0.383 ; p = 0.023 ) but not in lean control subjects ( r = 0.178 ; ns ) . changes in mbp , pulse pressure ( pp ) , and heart rate ( hr ) during a posture test ( 1 min standing , 1 min squatting [ gray zone ] , 1 min standing ) . a : 40 patients with type 1 diabetes ( ) versus 40 nondiabetic ( ) subjects , matched for age , sex , and bmi . b : 40 patients with type 2 diabetes ( ) versus 40 nondiabetic ( ) subjects matched for age , sex , and bmi . c : 40 patients with type 1 diabetes ( ) versus 40 patients with type 2 diabetes ( ) subjects , matched for age and sex . changes occurring during the transition from the initial standing position to the squatting position in middle - aged diabetic patients with type 1 diabetes , patients with type 2 diabetes , nondiabetic lean control subjects , and nondiabetic overweight / obese control subjects data are means sd . mean values of baroreflex gain as well as sqtv and sqts indices of cardiac autonomic neuropathy are also presented for the four groups . lc , nondiabetic lean control subjects ; oc , nondiabetic overweight / obese control subjects ; t1 dm , patients with type 1 diabetes ; t2 dm , patients with type 2 diabetes . compared with overweight / obese nondiabetic control subjects , patients with type 2 diabetes had similar mbp ( hypertension was considered as an exclusion criterion in the present study ) . however , they showed higher pulse pressure , heart rate , pulse pressure - to - mbp ratio , pphr , and sbphr levels throughout the test ( fig . 1b , table 1 ) . increases in pulse pressure , pulse pressure - to - mbp ratio , pphr , and sbphr when moving from standing to squatting were not significantly different in patients with type 2 diabetes and in overweight / obese nondiabetic control subjects ( table 2 ) . the baroreflex gain was significantly decreased in patients with type 2 diabetes compared with that in control subjects . the sqts index ( reflecting postsquatting tachycardia ) but not the sqtv index ( a marker of bradycardia during squatting ) was significantly lower in patients with type 2 diabetes than in overweight / obese nondiabetic control subjects ( table 2 ) . there was a highly significant inverse correlation between pulsatile stress and baroreflex gain in patients with type 2 diabetes ( r = 0.719 ; p = 0.0001 ) but not in overweight / obese control subjects ( r = 0.272 ; ns ) . no significant differences in pulsatile markers and can indexes were noticed between the patients with type 2 diabetes treated with insulin and those not treated with insulin . on average , mbp , pulse pressure , heart rate , pulse pressure - to - mbp ratio , pphr , and sbphr levels were comparable in middle - aged patients with type 1 and type 2 diabetes ( fig . 1c , table 1 ) . the transition from standing to squatting resulted in similar increases in mbp , pulse pressure , pulse pressure - to - mbp ratio , pphr , and sbphr in the two groups of diabetic patients ( table 2 ) . careful analysis of the two pulse pressure curves showed different kinetics in pulse pressure increases , with a second phase increase in pulse pressure in patients with type 1 diabetes that was not observed in patients with type 2 diabetes ; however , the between - group difference during the second part of squatting did not reach statistical significance ( fig . accordingly , sqtv and sqts indexes were not significantly different between the two diabetic groups ( table 2 ) . patients with type 1 diabetes had a much longer known disease duration ( 23 vs. 8 years ; p < 0.0001 ) but a much lower prevalence of metabolic syndrome ( 3% vs. 42% ; p < 0.01 ) than patients with type 2 diabetes . on average , mbp , pulse pressure , heart rate , pulse pressure - to - mbp ratio , pphr , and sbphr levels were comparable in obese and lean nondiabetic individuals in the present study ( table 1 ) . the postural change from standing to squatting resulted in similar increases in mbp , pulse pressure , pulse pressure - to - mbp ratio , and pphr in overweight / obese and lean subjects , with only a trend for a higher increase in sbphr ( + 963 1,178 vs. + 601 698 mmhg min ; p = 0.0991 ) in presence of obesity ( table 2 ) . the baroreflex gain was significantly lower in overweight / obese subjects than in lean individuals ( 2.97 2.18 vs. 4.11 2.26 mmhg min ; p = 0.0332 ) , even in absence of diabetes . the sqtv index was higher in obese subjects than in lean control subjects ( p = 0.0011 ) , whereas the sqts index was almost similar in the two nondiabetic groups ( table 2 ) . compared with control subjects , patients with type 1 diabetes had similar mbp but were characterized throughout the test by significantly higher pulse pressure , heart rate , pulse pressure / mbp , pphr , and sbphr levels ( fig . when squatting was compared with the initial standing position , a trend for higher increases in pulse pressure , pp / mbp , and pphr was observed in patients with type 1 diabetes than in control subjects , with a significantly higher increase in sbphr ( table 2 ) . the baroreflex gain calculated during the transition from squatting to standing was markedly decreased in patients with type 1 diabetes compared with that in control subjects . sqtv and sqts indexes were also significantly different in patients with type 1 diabetes compared with those in lean control subjects ( table 2 ) . there was a significant inverse correlation between pulsatile stress ( pphr ) and baroreflex gain in patients with type 1 diabetes ( r = 0.383 ; p = 0.023 ) but not in lean control subjects ( r = 0.178 ; ns ) . changes in mbp , pulse pressure ( pp ) , and heart rate ( hr ) during a posture test ( 1 min standing , 1 min squatting [ gray zone ] , 1 min standing ) . a : 40 patients with type 1 diabetes ( ) versus 40 nondiabetic ( ) subjects , matched for age , sex , and bmi . b : 40 patients with type 2 diabetes ( ) versus 40 nondiabetic ( ) subjects matched for age , sex , and bmi . c : 40 patients with type 1 diabetes ( ) versus 40 patients with type 2 diabetes ( ) subjects , matched for age and sex . changes occurring during the transition from the initial standing position to the squatting position in middle - aged diabetic patients with type 1 diabetes , patients with type 2 diabetes , nondiabetic lean control subjects , and nondiabetic overweight / obese control subjects data are means sd . mean values of baroreflex gain as well as sqtv and sqts indices of cardiac autonomic neuropathy are also presented for the four groups . lc , nondiabetic lean control subjects ; oc , nondiabetic overweight / obese control subjects ; t1 dm , patients with type 1 diabetes ; t2 dm , patients with type 2 diabetes . compared with overweight / obese nondiabetic control subjects , patients with type 2 diabetes had similar mbp ( hypertension was considered as an exclusion criterion in the present study ) . however , they showed higher pulse pressure , heart rate , pulse pressure - to - mbp ratio , pphr , and sbphr levels throughout the test ( fig . 1b , table 1 ) . increases in pulse pressure , pulse pressure - to - mbp ratio , pphr , and sbphr when moving from standing to squatting were not significantly different in patients with type 2 diabetes and in overweight / obese nondiabetic control subjects ( table 2 ) . the baroreflex gain was significantly decreased in patients with type 2 diabetes compared with that in control subjects . the sqts index ( reflecting postsquatting tachycardia ) but not the sqtv index ( a marker of bradycardia during squatting ) was significantly lower in patients with type 2 diabetes than in overweight / obese nondiabetic control subjects ( table 2 ) . there was a highly significant inverse correlation between pulsatile stress and baroreflex gain in patients with type 2 diabetes ( r = 0.719 ; p = 0.0001 ) but not in overweight / obese control subjects ( r = 0.272 ; ns ) . no significant differences in pulsatile markers and can indexes were noticed between the patients with type 2 diabetes treated with insulin and those not treated with insulin . on average , mbp , pulse pressure , heart rate , pulse pressure - to - mbp ratio , pphr , and sbphr levels were comparable in middle - aged patients with type 1 and type 2 diabetes ( fig . 1c , table 1 ) . the transition from standing to squatting resulted in similar increases in mbp , pulse pressure , pulse pressure - to - mbp ratio , pphr , and sbphr in the two groups of diabetic patients ( table 2 ) . careful analysis of the two pulse pressure curves showed different kinetics in pulse pressure increases , with a second phase increase in pulse pressure in patients with type 1 diabetes that was not observed in patients with type 2 diabetes ; however , the between - group difference during the second part of squatting did not reach statistical significance ( fig . accordingly , sqtv and sqts indexes were not significantly different between the two diabetic groups ( table 2 ) . patients with type 1 diabetes had a much longer known disease duration ( 23 vs. 8 years ; p < 0.0001 ) but a much lower prevalence of metabolic syndrome ( 3% vs. 42% ; p < 0.01 ) than patients with type 2 diabetes . on average , mbp , pulse pressure , heart rate , pulse pressure - to - mbp ratio , pphr , and sbphr levels were comparable in obese and lean nondiabetic individuals in the present study ( table 1 ) . the postural change from standing to squatting resulted in similar increases in mbp , pulse pressure , pulse pressure - to - mbp ratio , and pphr in overweight / obese and lean subjects , with only a trend for a higher increase in sbphr ( + 963 1,178 vs. + 601 698 mmhg min ; p = 0.0991 ) in presence of obesity ( table 2 ) . the baroreflex gain was significantly lower in overweight / obese subjects than in lean individuals ( 2.97 2.18 vs. 4.11 2.26 mmhg min ; p = 0.0332 ) , even in absence of diabetes . the sqtv index was higher in obese subjects than in lean control subjects ( p = 0.0011 ) , whereas the sqts index was almost similar in the two nondiabetic groups ( table 2 ) . the main findings of the present study are 1 ) higher pulse pressure , pulse pressure - to - mbp ratio , pphr , and sbphr levels in middle - aged patients with type 1 diabetes compared with those in lean control subjects , in agreement with higher pulsatile stress and cardiac workload in patients with long - standing type 1 diabetes , 2 ) similarly , higher pulse pressure , pulse pressure - to - mbp ratio , pphr , and sbphr levels in middle - aged nonhypertensive patients with type 2 diabetes compared with those in overweight / obese nondiabetic control subjects , 3 ) the absence of significant differences in pulse pressure , pulsatile index , pulsatile stress , and double product between patients with type 1 diabetes and with type 2 diabetes matched for age ( 50 years on average ) ; and 4 ) indexes of can as shown by lower baroreflex gain and altered sqt indexes during squatting in both patients with type 1 diabetes and type 2 diabetes compared with those in nondiabetic control subjects . therefore , middle - aged patients with type 1 diabetes or with type 2 diabetes are exposed to comparable pulsatile stress , a known cardiovascular and renal risk marker ( 15,15,16 ) . in patients with type 1 diabetes , the negative influence of a much longer diabetes duration ( 23 years on average in the present study ) might be at least partially compensated for by the positive influence of lower bmi , less insulin resistance , and a much lower prevalence of metabolic syndrome compared with those for patients with type 2 diabetes . on the contrary , middle - aged patients with type 2 diabetes are exposed to high pulsatile stress despite a shorter known duration of diabetes ( 8 years on average in our population ) , presumably because of the presence of other concomitant cardiovascular risk factors ( even if hypertension were excluded in the present study ) , as shown by a much higher prevalence of metabolic syndrome compared with that for patients with type 1 diabetes . the observation of higher pulse pressure levels in patients with type 1 diabetes compared with control subjects in the age range 4060 years is in agreement with previous studies from our group , demonstrating an earlier pulse pressure increase with age in this population ( 7,8 ) and with the observational data of the large cross - sectional , case - control finndiane study ( 3 ) . because pulse pressure is considered an indirect marker of arterial stiffness , these higher pulse pressure results are in agreement with accelerated vascular aging in the population with type 1 diabetes ( 6 ) , especially patients with chronic poor glucose control ( 18 ) . in the finndiane study , the ambient level of glucose was not associated with increased pulse pressure , but the time of exposure to hyperglycemia seemed to play a fundamental role in the process of premature arterial stiffening in patients with type 1 diabetes ( 3 ) . in the eurodiab prospective complications study , pulse pressure was significantly associated with all - cause mortality and a mean 12 mmhg higher pulse pressure was observed in patients with type 1 diabetes who died compared with that of those who survived ( 5 ) . decreased baroreflex gain was observed in our patients with type 1 diabetes , reflecting the presence of can after > 20 years of diabetes ( 19 ) . this result was confirmed by altered sqts and sqtv indexes during the squatting test , markers of parasympathetic and sympathetic dysfunction , respectively ( 10 ) . can exposes diabetic patients to an increased mortality risk ( 19 ) . there may be some connection between pulse pressure and can ( 8) , between aortic stiffness and can ( 20 ) , and between arterial stiffness , cardiovagal baroreflex sensitivity , and postural blood pressure changes ( 21 ) . increased sbp was identified as a factor associated with an increased risk of developing can in the cohort of patients with type 1 diabetes of the eurodiab prospective complications study ( 22 ) . the pathophysiological mechanism linking can to arterial stiffness in patients with type 1 diabetes remains unknown , but this association persisted after adjustment for potential confounders such as baseline a1c , hdl cholesterol , and smoking history ( 23 ) . in the present study , we found a significant relationship between pulsatile stress and baroreflex gain as a marker of can in patients with type 1 diabetes . in patients with type 2 diabetes , markers of can are also present ( 11 ) , although less marked than in patients with prolonged type 1 diabetes ( 7 ) . the relationship between pulse pressure and can is less well known in patients with type 2 diabetes even if associations between autonomic neuropathy , vascular dysfunction , and hyperinsulinemia have been demonstrated ( 24 ) . interestingly , a remarkable significant inverse correlation was noted between pulsatile stress and baroreflex gain in the group of patients with type 2 diabetes in our study . the stiffness of the carotid arteries and the aorta , where the arterial baroreceptors are located , may affect the stretch - sensitive receptors and hence baroreflex sensitivity . in addition to structural vascular changes , functional mechanisms associated with endothelial dysfunction may also contribute to the impairment of baroreflex sensitivity associated with arterial stiffness ( 21 ) . patients with type 2 diabetes also showed increased pulse pressure , pulsatility index , and pulsatile stress compared with those for overweight / obese nondiabetic individuals matched for bmi , age , and sex . this result was observed despite the absence of hypertension and a much shorter duration of diabetes compared with those in the population with type 1 diabetes analyzed in the present study . it is well known , however , that type 2 diabetes remains silent during an average of 10 years before diagnosis and initiation of treatment in most cases . thus , selected patients may have a longer duration of type 2 diabetes than the average 8-year known duration noted in the present population . to avoid the potential bias of hypertension and the interferences of antihypertensive agents , we deliberately selected type 2 diabetic patients without hypertension . despite normal mbp , middle - aged patients with type 2 diabetes had higher pulse pressure and pulsatile stress and higher sbphr , two cvd risk markers ( 16 ) . increased pulse pressure levels have been repeatedly demonstrated in large longitudinal studies in patients with type 2 diabetes and shown to be associated with a higher incidence of cardiovascular events ( 1,2 ) . some limitations of the present study should be discussed . several studies have demonstrated that absolute brachial and finger pulse pressure measurements are not identical with larger differences in sbp . however , the differences were generally small and not considered of clinical relevance ( 13 ) . furthermore , some studies have shown a good concordance between periphery ( finger , as in the present study ) and central ( aortic , now recognized as the most important risk factor ) blood pressure measurements ( 25 ) . nevertheless , pulse pressure measured at the finger site may not necessarily reflect central pulse pressure because of the amplification phenomenon . second , glucose control of patients with type 1 diabetes evaluated in the present study was far from optimal , despite intensified insulin therapy . therefore , our results could not necessarily be extrapolated to patients with near normoglycemia for many years because chronic hyperglycemia seems to play a major role in accelerating arterial stiffness ( 18 ) . third , patients with type 2 diabetes selected in the present study did not have hypertension . therefore , the similar results in markers of pulsatile stress in middle - aged patients with type 1 and type 2 diabetes should be interpreted in this context . we can not exclude the possibility that overweight / obese patients with type 2 diabetes and hypertension may be exposed to higher vascular stress than lean normotensive patients with type 1 diabetes at the same age . this would certainly be the case for sbphr but may also be true for the various pulsatility markers . fourth , very few patients had positive microalbuminuria in the two diabetic cohorts analyzed in the present study , because we excluded patients with hypertension or those taking antihypertensive agents . therefore , we were not in a position to study the possible relationship between pulsatile stress and early renal alterations as shown in previous studies ( 15 ) . in summary , middle - aged patients with a long duration of type 1 diabetes have similarly increased pulsatile stress compared with age - matched patients with type 2 diabetes characterized by a shorter duration of the disease , but the presence of other vascular risk factors such as obesity and insulin resistance and no hypertension . in addition , both diabetic groups have markers of can with a reduced baroreflex gain compared with nondiabetic control subjects . the combination of these risk factors may contribute to increase the cvd risk in type 1 diabetic patients with a long exposure to chronic hyperglycemia in a fashion similar to that of patients with type 2 diabetes whose high cvd risk is well known .

objectivearterial pulse pressure is considered to be an independent cardiovascular risk factor . we compared pulse pressure during an active orthostatic test in middle - aged patients with type 1 diabetes and with type 2 diabetes and corresponding nondiabetic control subjects.research design and methodsforty patients with type 1 diabetes ( mean age 50 years , diabetes duration 23 years , and bmi 23.0 kg / m2 ) were compared with 40 nonhypertensive patients with type 2 diabetes ( respectively , 50 years , 8 years , and 29.7 kg / m2 ) . patients taking antihypertensive agents or with renal insufficiency were excluded . all patients were evaluated with a continuous noninvasive arterial blood pressure monitoring ( finapres ) in standing ( 1 min ) , squatting ( 1 min ) , and again standing position ( 1 min ) . patients with type 1 or type 2 diabetes were compared with two groups of 40 age- , sex- and bmi - matched healthy subjects.resultspatients with type 1 diabetes and patients with type 2 diabetes showed significantly higher pulse pressure , heart rate , and double product of pulse pressure and heart rate ( pphr ) ( type 1 : 5,263 vs. 4,121 mmhg / min , p = 0.0004 ; type 2 : 5,359 vs. 4,321 mmhg , p = 0.0023 ) levels than corresponding control subjects . there were no significant differences between patients with type 1 diabetes and type 2 diabetes regarding pulse pressure ( 59 vs. 58 mmhg ) , heart rate ( 89 vs. 88/min ) , and pphr ( 5,263 vs. 5,359 mmhg / min).conclusionspatients with type 1 diabetes have increased levels of peripheral pp , an indirect marker of arterial stiffness , and pphr , an index of pulsatile stress , comparable to those of nonhypertensive patients with type 2 diabetes at similar mean age of 50 years .

RESEARCH DESIGN AND METHODS Orthostatic test Measurements Statistical analysis RESULTS Patients with type 1 diabetes versus nondiabetic lean subjects Patients with type 2 diabetes versus nondiabetic overweight/obese patients Patients with type 1 diabetes versus patients with type 2 diabetes Overweight/obese versus lean subjects without diabetes CONCLUSIONS

forty patients ( 20 men and 20 women ) with type 1 diabetes and 40 patients ( 20 men and 20 women ) with type 2 diabetes were recruited among the patients followed in our department . patients with type 2 diabetes received various types of oral glucose - lowering therapies ( metformin alone , sulfonylurea alone , or metformin - sulfonylurea combination ) ( n = 25 ) or insulin alone ( n = 5 ) or combined with metformin ( n = 10 ) . two groups of healthy subjects were used as control subjects and matched for bmi with either type 1 diabetic patients or type 2 diabetic patients ( table 1 ) . characteristics of middle - aged diabetic patients with type 1 diabetes , patients with type 2 diabetes , nondiabetic lean control subjects , and nondiabetic overweight / obese control subjects and average values recorded during the whole 3-min squatting test data are means sd . lc , nondiabetic lean control subjects ; na , not applicable ; oc , nondiabetic overweight / obese control subjects ; t1 dm , patients with type 1 diabetes , t2 dm , patients with type 2 diabetes . these changes result in a significant increase in mean arterial blood pressure and pulse pressure ( 7,8 ) , which is accompanied by an immediate decrease in heart rate and forearm vascular resistance , probably due to activation of cardiopulmonary and arterial baroreflexes , implicating the autonomic nervous system . changes in systolic blood pressure ( sbp ) , diastolic blood pressure ( dbp ) , and heart rate were measured continuously with a finapres instrument ( ohmeda ) that allows careful study of cardiovascular reflexes , especially during an orthostatic maneuver ( 13 ) . pulsatile stress was defined as the double product of pulse pressure and heart rate ( pphr ) ) ; it has been shown to be largely regulated by arterial stiffness and by sympathetic nerve activity and to be associated with a higher risk of ( micro)albuminuria ( 15 ) . lipid profiles were also collected in diabetic patients and the prevalence of the metabolic syndrome ( national cholesterol education program adult treatment panel iii criteria ) was calculated in patients with type 1 diabetes and in patients with type 2 diabetes . a difference of 10 mmhg was chosen as clinically significant because it has been shown to be associated with increased cardiovascular mortality in type 2 diabetes ( 1 ) and total mortality in the large eurodiab cohort of patients with type 1 diabetes ( 5 ) . these changes result in a significant increase in mean arterial blood pressure and pulse pressure ( 7,8 ) , which is accompanied by an immediate decrease in heart rate and forearm vascular resistance , probably due to activation of cardiopulmonary and arterial baroreflexes , implicating the autonomic nervous system . changes in systolic blood pressure ( sbp ) , diastolic blood pressure ( dbp ) , and heart rate were measured continuously with a finapres instrument ( ohmeda ) that allows careful study of cardiovascular reflexes , especially during an orthostatic maneuver ( 13 ) . pulsatile stress was defined as the double product of pulse pressure and heart rate ( pphr ) ) ; it has been shown to be largely regulated by arterial stiffness and by sympathetic nerve activity and to be associated with a higher risk of ( micro)albuminuria ( 15 ) . lipid profiles were also collected in diabetic patients and the prevalence of the metabolic syndrome ( national cholesterol education program adult treatment panel iii criteria ) was calculated in patients with type 1 diabetes and in patients with type 2 diabetes . a difference of 10 mmhg was chosen as clinically significant because it has been shown to be associated with increased cardiovascular mortality in type 2 diabetes ( 1 ) and total mortality in the large eurodiab cohort of patients with type 1 diabetes ( 5 ) . compared with control subjects , patients with type 1 diabetes had similar mbp but were characterized throughout the test by significantly higher pulse pressure , heart rate , pulse pressure / mbp , pphr , and sbphr levels ( fig . when squatting was compared with the initial standing position , a trend for higher increases in pulse pressure , pp / mbp , and pphr was observed in patients with type 1 diabetes than in control subjects , with a significantly higher increase in sbphr ( table 2 ) . the baroreflex gain calculated during the transition from squatting to standing was markedly decreased in patients with type 1 diabetes compared with that in control subjects . sqtv and sqts indexes were also significantly different in patients with type 1 diabetes compared with those in lean control subjects ( table 2 ) . there was a significant inverse correlation between pulsatile stress ( pphr ) and baroreflex gain in patients with type 1 diabetes ( r = 0.383 ; p = 0.023 ) but not in lean control subjects ( r = 0.178 ; ns ) . changes in mbp , pulse pressure ( pp ) , and heart rate ( hr ) during a posture test ( 1 min standing , 1 min squatting [ gray zone ] , 1 min standing ) . a : 40 patients with type 1 diabetes ( ) versus 40 nondiabetic ( ) subjects , matched for age , sex , and bmi . b : 40 patients with type 2 diabetes ( ) versus 40 nondiabetic ( ) subjects matched for age , sex , and bmi . c : 40 patients with type 1 diabetes ( ) versus 40 patients with type 2 diabetes ( ) subjects , matched for age and sex . changes occurring during the transition from the initial standing position to the squatting position in middle - aged diabetic patients with type 1 diabetes , patients with type 2 diabetes , nondiabetic lean control subjects , and nondiabetic overweight / obese control subjects data are means sd . lc , nondiabetic lean control subjects ; oc , nondiabetic overweight / obese control subjects ; t1 dm , patients with type 1 diabetes ; t2 dm , patients with type 2 diabetes . compared with overweight / obese nondiabetic control subjects , patients with type 2 diabetes had similar mbp ( hypertension was considered as an exclusion criterion in the present study ) . however , they showed higher pulse pressure , heart rate , pulse pressure - to - mbp ratio , pphr , and sbphr levels throughout the test ( fig . increases in pulse pressure , pulse pressure - to - mbp ratio , pphr , and sbphr when moving from standing to squatting were not significantly different in patients with type 2 diabetes and in overweight / obese nondiabetic control subjects ( table 2 ) . the baroreflex gain was significantly decreased in patients with type 2 diabetes compared with that in control subjects . the sqts index ( reflecting postsquatting tachycardia ) but not the sqtv index ( a marker of bradycardia during squatting ) was significantly lower in patients with type 2 diabetes than in overweight / obese nondiabetic control subjects ( table 2 ) . there was a highly significant inverse correlation between pulsatile stress and baroreflex gain in patients with type 2 diabetes ( r = 0.719 ; p = 0.0001 ) but not in overweight / obese control subjects ( r = 0.272 ; ns ) . no significant differences in pulsatile markers and can indexes were noticed between the patients with type 2 diabetes treated with insulin and those not treated with insulin . on average , mbp , pulse pressure , heart rate , pulse pressure - to - mbp ratio , pphr , and sbphr levels were comparable in middle - aged patients with type 1 and type 2 diabetes ( fig . the transition from standing to squatting resulted in similar increases in mbp , pulse pressure , pulse pressure - to - mbp ratio , pphr , and sbphr in the two groups of diabetic patients ( table 2 ) . careful analysis of the two pulse pressure curves showed different kinetics in pulse pressure increases , with a second phase increase in pulse pressure in patients with type 1 diabetes that was not observed in patients with type 2 diabetes ; however , the between - group difference during the second part of squatting did not reach statistical significance ( fig . patients with type 1 diabetes had a much longer known disease duration ( 23 vs. 8 years ; p < 0.0001 ) but a much lower prevalence of metabolic syndrome ( 3% vs. 42% ; p < 0.01 ) than patients with type 2 diabetes . on average , mbp , pulse pressure , heart rate , pulse pressure - to - mbp ratio , pphr , and sbphr levels were comparable in obese and lean nondiabetic individuals in the present study ( table 1 ) . the postural change from standing to squatting resulted in similar increases in mbp , pulse pressure , pulse pressure - to - mbp ratio , and pphr in overweight / obese and lean subjects , with only a trend for a higher increase in sbphr ( + 963 1,178 vs. + 601 698 mmhg min ; p = 0.0991 ) in presence of obesity ( table 2 ) . compared with control subjects , patients with type 1 diabetes had similar mbp but were characterized throughout the test by significantly higher pulse pressure , heart rate , pulse pressure / mbp , pphr , and sbphr levels ( fig . when squatting was compared with the initial standing position , a trend for higher increases in pulse pressure , pp / mbp , and pphr was observed in patients with type 1 diabetes than in control subjects , with a significantly higher increase in sbphr ( table 2 ) . the baroreflex gain calculated during the transition from squatting to standing was markedly decreased in patients with type 1 diabetes compared with that in control subjects . sqtv and sqts indexes were also significantly different in patients with type 1 diabetes compared with those in lean control subjects ( table 2 ) . there was a significant inverse correlation between pulsatile stress ( pphr ) and baroreflex gain in patients with type 1 diabetes ( r = 0.383 ; p = 0.023 ) but not in lean control subjects ( r = 0.178 ; ns ) . changes in mbp , pulse pressure ( pp ) , and heart rate ( hr ) during a posture test ( 1 min standing , 1 min squatting [ gray zone ] , 1 min standing ) . a : 40 patients with type 1 diabetes ( ) versus 40 nondiabetic ( ) subjects , matched for age , sex , and bmi . b : 40 patients with type 2 diabetes ( ) versus 40 nondiabetic ( ) subjects matched for age , sex , and bmi . c : 40 patients with type 1 diabetes ( ) versus 40 patients with type 2 diabetes ( ) subjects , matched for age and sex . changes occurring during the transition from the initial standing position to the squatting position in middle - aged diabetic patients with type 1 diabetes , patients with type 2 diabetes , nondiabetic lean control subjects , and nondiabetic overweight / obese control subjects data are means sd . lc , nondiabetic lean control subjects ; oc , nondiabetic overweight / obese control subjects ; t1 dm , patients with type 1 diabetes ; t2 dm , patients with type 2 diabetes . compared with overweight / obese nondiabetic control subjects , patients with type 2 diabetes had similar mbp ( hypertension was considered as an exclusion criterion in the present study ) . however , they showed higher pulse pressure , heart rate , pulse pressure - to - mbp ratio , pphr , and sbphr levels throughout the test ( fig . increases in pulse pressure , pulse pressure - to - mbp ratio , pphr , and sbphr when moving from standing to squatting were not significantly different in patients with type 2 diabetes and in overweight / obese nondiabetic control subjects ( table 2 ) . the baroreflex gain was significantly decreased in patients with type 2 diabetes compared with that in control subjects . the sqts index ( reflecting postsquatting tachycardia ) but not the sqtv index ( a marker of bradycardia during squatting ) was significantly lower in patients with type 2 diabetes than in overweight / obese nondiabetic control subjects ( table 2 ) . there was a highly significant inverse correlation between pulsatile stress and baroreflex gain in patients with type 2 diabetes ( r = 0.719 ; p = 0.0001 ) but not in overweight / obese control subjects ( r = 0.272 ; ns ) . no significant differences in pulsatile markers and can indexes were noticed between the patients with type 2 diabetes treated with insulin and those not treated with insulin . on average , mbp , pulse pressure , heart rate , pulse pressure - to - mbp ratio , pphr , and sbphr levels were comparable in middle - aged patients with type 1 and type 2 diabetes ( fig . the transition from standing to squatting resulted in similar increases in mbp , pulse pressure , pulse pressure - to - mbp ratio , pphr , and sbphr in the two groups of diabetic patients ( table 2 ) . careful analysis of the two pulse pressure curves showed different kinetics in pulse pressure increases , with a second phase increase in pulse pressure in patients with type 1 diabetes that was not observed in patients with type 2 diabetes ; however , the between - group difference during the second part of squatting did not reach statistical significance ( fig . patients with type 1 diabetes had a much longer known disease duration ( 23 vs. 8 years ; p < 0.0001 ) but a much lower prevalence of metabolic syndrome ( 3% vs. 42% ; p < 0.01 ) than patients with type 2 diabetes . on average , mbp , pulse pressure , heart rate , pulse pressure - to - mbp ratio , pphr , and sbphr levels were comparable in obese and lean nondiabetic individuals in the present study ( table 1 ) . the postural change from standing to squatting resulted in similar increases in mbp , pulse pressure , pulse pressure - to - mbp ratio , and pphr in overweight / obese and lean subjects , with only a trend for a higher increase in sbphr ( + 963 1,178 vs. + 601 698 mmhg min ; p = 0.0991 ) in presence of obesity ( table 2 ) . the main findings of the present study are 1 ) higher pulse pressure , pulse pressure - to - mbp ratio , pphr , and sbphr levels in middle - aged patients with type 1 diabetes compared with those in lean control subjects , in agreement with higher pulsatile stress and cardiac workload in patients with long - standing type 1 diabetes , 2 ) similarly , higher pulse pressure , pulse pressure - to - mbp ratio , pphr , and sbphr levels in middle - aged nonhypertensive patients with type 2 diabetes compared with those in overweight / obese nondiabetic control subjects , 3 ) the absence of significant differences in pulse pressure , pulsatile index , pulsatile stress , and double product between patients with type 1 diabetes and with type 2 diabetes matched for age ( 50 years on average ) ; and 4 ) indexes of can as shown by lower baroreflex gain and altered sqt indexes during squatting in both patients with type 1 diabetes and type 2 diabetes compared with those in nondiabetic control subjects . therefore , middle - aged patients with type 1 diabetes or with type 2 diabetes are exposed to comparable pulsatile stress , a known cardiovascular and renal risk marker ( 15,15,16 ) . in patients with type 1 diabetes , the negative influence of a much longer diabetes duration ( 23 years on average in the present study ) might be at least partially compensated for by the positive influence of lower bmi , less insulin resistance , and a much lower prevalence of metabolic syndrome compared with those for patients with type 2 diabetes . on the contrary , middle - aged patients with type 2 diabetes are exposed to high pulsatile stress despite a shorter known duration of diabetes ( 8 years on average in our population ) , presumably because of the presence of other concomitant cardiovascular risk factors ( even if hypertension were excluded in the present study ) , as shown by a much higher prevalence of metabolic syndrome compared with that for patients with type 1 diabetes . the observation of higher pulse pressure levels in patients with type 1 diabetes compared with control subjects in the age range 4060 years is in agreement with previous studies from our group , demonstrating an earlier pulse pressure increase with age in this population ( 7,8 ) and with the observational data of the large cross - sectional , case - control finndiane study ( 3 ) . because pulse pressure is considered an indirect marker of arterial stiffness , these higher pulse pressure results are in agreement with accelerated vascular aging in the population with type 1 diabetes ( 6 ) , especially patients with chronic poor glucose control ( 18 ) . in the finndiane study , the ambient level of glucose was not associated with increased pulse pressure , but the time of exposure to hyperglycemia seemed to play a fundamental role in the process of premature arterial stiffening in patients with type 1 diabetes ( 3 ) . in the eurodiab prospective complications study , pulse pressure was significantly associated with all - cause mortality and a mean 12 mmhg higher pulse pressure was observed in patients with type 1 diabetes who died compared with that of those who survived ( 5 ) . there may be some connection between pulse pressure and can ( 8) , between aortic stiffness and can ( 20 ) , and between arterial stiffness , cardiovagal baroreflex sensitivity , and postural blood pressure changes ( 21 ) . the pathophysiological mechanism linking can to arterial stiffness in patients with type 1 diabetes remains unknown , but this association persisted after adjustment for potential confounders such as baseline a1c , hdl cholesterol , and smoking history ( 23 ) . in the present study , we found a significant relationship between pulsatile stress and baroreflex gain as a marker of can in patients with type 1 diabetes . in patients with type 2 diabetes , markers of can are also present ( 11 ) , although less marked than in patients with prolonged type 1 diabetes ( 7 ) . the relationship between pulse pressure and can is less well known in patients with type 2 diabetes even if associations between autonomic neuropathy , vascular dysfunction , and hyperinsulinemia have been demonstrated ( 24 ) . patients with type 2 diabetes also showed increased pulse pressure , pulsatility index , and pulsatile stress compared with those for overweight / obese nondiabetic individuals matched for bmi , age , and sex . despite normal mbp , middle - aged patients with type 2 diabetes had higher pulse pressure and pulsatile stress and higher sbphr , two cvd risk markers ( 16 ) . increased pulse pressure levels have been repeatedly demonstrated in large longitudinal studies in patients with type 2 diabetes and shown to be associated with a higher incidence of cardiovascular events ( 1,2 ) . therefore , the similar results in markers of pulsatile stress in middle - aged patients with type 1 and type 2 diabetes should be interpreted in this context . we can not exclude the possibility that overweight / obese patients with type 2 diabetes and hypertension may be exposed to higher vascular stress than lean normotensive patients with type 1 diabetes at the same age . in summary , middle - aged patients with a long duration of type 1 diabetes have similarly increased pulsatile stress compared with age - matched patients with type 2 diabetes characterized by a shorter duration of the disease , but the presence of other vascular risk factors such as obesity and insulin resistance and no hypertension . the combination of these risk factors may contribute to increase the cvd risk in type 1 diabetic patients with a long exposure to chronic hyperglycemia in a fashion similar to that of patients with type 2 diabetes whose high cvd risk is well known .

kidney transplantation ( kt ) is the treatment of choice for end stage renal failure . ischemia leads to deprivation of nutrients and oxygen resulting in atp depletion , loss of ion gradients , cell swelling , and increase of toxic by - products . although contradictive , reperfusion enhances the damage by flow of oxygen rich blood , production of oxygen - free radicals , and activation of an inflammatory response . after kt , iri may induce delayed graft function ( dgf ) , which is associated with increased comorbidity and longer hospital stay and is associated with acute and chronic rejection . despite extensive research , the specific mechanisms behind iri are still not fully understood and no specific therapy is available . preventing or treating iri at an early stage could prevent dgf and thereby contribute to fast patient recovery , shortened hospital stay , and improved graft function . micrornas ( mirnas ) are small , ~1925 nucleotide long , single - stranded rna molecules which play an important role in posttranscriptional regulation of gene expression by inhibiting translation of target mrnas . lin-4 were the first mirnas discovered in caenorhabditis elegans in 1993 by lee and ambros . five years later fire and mello reported on double - stranded rna ( dsrna ) that could silence genes through rna interference ( rnai ) . since then , mirnas have been found in a wide variety of species from plants to viruses and up till now several hundred types are known in humans . other types of small noncoding rnas have been found in plants and animals , like small - interfering rnas and piwi - interacting rnas ( pirnas ) . in this review their small size and their stability and presence in body fluids make them promising candidates , both as therapeutical targets and as biomarkers . in the last years , much progress has been made in the understanding of the role of mirnas in ( patho)physiologic mechanisms . the goal of this review is to summarize the current knowledge of the role of mirnas in transplant related iri and kidney transplantation , with emphasis on their mechanistic role and use as biomarker and as either therapeutic target or agent . we performed a pubmed - search containing the mesh - terms microrna and renal ischemia reperfusion injury . all abstracts were read and only those reports were selected when the main topic was microrna - expression after renal ischemia reperfusion injury . again only those reports were selected where the main topic was microrna - expression and after kidney transplantation . non - english papers were excluded . one report on microrna and ischemia reperfusion injury was focused on a new method of analysis and was therefore excluded . as mirnas play an important role in the regulation of protein synthesis , measurement of their expression may provide information about the physiology of organs in normal and diseased states . since organs have a different spectrum of mirna - expression , it was suggested that tissues contained specific mirnas . however , in an expression atlas of human and mouse mirnas , great similarity between the two species was found , and exclusive expression in an organ appeared to be very rare . expression of most mirnas was ubiquitous , and some showed some degree of tissue specificity . it was found that mirnas that were previously considered as tissue specific were actually present in low concentrations in other tissue types as well . therefore , it was suggested to redefine tissue specific mirnas as preferentially expressed or organ - enriched mirnas . several organ - enriched mirnas were found in the human kidney ; mirna-192 , mirna-194 , mirna-204 , mirna-215 , and mirna-216 were more abundant in kidney as compared to heart , lung , spleen , muscle , and prostate . in the rat kidney , different expression profiles between the cortex and medulla have been described . eleven mirnas were found with a higher expression in the medulla , among them mir-30c and mir-200c . studies on mirnas in renal iri and kt have mainly focused on finding an early and specific biomarker for graft function . serum creatinine is now used to monitor the function of the graft but is a late biomarker with low specificity and sensitivity . seven studies have used human kidney biopsies to study mirna expression in normal grafts compared to those with acute rejection or interstitial fibrosis and tubular atrophy ( if / ta ) [ 914 ] . one study focused on expression differences between operationally tolerant patients and patients with stable graft function under immunosuppression . in five studies , animal experiments were used to investigate the involvement of mirnas in renal iri [ 1620 ] . most studies first perform genome - wide expression profiling using kidney tissue , either after renal iri in animals or from clinical biopsies or urine samples . to verify expression levels of a mirna of interest , qrt - pcr is used . to further investigate their function , the majority of the reports use kidney cell lines such as hk-2 , hrc , or ptec , or pbmcs . dicer is an enzyme found in the cytosol and cleaves precursor mirna to produce mature mirnas . deletion of dicer is used to study the role of mirnas in different organ systems . mice with targeted deletion of dicer in renal proximal tubule develop normally with no abnormalities in the kidney . under normal conditions 80% of 173 measured mirnas in these mice had a significantly lower expression in the kidney compared to wild type controls . after bilateral renal ischemia for 30 minutes survival was significantly higher in the dicer - knockout group , and damage to the proximal tubule decreased . mirna - profiling in kidney after 12 and 48 hours of reperfusion showed several up- and downregulated mirnas in the cortex ( with a log2 fold change > 2 ) ( table 1 ) . of the 13 upregulated mirnas , only two ( mir-132 , mir-362 ) were both upregulated after 12 and after 48 hours . in the downregulated mirnas only one ( mir-379 ) was found at both time points . both upregulated mir-132 and downregulated mir-379 are thought to play a role in the mapk - pathway , which is known to be involved in renal iri [ 22 , 23 ] . to study the effect of lymphocyte infiltration on mirna expression , r/cmice that lack nk cells , nkt cells , b cells , and t cells were subjected to 30 minutes of renal iri , and mirna expression was compared to wild type mice . differential expression of mir-21 , mir-20a , mir-146a , mir-199a-3p , mir-214 , mir-192 , mir-187 , mir-805 , and mir-194 in the kidney of both groups was found . the mirna expression pattern over a time course after iri was similar between wild type and r/cmice , suggesting that the mirna profile after iri seems to be independent of infiltrating lymphocytes . when iri was compared to sham - operated mice , 5 differentially expressed mirnas were identified in the kidney . three of them , mir-17 - 5 , mir-21 , and mir-106 , had fold changes of more than 30% at 24 hours after reperfusion . mir-17 - 5 and mir-106 belong to the same mir-17 family and had similar expression patterns . in a time course , it was seen that mir-17 - 5 was upregulated from 24 hours until 3 days after reperfusion . mir-21 was upregulated at 3 and 4 days after reperfusion . at all measured time points ( 1 , 3 , and 4 days after reperfusion ) , a significant correlation was found between mir-17 - 5 and mir-21 . the time points at which these mirnas were upregulated may give some insight in which mechanism they are involved , either the injury and maintenance phase ( mir-17 - 5 ) or recovery phase ( mir-21 ) . their correlation suggests that these mirnas influence each other 's expression . to study mirna profiles after renal iri in rats , renal pedicles were clamped for 30 minutes and mirna expression in kidney , blood , and urine was compared to sham - operated rats at 24 , 72 , and 120 hours after reperfusion . a > 2-fold difference in expression was considered significant . among the mirnas which were differentially expressed at all three time points , three mirnas of interest expression data were validated using qrt - pcr and compared between time points but also between cortex and medulla . mir-21 expression in the cortex increased after reperfusion and remained stable during the time course . in the medulla however expression continued to rise from a fold change of ~2.5 at 24 hours to a fold change of ~13 at 120 hours . mir-155 had a high expression ( ~6-fold change ) at 24 hours in the cortex , which decreased to a fold change of ~3 at 120 hours . in the medulla , expression showed a reversed course , with low expression at 24 hours and a fold change of ~5 at 120 hours . furthermore , expression of these mirnas did not change in other tissues ( heart , liver , lung , and spleen ) after renal iri . expression in whole blood showed a significant decrease of the three mirnas after reperfusion . in urine , only mir-21 showed a significant increase at 72 hours . mir-18a was not detectable in urine . using target scan , 29 mrna targets involved in apoptosis and cell proliferation were found for the three mirnas , but their precise role was not investigated any further . ischemic preconditioning ( ipc ) has been shown in some models to be an efficient technique to ameliorate damage by iri in different organs like heart , brain , liver , and kidney [ 2527 ] . the possible involvement of mir-21 in its protection against iri was studied in a rat model . this corresponded with lower serum creatinine levels at 24 hours after iri . when anti - mir-21 was administered before ipc , no rise in mir-21 was seen , and protection by ipc was abolished . when anti - mir-21 was administered before iri without ipc , no effect on the severity of iri was seen . this suggests that mir-21 is involved in induction of the protective effect of ipc and that administration of mir-21 before iri may mimic the effects of ipc . these authors also showed that preconditioning with xenon provides similar effects on mir-21 expression as ipc . pretreatment with xenon , a noble gas with volatile anaesthetic properties , resulted in protection against iri . again , this effect was dependent on mir-21 and protection was lost when anti - mir-21 was administered . microvesicles derived from endothelial progenitor cells have the ability to induce resistance against renal iri . this was not seen after administering microvesicles from fibroblasts . when treated with endothelial microvesicles , creatinine and bun levels were significantly lower compared to untreated controls . via microarray analysis , 26 mirnas were found to be expressed in these microvesicles . mir-126 and mir-296 were chosen for further analysis because of their proangiogenic and antiapoptotic effects . using qrt - pcr , it was found that mir-126 and mir-296 were expressed in endothelial progenitor microvesicles , but not in microvesicles derived from fibroblasts . when anti - mir-126 and anti - mir-296 were added to the microvesicles , the protective effect was lost , showing that the protective effect was dependent on mir-126 and mir-296 . because the aims , models , methods , and time points used in these studies differ considerably , it is difficult to draw an overall conclusion on the role and relevance of specific mirnas in iri . mirna expression associated with protection against iri can not be compared to the profiled mirnas associated with iri itself . in the former the emphasis is to find protective mirnas , while in the latter the pathophysiological response of mirnas after iri is measured . another reason for the lack in overlap between mirnas is that initial profiling experiments later randomly select particular mirnas of interest and neglect the role of other mirnas found in these experiments . godwin et al . found a downregulation of mir-194 after renal iri , which suggests that low expression of mir-194 is involved in protection against iri ( table 1 ) . already in 2007 , a mirna profile in patients with acute rejection was reported . cortical biopsies from three kidneys with acute rejection after live donor kt were compared to 3 biopsies retrieved from kidney tissue after nephrectomy because of a renal tumour . results showed 8 upregulated and 12 downregulated mirnas , with a ratio ( rejection / normal ) < 0.5 or > 2.0 considered significant ( table 1 ) . another profiling study compared mirna expression in biopsies suffering from dgf , acute rejection , or antibody - mediated rejection ( amr ) with normal allograft biopsies . in the dgf group , all upregulated mirnas ( table 1 ) only one mirna was also upregulated in amr , mir-182 , which was found to be upregulated in a murine iri model as well . mir-182 inhibits foxo1 , which in turn causes proliferation of t - helper cells and is associated with cardiac allograft rejection in mice and ischemic damage in the brain [ 16 , 29 , 30 ] . furthermore , an overlap of six mirnas was found ( mir-155 , mir-125a , mir-30c , mir-27b , mir-193b , and mir-125b ) in the acute rejection set of another study , in which biopsies of 17 normal allografts were compared with 12 allografts with acute rejection . in this study , mir-142 - 5p , mir-155 , and mir-223 were the most significantly upregulated mirnas in acutely rejected kidneys ( p < 0.0001 ) . a positive correlation between the expression of the t - cell marker cd3 and mir-142 - 5p was found . since mir-142 - 5p is mostly expressed in hematopoietic cells , its presence can be attributed to the influx of inflammatory cells in the kidney . in pbmcs , expression levels of mir-142 - 5p may vary and are significantly increased in chronic antibody - mediated rejection compared to stable graft function but are not altered during acute rejection . this suggests that mirna-142 - 5p in pbmc may be a marker for antibody - mediated chronic rejection . since biopsies are invasive and bear a high risk for complications , a noninvasive method to measure rejection is preferred . mirnas can be measured in the urine , which could be a promising way to find biomarkers for acute rejection . as mentioned before , mir-21 and mir-155 urine samples of 22 patients with acute kidney injury admitted to the icu with high serum creatinine levels and elevated levels of kim-1 in the urine were collected and mirna expression was compared to urine samples of 25 healthy volunteers . among the 22 icu - patients , 9 were kt - recipients , and 13 had acute kidney injury of their native kidneys . in the patients with aki ( both native and transplant kidneys ) an increased expression of mir-21 and decreased expression of mir-155 were found compared to healthy individuals . compared 68 urine samples of 62 patients with biopsy proven acute rejection with 20 samples of 19 patients with stable graft function . as a disease control group , 13 urine samples of stable transplant recipients with a urinary tract infection twenty - one mirnas were found with a > 10-fold difference in expression between patients with acute rejection and stable controls . an increase of mir-10a and a decrease of mir-10b and mir-210 were found in urinary samples from patients with acute rejection . mir-210 correlated with glomerular filtration rate over time and a normalization of mir-210 levels was seen after successful antirejection therapy . compared to the expression values in urinary samples with urinary tract infection only mir-210 showed a differential expression , suggesting mir-210 could act as a urinary biomarker and predictor for acute rejection and long term graft function , respectively . a similar study was done to find a urinary biomarker for if / ta . thirteen biopsies with chronic allograft dysfunction and if / ta were compared to 5 normal biopsies , all from recipients of a kidney from a deceased donor . with use of a target prediction program , fold change , and statistical significance they focused on 5 mirnas ( mir-142 - 3p , mir-204 , mir-107 , mir-211 , and mir-32 ) for further research . in urinary samples , a significant difference was found for three of the five mirnas , namely , mir-142 - 3p , mir-204 , and mir-211 . although the predictive value still has to be established , this mirna signature is a possible biomarker for if / ta . with an in silico model , targets of these mirnas were predicted and were found to be the costimulatory molecule cd28 on t - helper cells , b - cell development , and allograft rejection signalling . other biological functions regulated by this mirna signature are apoptosis , lymphocyte proliferation , and differentiation of natural killer ( nk- ) cells , b - cells , and t - cells . furthermore , a decrease in mir-204 is associated with increased apoptosis in hela cells . using sequencing to profile mirna expression , biopsies with if / ta and 4 normal biopsies were compared . in biopsies with if / ta a differential expression ( > 2-fold ) of several mirnas ( table 1 ) was found , including mir-223 which was increased and the mir-30 family , which was decreased . they found 22 differentially expressed mirnas ( p < 0.01 with a false discovery rate < 15% ) ( table 1 ) . differential expression of mir-99a , mir-140 - 3p , mir-200b , and mir-200 was seen both early and late after transplantation ( 3.73 1.30 and 20 4 months , resp . ) . the limited number of human studies that has been conducted so far has yielded hardly any overlap in mirnas . comparison of these studies is limited by the use of different patient populations and tissues , like biopsies from cortex , percutaneous core needle biopsies , or urine . other limitations are the different time points when samples were collected , even within one study . in addition , the focus of the studies differed between acute rejection and if / ta in which involvement of mirna undoubtedly differs . mir-142 - 3p is upregulated in both reports on acute rejection and if / ta [ 12 , 13 ] . mir-142 - 3p is mostly found in hematopoietic cells and is associated with upregulation of cd25 + cd4 t - cells and downregulation of heat shock protein ( hsp)70 . another overlapping mirna , mir-223 , is also associated with inflammation , regarded as modulator of neutrophil maturation and fine tuner of granulocyte function and is also found in monocytes . the mir-30 family is shown to be needed in kidney development and mir-30 suppresses apoptosis by targeting the mitochondrial fission machinery ( table 2 ) . in mice , mir-21 was found to play a role in renal iri . since mir-21 is associated with cell death , proliferation , and fibrosis [ 4042 ] , the role of mir-21 in proliferation and renal fibrosis knockdown of mir-21 led to increased cell death , whereas overexpression resulted in decreased cell death . hif-1 is protective against iri as it promotes alterations in expression of genes involved in tissue repair after ischemia . mirna-127 was found to be involved in the hif-1 pathway , and iri in nrk52e rat kidney cells and hk-2 cells stabilized hif-1 expression , which upregulated mir-127 . following renal ischemia and reperfusion in rats , an increased expression of rno - mir-127 was found , and kinesin family member 3b ( kif3b ) , which is involved in cellular endocytosis , was identified as a novel target of mir-127 . in human pbmcs from recipients with stable graft function under immunosuppression , expression of mir-106b , mir-142 - 3p , mir-450b-5p , and mir-876 - 3p was significantly lower and mir-98 , mir-148b , mir-324 - 5p , and mir-508 - 3p were significantly higher expressed , compared to operationally tolerant recipients . when pbmcs of healthy volunteers were stimulated with phytohemagglutinin a ( pha ) and il-2 , expression of mir-142 - 3p , mir-324 - 5p , and mir-450b-5p decreased and expression of mir-876 - 3p increased . further investigation showed higher expression of mir-142 - 3p in b - cells in operationally tolerant patients . pha stimulation of pbmcs was also performed to study the presence of mirnas found earlier in human kidney biopsies . of the 7 upregulated mirnas in the kidney , only mir-155 was increased in stimulated pbmcs , whereas let-7c was decreased . mir-223 and mir-142 - 5p , however , were decreased in pbmcs in contrast to their upregulation in kidney tissue . in hk-2 cells iri was induced by culturing cells in low oxygen ( 0.1% ) for 16 hours , followed by 3 or 10 hours of reoxygenation . lower expression of mir-205 was associated with a decreased cell survival . in cells overexpressing mir-205 this protection was associated with the suppression of egln2 and an increase of hif-1 and ho-1 . one in vitro model focused on if / ta induced by hypoxia in renal epithelial cells . mir-124 seems to regulate mmp-2 , a fibrosis associated gene , and overexpression of mir-124 reversed increased proliferation after hypoxia , although this was independent of mmp-2 ( table 3 ) . well - recognized limitations of these in vitro studies are that the pathology of iri is difficult to capture in vitro , that mostly only one cell line is studied and the focus is on the role of one particular mirna found previously in literature or previous investigation . despite the great interest in mirnas in biomedical research , there are only few experimental and clinical studies on mirnas in renal iri and kt . these studies focus on the feasibility of measuring mirnas and exploring their role in iri . comparing clinical studies with iri experiments in small animals is difficult . in animal experiments , kidneys underwent substantial cold ischemia , which may influence mirna expression , and were subjected to alloreactivity in the recipient . furthermore , animal experiments focus on iri whilst clinical studies study biomarker potential for rejection or if / ta . hitherto , no specific early biomarker is available for ischemic kidney damage after transplantation . in recent years it has been found that the kinetics of mirnas in regulating mrna translation is more complex than previously thought . regulation succeeds through binding of a mirna to the mrna , but feedback loops are also involved as shown in tgf-1 regulation in the kidney . that and the fact that one single mirna is able to suppress translation of multiple mrna make it even more difficult to interpret mirna expression levels alone without studying the effects on target pathways . mirnas have the potential to act as specific biomarkers , since it has been shown that mirnas are dysregulated in disease states as found first in b - cell leukemia . unfortunately , the different studies revealed different mirnas as a possible biomarker with no or little overlap between these studies . this may be the result of using different models , biomaterials , or different time points . when all reports are taken together , no single mirna has been reproducibility identified as biomarker . for sensitivity even more , the lack of ready - to - use , reproducible assays could bias the results of the studies . when micrornas found in microarrays are validated with pcr , only those micrornas which give comparable expression levels are investigated further . this could result in a bias , where only those micrornas will be examined further , which are validated through pcr . future studies using rna sequencing techniques which provide both qualitative and quantitative data could overcome these problems . because of their small size and stability , mirnas could also be used as a therapeutic target by inactivating them using antagomirs or as a therapeutic agent as locked nucleic acids [ 48 , 49 ] . two reports studied differences in mirna expression between untreated renal iri and iri ameliorated by known protective mechanisms , in an attempt to find mirnas , which are involved in protection against iri . mir-21 was found to be upregulated after ipc , and knockdown diminished the protective effect of ipc . nevertheless , overexpression of mir-21 did not result in increased cell survival after oxidative stress . thus , although mir-21 is obviously involved in protection against iri , it seems to be part of a more complex mechanism . this mechanism needs to be unravelled to understand the interactions between mirna-21 and other factors in renal iri . although mirnas have a great potential as biomarker , therapeutic target , and therapeutic agent in kt , the most important conclusion to be drawn at present is that mirna research has not led to significant new insights into the pathophysiology of renal iri , graft rejection , or tolerance and has failed to come up with clinically applicable biomarkers , therapeutic targets , or agents . this could be the result of differences in design of the studies , different tissues used to measure mirna expression levels , different time points used , and different platforms to determine expression levels of mirnas . therefore , studies using more comparable aims , models , patient populations , and expression platforms are needed to determine if mirnas are able to live up to their expectations in kidney transplantation .

since the discovery of micrornas , ample research has been conducted to elucidate their involvement in an array of ( patho)physiological conditions . ischemia reperfusion injury is a major problem in kidney transplantation and its mechanism is still not fully known , nor is there an effective therapy . furthermore , no biomarker is available to specifically measure ( ischemic ) damage after kidney transplantation or predict transplantation outcome . in this review , we summarize studies conducted on micrornas in renal ischemia reperfusion injury and kidney transplantation . although the number of publications on mirnas in different areas of nephrology is increasing every year , only a limited number of reports that address the role of mirnas in relation to ischemia reperfusion injury or kidney transplantation are available . all reports up to june 2014 on micrornas in renal iri , kidney transplantation , and renal allograft status were included . design of the studies was highly variable and there was limited overlap between micrornas found in these reports . no single microrna expression pattern could be found , although multiple micrornas involved in the immune response seem to be altered after ischemia reperfusion injury and kidney transplantation . although there is a growing interest in microrna research in kidney transplantation aiming to identify biomarkers and therapeutical targets , to date , no specific microrna has been demonstrated to be applicable as either one , mostly because of lack of specificity . more systematical research is needed to determine whether micrornas can be applied as biomarker , therapeutic target , or therapeutic agent in kidney transplantation .

1. Introduction 2. Methods 3. Discussion

kidney transplantation ( kt ) is the treatment of choice for end stage renal failure . ischemia leads to deprivation of nutrients and oxygen resulting in atp depletion , loss of ion gradients , cell swelling , and increase of toxic by - products . although contradictive , reperfusion enhances the damage by flow of oxygen rich blood , production of oxygen - free radicals , and activation of an inflammatory response . despite extensive research , the specific mechanisms behind iri are still not fully understood and no specific therapy is available . preventing or treating iri at an early stage could prevent dgf and thereby contribute to fast patient recovery , shortened hospital stay , and improved graft function . since then , mirnas have been found in a wide variety of species from plants to viruses and up till now several hundred types are known in humans . other types of small noncoding rnas have been found in plants and animals , like small - interfering rnas and piwi - interacting rnas ( pirnas ) . in this review their small size and their stability and presence in body fluids make them promising candidates , both as therapeutical targets and as biomarkers . in the last years , much progress has been made in the understanding of the role of mirnas in ( patho)physiologic mechanisms . the goal of this review is to summarize the current knowledge of the role of mirnas in transplant related iri and kidney transplantation , with emphasis on their mechanistic role and use as biomarker and as either therapeutic target or agent . we performed a pubmed - search containing the mesh - terms microrna and renal ischemia reperfusion injury . all abstracts were read and only those reports were selected when the main topic was microrna - expression after renal ischemia reperfusion injury . again only those reports were selected where the main topic was microrna - expression and after kidney transplantation . one report on microrna and ischemia reperfusion injury was focused on a new method of analysis and was therefore excluded . as mirnas play an important role in the regulation of protein synthesis , measurement of their expression may provide information about the physiology of organs in normal and diseased states . however , in an expression atlas of human and mouse mirnas , great similarity between the two species was found , and exclusive expression in an organ appeared to be very rare . expression of most mirnas was ubiquitous , and some showed some degree of tissue specificity . several organ - enriched mirnas were found in the human kidney ; mirna-192 , mirna-194 , mirna-204 , mirna-215 , and mirna-216 were more abundant in kidney as compared to heart , lung , spleen , muscle , and prostate . in the rat kidney , different expression profiles between the cortex and medulla have been described . eleven mirnas were found with a higher expression in the medulla , among them mir-30c and mir-200c . studies on mirnas in renal iri and kt have mainly focused on finding an early and specific biomarker for graft function . serum creatinine is now used to monitor the function of the graft but is a late biomarker with low specificity and sensitivity . in five studies , animal experiments were used to investigate the involvement of mirnas in renal iri [ 1620 ] . most studies first perform genome - wide expression profiling using kidney tissue , either after renal iri in animals or from clinical biopsies or urine samples . to further investigate their function , the majority of the reports use kidney cell lines such as hk-2 , hrc , or ptec , or pbmcs . dicer is an enzyme found in the cytosol and cleaves precursor mirna to produce mature mirnas . deletion of dicer is used to study the role of mirnas in different organ systems . mice with targeted deletion of dicer in renal proximal tubule develop normally with no abnormalities in the kidney . under normal conditions 80% of 173 measured mirnas in these mice had a significantly lower expression in the kidney compared to wild type controls . after bilateral renal ischemia for 30 minutes survival was significantly higher in the dicer - knockout group , and damage to the proximal tubule decreased . mirna - profiling in kidney after 12 and 48 hours of reperfusion showed several up- and downregulated mirnas in the cortex ( with a log2 fold change > 2 ) ( table 1 ) . of the 13 upregulated mirnas , only two ( mir-132 , mir-362 ) were both upregulated after 12 and after 48 hours . in the downregulated mirnas only one ( mir-379 ) was found at both time points . both upregulated mir-132 and downregulated mir-379 are thought to play a role in the mapk - pathway , which is known to be involved in renal iri [ 22 , 23 ] . to study the effect of lymphocyte infiltration on mirna expression , r/cmice that lack nk cells , nkt cells , b cells , and t cells were subjected to 30 minutes of renal iri , and mirna expression was compared to wild type mice . differential expression of mir-21 , mir-20a , mir-146a , mir-199a-3p , mir-214 , mir-192 , mir-187 , mir-805 , and mir-194 in the kidney of both groups was found . the mirna expression pattern over a time course after iri was similar between wild type and r/cmice , suggesting that the mirna profile after iri seems to be independent of infiltrating lymphocytes . when iri was compared to sham - operated mice , 5 differentially expressed mirnas were identified in the kidney . three of them , mir-17 - 5 , mir-21 , and mir-106 , had fold changes of more than 30% at 24 hours after reperfusion . at all measured time points ( 1 , 3 , and 4 days after reperfusion ) , a significant correlation was found between mir-17 - 5 and mir-21 . the time points at which these mirnas were upregulated may give some insight in which mechanism they are involved , either the injury and maintenance phase ( mir-17 - 5 ) or recovery phase ( mir-21 ) . to study mirna profiles after renal iri in rats , renal pedicles were clamped for 30 minutes and mirna expression in kidney , blood , and urine was compared to sham - operated rats at 24 , 72 , and 120 hours after reperfusion . mir-21 expression in the cortex increased after reperfusion and remained stable during the time course . in the medulla however expression continued to rise from a fold change of ~2.5 at 24 hours to a fold change of ~13 at 120 hours . mir-155 had a high expression ( ~6-fold change ) at 24 hours in the cortex , which decreased to a fold change of ~3 at 120 hours . in the medulla , expression showed a reversed course , with low expression at 24 hours and a fold change of ~5 at 120 hours . furthermore , expression of these mirnas did not change in other tissues ( heart , liver , lung , and spleen ) after renal iri . expression in whole blood showed a significant decrease of the three mirnas after reperfusion . in urine , only mir-21 showed a significant increase at 72 hours . using target scan , 29 mrna targets involved in apoptosis and cell proliferation were found for the three mirnas , but their precise role was not investigated any further . ischemic preconditioning ( ipc ) has been shown in some models to be an efficient technique to ameliorate damage by iri in different organs like heart , brain , liver , and kidney [ 2527 ] . when anti - mir-21 was administered before ipc , no rise in mir-21 was seen , and protection by ipc was abolished . when anti - mir-21 was administered before iri without ipc , no effect on the severity of iri was seen . this suggests that mir-21 is involved in induction of the protective effect of ipc and that administration of mir-21 before iri may mimic the effects of ipc . microvesicles derived from endothelial progenitor cells have the ability to induce resistance against renal iri . via microarray analysis , 26 mirnas were found to be expressed in these microvesicles . because the aims , models , methods , and time points used in these studies differ considerably , it is difficult to draw an overall conclusion on the role and relevance of specific mirnas in iri . in the former the emphasis is to find protective mirnas , while in the latter the pathophysiological response of mirnas after iri is measured . another reason for the lack in overlap between mirnas is that initial profiling experiments later randomly select particular mirnas of interest and neglect the role of other mirnas found in these experiments . found a downregulation of mir-194 after renal iri , which suggests that low expression of mir-194 is involved in protection against iri ( table 1 ) . cortical biopsies from three kidneys with acute rejection after live donor kt were compared to 3 biopsies retrieved from kidney tissue after nephrectomy because of a renal tumour . another profiling study compared mirna expression in biopsies suffering from dgf , acute rejection , or antibody - mediated rejection ( amr ) with normal allograft biopsies . in the dgf group , all upregulated mirnas ( table 1 ) only one mirna was also upregulated in amr , mir-182 , which was found to be upregulated in a murine iri model as well . mir-182 inhibits foxo1 , which in turn causes proliferation of t - helper cells and is associated with cardiac allograft rejection in mice and ischemic damage in the brain [ 16 , 29 , 30 ] . furthermore , an overlap of six mirnas was found ( mir-155 , mir-125a , mir-30c , mir-27b , mir-193b , and mir-125b ) in the acute rejection set of another study , in which biopsies of 17 normal allografts were compared with 12 allografts with acute rejection . in this study , mir-142 - 5p , mir-155 , and mir-223 were the most significantly upregulated mirnas in acutely rejected kidneys ( p < 0.0001 ) . a positive correlation between the expression of the t - cell marker cd3 and mir-142 - 5p was found . since mir-142 - 5p is mostly expressed in hematopoietic cells , its presence can be attributed to the influx of inflammatory cells in the kidney . mirnas can be measured in the urine , which could be a promising way to find biomarkers for acute rejection . as mentioned before , mir-21 and mir-155 urine samples of 22 patients with acute kidney injury admitted to the icu with high serum creatinine levels and elevated levels of kim-1 in the urine were collected and mirna expression was compared to urine samples of 25 healthy volunteers . in the patients with aki ( both native and transplant kidneys ) an increased expression of mir-21 and decreased expression of mir-155 were found compared to healthy individuals . an increase of mir-10a and a decrease of mir-10b and mir-210 were found in urinary samples from patients with acute rejection . with use of a target prediction program , fold change , and statistical significance they focused on 5 mirnas ( mir-142 - 3p , mir-204 , mir-107 , mir-211 , and mir-32 ) for further research . in urinary samples , a significant difference was found for three of the five mirnas , namely , mir-142 - 3p , mir-204 , and mir-211 . although the predictive value still has to be established , this mirna signature is a possible biomarker for if / ta . with an in silico model , targets of these mirnas were predicted and were found to be the costimulatory molecule cd28 on t - helper cells , b - cell development , and allograft rejection signalling . other biological functions regulated by this mirna signature are apoptosis , lymphocyte proliferation , and differentiation of natural killer ( nk- ) cells , b - cells , and t - cells . furthermore , a decrease in mir-204 is associated with increased apoptosis in hela cells . in biopsies with if / ta a differential expression ( > 2-fold ) of several mirnas ( table 1 ) was found , including mir-223 which was increased and the mir-30 family , which was decreased . differential expression of mir-99a , mir-140 - 3p , mir-200b , and mir-200 was seen both early and late after transplantation ( 3.73 1.30 and 20 4 months , resp . ) the limited number of human studies that has been conducted so far has yielded hardly any overlap in mirnas . comparison of these studies is limited by the use of different patient populations and tissues , like biopsies from cortex , percutaneous core needle biopsies , or urine . in addition , the focus of the studies differed between acute rejection and if / ta in which involvement of mirna undoubtedly differs . mir-142 - 3p is mostly found in hematopoietic cells and is associated with upregulation of cd25 + cd4 t - cells and downregulation of heat shock protein ( hsp)70 . another overlapping mirna , mir-223 , is also associated with inflammation , regarded as modulator of neutrophil maturation and fine tuner of granulocyte function and is also found in monocytes . the mir-30 family is shown to be needed in kidney development and mir-30 suppresses apoptosis by targeting the mitochondrial fission machinery ( table 2 ) . in mice , mir-21 was found to play a role in renal iri . since mir-21 is associated with cell death , proliferation , and fibrosis [ 4042 ] , the role of mir-21 in proliferation and renal fibrosis knockdown of mir-21 led to increased cell death , whereas overexpression resulted in decreased cell death . hif-1 is protective against iri as it promotes alterations in expression of genes involved in tissue repair after ischemia . mirna-127 was found to be involved in the hif-1 pathway , and iri in nrk52e rat kidney cells and hk-2 cells stabilized hif-1 expression , which upregulated mir-127 . following renal ischemia and reperfusion in rats , an increased expression of rno - mir-127 was found , and kinesin family member 3b ( kif3b ) , which is involved in cellular endocytosis , was identified as a novel target of mir-127 . in human pbmcs from recipients with stable graft function under immunosuppression , expression of mir-106b , mir-142 - 3p , mir-450b-5p , and mir-876 - 3p was significantly lower and mir-98 , mir-148b , mir-324 - 5p , and mir-508 - 3p were significantly higher expressed , compared to operationally tolerant recipients . when pbmcs of healthy volunteers were stimulated with phytohemagglutinin a ( pha ) and il-2 , expression of mir-142 - 3p , mir-324 - 5p , and mir-450b-5p decreased and expression of mir-876 - 3p increased . of the 7 upregulated mirnas in the kidney , only mir-155 was increased in stimulated pbmcs , whereas let-7c was decreased . mir-223 and mir-142 - 5p , however , were decreased in pbmcs in contrast to their upregulation in kidney tissue . one in vitro model focused on if / ta induced by hypoxia in renal epithelial cells . mir-124 seems to regulate mmp-2 , a fibrosis associated gene , and overexpression of mir-124 reversed increased proliferation after hypoxia , although this was independent of mmp-2 ( table 3 ) . well - recognized limitations of these in vitro studies are that the pathology of iri is difficult to capture in vitro , that mostly only one cell line is studied and the focus is on the role of one particular mirna found previously in literature or previous investigation . despite the great interest in mirnas in biomedical research , there are only few experimental and clinical studies on mirnas in renal iri and kt . in animal experiments , kidneys underwent substantial cold ischemia , which may influence mirna expression , and were subjected to alloreactivity in the recipient . furthermore , animal experiments focus on iri whilst clinical studies study biomarker potential for rejection or if / ta . hitherto , no specific early biomarker is available for ischemic kidney damage after transplantation . in recent years it has been found that the kinetics of mirnas in regulating mrna translation is more complex than previously thought . regulation succeeds through binding of a mirna to the mrna , but feedback loops are also involved as shown in tgf-1 regulation in the kidney . mirnas have the potential to act as specific biomarkers , since it has been shown that mirnas are dysregulated in disease states as found first in b - cell leukemia . unfortunately , the different studies revealed different mirnas as a possible biomarker with no or little overlap between these studies . this may be the result of using different models , biomaterials , or different time points . when all reports are taken together , no single mirna has been reproducibility identified as biomarker . for sensitivity even more , the lack of ready - to - use , reproducible assays could bias the results of the studies . when micrornas found in microarrays are validated with pcr , only those micrornas which give comparable expression levels are investigated further . because of their small size and stability , mirnas could also be used as a therapeutic target by inactivating them using antagomirs or as a therapeutic agent as locked nucleic acids [ 48 , 49 ] . two reports studied differences in mirna expression between untreated renal iri and iri ameliorated by known protective mechanisms , in an attempt to find mirnas , which are involved in protection against iri . mir-21 was found to be upregulated after ipc , and knockdown diminished the protective effect of ipc . thus , although mir-21 is obviously involved in protection against iri , it seems to be part of a more complex mechanism . this mechanism needs to be unravelled to understand the interactions between mirna-21 and other factors in renal iri . although mirnas have a great potential as biomarker , therapeutic target , and therapeutic agent in kt , the most important conclusion to be drawn at present is that mirna research has not led to significant new insights into the pathophysiology of renal iri , graft rejection , or tolerance and has failed to come up with clinically applicable biomarkers , therapeutic targets , or agents . this could be the result of differences in design of the studies , different tissues used to measure mirna expression levels , different time points used , and different platforms to determine expression levels of mirnas . therefore , studies using more comparable aims , models , patient populations , and expression platforms are needed to determine if mirnas are able to live up to their expectations in kidney transplantation .

that life - saving organ transplantation is a good thing is fairly uncontroversial , but it relies upon a supply of donated organs . not all potential organ donations are considered ethically acceptable , however , and the reasons for donation have been regarded as significant in determining the overall acceptability of particular donations ( p.129).1 suspect motivations in deceased organ donation include profit ( financial or otherwise)2 and seeking to prevent specific recipients from benefiting or , conversely , ensuring that they do.3 the apparent absence of altruistic motivation has been considered sufficient to exclude donations that have conditions or directions attached or involve any kind of transaction,3 and for an otherwise potentially lifesaving / enhancing donation to be rejected . the nuffield council on bioethics 2011 report ( henceforth nuffield report ) emphasises the central position of altruism in the uk : altruism , long promulgated as the only ethical basis for donation of bodily material , should continue to play a central role in ethical thinking in this field . while some of the claims made for altruism may be overblown , the notion of altruism as underpinning important communal values expresses something very significant about the kind of society in which we wish to live ( p.5 ) . altruism , long promulgated as the only ethical basis for donation of bodily material , should continue to play a central role in ethical thinking in this field . while some of the claims made for altruism may be overblown , the notion of altruism as underpinning important communal values expresses something very significant about the kind of society in which we wish to live ( p.5 ) . altruism is often referred to as one of the fundamental principles of transplantation ( p.2535 , p.46 ) but mostly without clear definition . altruism can refer to both motivation and action / practice.4 the distinction between the two is the difference between what motivates an action , behaviour or practice and the actual outcome of that action , behaviour or practice . it is difficult to determine in advance whether a specific organ donation is an altruistic action because while most recipients will benefit from their transplantation , if an organ is rejected or fails the recipient may be worse off . since altruistic action / practice is judged on the basis of outcome it is a poor guide to determining in advance of surgery whether any particular offer of donation is acceptable . aside from this practical point , it is clearly motivational altruism that is currently considered important in organ donation . what are of concern to transplant authorities are the ( altruistic ) reasons that motivate a donation , rather than the contingent ( altruistic ) consequence ( it is reasonable to suppose that transplant surgery would not be offered unless it was thought on balance to be of benefit to the recipient ) . it is therefore motivational altruism that is the focus of this paper . as we shall see , it may nonetheless be difficult to be certain of an individual 's motives . this paper , however , explores the way in which altruistic motivation has been , and could be , used as a guiding principle when attempting to determine whether individual offers of donation are acceptable . the nuffield report defines altruism as entailing a selfless gift to others without expectation of remuneration ( p.1204 ) and this definition is provided as though it were uncontroversial . there are , however , long - standing philosophical debates over whether altruism really exists , and if so , what it means and how it manifests itself . despite some superficial similarities , the term altruism as used in uk transplantation is quite different from the mainstream views in philosophy , and has given rise to a form of organ donation altruism ( oda ) that has specific purposes and more limited scope . oda is used throughout this paper to refer to the account of altruism seemingly endorsed by organ donation policy and guidance in the uk . the nuffield report has repositioned altruism at the forefront of organ donation and transplantation ethics , so it is timely to reconsider what it means to say organ donation must be altruistic. since the application of altruism results in the rejection of potentially life - saving ( and life - enhancing ) donations , a precise , rigorous and justifiable definition is required . it will be argued that the oda account lacks the required precision and rigour . although the nuffield report 's discussion of altruism is arguably a step forward , it will be maintained that even it offers a severely restricted assessment against which to measure potential donors motivations . organ donation has been described as the gift of life,7 and the gift relationship has long underpinned organ donation systems . titmuss 's analysis of the gift relationship focused on blood donation , and concluded that there were many advantages to having a voluntary system based upon altruistic giving . titmuss acknowledges , however , that no donor type can be characterised by complete , disinterested , spontaneous altruism ( p.898 ) . a problem with motivational altruism is that it is difficult ( if not impossible ) for transplant staff to establish the motivation of donors . it may be easier to establish that something is not altruistically motivated than to decisively conclude that it is ; for instance , demanding reward for donating suggests a non - altruistic motivation , but the mere absence of this sort of demand does not mean that the donation is altruistically motivated . given this , the suitability of altruistic motivation as a fundamental requirement for acceptable organ donation is immediately questionable . oda is sometimes taken to mean giving without expectation of reward ( ordinarily financial reward).9 for example , regardless of the legal position , trading in organs ( from living / deceased donors ) has been rejected on the grounds that it is non - altruistic ( p.7410 ) . oda is sometimes taken to mean simply the opposite of a commerce - based system ( p.1311 ) . although oda may have started as a means of prohibiting organ trade , it has since been used to determine the acceptability of other practices relating to organ donation . conditional and directed deceased donations are distinguished from one another by the human tissue authority , according to which : a condition would serve to exclude certain recipients ; a direction would identify one or more recipients.12 both sorts of donation are generally prohibited . the incident that led to the ban occurred in 1998 when a white man 's next of kin requested his organs were only given to white people ( p.13 ) . a subsequent department of health ( dh ) report specifically criticised this donation for being non - altruistic ( p.253 ) . this suggests that oda entails more than just giving without anticipation of financial or other rewards , since there were no clear benefits to the donor or donor family that could not also apply to most , if not all , deceased organ donation ( some comfort for the bereaved , sense of some good coming out of tragedy , etc ) . the relationship between conditions and altruism was further refined in guidance issued in march 2010 in relation to requests for directed allocation of organs.13 the dh report 2000 states that conditional donation offends against the fundamental principle that organs are donated altruistically and should go to patients in the greatest need ( p.253 ) , whereas the 2010 guidance states that conditional donation offends against the fundamental principle that organs are donated voluntarily and freely and should go to patients according to the agreed criteria ( p.413 ) . this suggests that for a deceased donation to be altruistic it must be donated without coercion or constraints on who receives the organ . regarding conditional donations as non - altruistic suggests that donation is only altruistic if it is without any constraints or conditions . this is problematic though , because organs are never donated absolutely freely and unconditionally ; at the very least organs are donated after death on the understanding that they will be used for the purposes consented to ( be it transplantation or research ) . the fact that donors can choose whether their organs are used for transplantation or research suggests that some constraints are considered acceptable . in addition , the organ donor register permits potential donors to specify which organs they are willing to give , implying that these organs will be given on condition that others are not removed . all this suggests that constraints on donations are only considered contrary to altruism if they fall outside the parameters already deemed acceptable by reference to other principles . the objection to conditional deceased donation must go deeper than the fact that conditional donations interfere with traditional allocation models based on medical need and clinical judgement , since the racial constraints in the 1998 case did not alter the allocation . the recipients who topped the waiting lists were all white and as such the conditions were met without disadvantaging any non - white recipients.14 even so , the dh concluded the donation was non - altruistic . the without constraint element of altruism employed here appears to be linked to impartial allocation ( where impartial means according to medical criteria ) . this suggests that to be altruistic , a donation must be motivated by a desire to help other people in general , rather than a specific person or group of people . this definition of altruism as impartial and undirected runs into trouble when the 2010 guidance13 is explored further . the guidance was issued following the case of a laura ashworth.15 the ban on conditional and directed deceased donations prevented her organs being donated to her mother who needed a kidney transplant . instead they were donated to the general pool and allocated accordingly . following public and media reaction to this case , the march 2010 guidance was issued specifically to permit the kinds of requests for directed donation to family members in deceased donation that are common in living donation . if , however , a decision to donate is strongly motivated by a desire to help a specific family member , this can not be considered impartial . as though to anticipate this objection , the guidance requires that deceased donation must not be contingent upon the request being followed ( p.413 ) ; so in effect the donation is unconstrained and can still be considered altruistic in this sense . this understanding of altruism is not consistently applied in living donation , where the vast majority of donations are contingent upon the donated organ being transplanted to a specific individual . it also contrasts with altruism as understood by nagel,16 which depends much more heavily on rigorously applied impartiality . for nagel altruism is not to be confused with a generalised affection for the human race , and is instead merely a willingness to act in consideration of the interests of other persons , without the need of ulterior motives ( p.116 ) . nagel is interested in the fundamental reasons for action and the possibility that others interests can give one reasons to act . he argues that we can have a direct reason to promote the interests of others ( one that does not depend upon one 's own interests or antecedent sentiments of sympathy and benevolence ( p.1616 ) ) . for nagel , in order to act altruistically , the interests of others must provide the reason for action , and this requires that one must consider oneself to be just one person amongst many:[i]n any situation in which there is reason for one person to promote some end , we must be able to discover an end which there is reason for anyone to promote , should he be in a position to do so . ( p.9016 ) [ i]n any situation in which there is reason for one person to promote some end , we must be able to discover an end which there is reason for anyone to promote , should he be in a position to do so . ( p.9016 ) for nagel , then , we can have a rational interest in helping others regardless of things like sympathy or reward . accordingly , altruism must be objective and impartial , which means that acting in the interests of a friend because he is a friend is not altruistic . the sort of directed but unconditional donation permitted in the 2010 guidance would not be altruistic according to nagel , because it would express unacceptable partiality , as would directed living related donation . some partiality could , however , be accommodated in altruism according to blum , for whom these kinds of emotions are central to altruistic behaviour because it is good to be sympathetic , compassionate , concerned , and caring for other human beings ( p.717 ) . often these sorts of emotions will be most strongly felt towards family or friends , and blum 's account of altruism accommodates the partiality these relationships might entail . importantly , blum does not argue that favouring friends or family is always permissible and altruistic merely that it is acceptable to give some additional weight to their interests . accordingly , blum 's account would allow directed donations to count as altruistic in circumstances like those outlined in the 2010 guidance . before we conclude that altruism in transplantation can be likened to that justified by blum we must consider how living - related donation is differentiated from altruistic living donation . the latter , sometimes referred to as non - directed altruistic living donation,18 refers to cases where living individuals donate an organ usually a kidney or part of an organ , ordinarily to be allocated according to the usual criteria applied to deceased donation that is , they are not donating to a specific known / loved individual . that altruism features in the name of non - directed living donation , but not living - related donation , suggests that organ donation is only considered altruistically motivated if the organs are available for allocation to recipients with whom the donor does not have a genetic or longstanding personal relationship , which is contrary to blum 's account . living - related donation could be considered with reference to miller 's account of altruism which distinguishes it from that which we have an interest in doing or which we are obliged for other reasons to do . for miller , altruism is behaviour that is intended to meet the needs of others , where there is no immediate self - interested reason to help , and where there is no institutional requirement that one should ( p.10819 ) . just as feeding one 's children would not be altruistic ( p.10819 ) , nor presumably would giving them one 's kidney . living - related donation would not , therefore , be considered altruistically motivated even though it is nonetheless acceptable . part of the problem for altruism in transplantation is that the link between altruism and the ethical acceptability of something is not always clear cut . depending upon which account of altruism one uses moral myopia ( p.1720 ) ; altruism can tell us whether an act is altruistic or not , but not always whether it is right or wrong , especially when motives may be mixed . for instance , a person may be motivated to donate partly by a desire to help people and partly because her family disapproves . the relationship between altruism and rightness is not a problem for nagel 's account which , owing to its strictly rational demands and kantian grounding , aligns altruistically motivated acts with right acts . blum 's account though , and the account apparently used in transplantation , are more vulnerable to this criticism . she jumps in and saves as many of the children as she can but some drown . on the face of it this seems altruistic and praiseworthy . but suppose the rescuer , calculating that only three children could be saved in the time available , targeted her own children . it would also not be altruism according to miller , as one could argue that parents have a responsibility to save their children . it could be altruism according to blum , if it was motivated by altruistic emotions . it would also be altruism according to oda , if the mother would still have attempted to rescue other children if she were unable to save her own . if , however , she had not been prepared to save other children , and only ever intended to save her own , she would not be altruistically motivated according to the 2010 guidance . this shows us that even taking competing philosophical definitions into account , altruism as used to guide transplantation practice is confused . if one does not have a relative on a transplant waiting list , then one 's altruism must be impartial , much like nagel 's . this prevents people from directing organs in cases like the racist donation in 1998 , or from directing organs in response to media - facilitated appeal by individuals.21 yet if one does have a relative on a transplant waiting list , then one 's altruism can be partial and directed , more like blum 's . unlike the 2010 guidance , however , blum does not insist that we must be willing to follow through with an action even if our family member can not after all benefit from it , but then neither does usual practice in relation to living - related donation . neither blum 's nor nagel 's account is open to a pick and mix approach : owing to their fundamentally different theoretical groundings , one can not endorse one while also endorsing the other without contradiction . if one were to adopt any of these philosophical accounts of altruism as the basis for ethical organ donation , significantly more potential donations would have to be rejected . nagel 's altruism is particularly demanding , and would require the rejection of any organ not donated completely impartially . seeking to give meaning to the death of one 's loved one , being comforted in one 's bereavement , wanting to help particular kinds of research efforts ( like those trying to find effective treatments for the condition from which one 's loved one died ) , wanting to save the life of one 's child / friend / partner , being moved by the story of a particular patient and even fulfilling the wishes of the deceased person would all be incompatible with nagel 's altruism and the organs would have to be rejected . blum 's altruism is still demanding , but in a different way from nagel 's . where nagel 's altruism is strictly rational , blum allows altruistic emotions to play a significant role and would potentially be compatible with donations directed towards family and friends . it seems more likely that altruistic emotions would exist towards family members than strangers , since one is most likely to feel sympathy and compassion for those who one cares most about . moreover , although blum 's altruism focuses on altruistic emotions , it does not overly concern itself with their origins ; if a racist person felt altruistic emotions for another person of his own race purely by virtue of their skin colour , this could be compatible with blum 's altruism . this means that blum 's altruism features the moral myopia described de wispelaere , as although a racist donation may be altruistic , this does not make it ethical . the nuffield report discusses altruism in detail , and presents a distinctive account that has elements in common with oda and accounts found in philosophical literature . it defines altruism as behaviour that is motivated by concern for the welfare of the recipient of some beneficent behaviour , rather than by concern for the welfare of the person carrying out the action ( p.1394 ) . in a departure from oda , however , the recipient of the beneficent behaviour need not necessarily be the eventual organ recipient . much media attention was given to the nuffield report 's proposal to pay a donor 's funeral expenses and the claim that this is compatible with altruism4 ( ch . 5 22 ) . it argues that some forms of reward may be compatible with altruistic intent , since a single person may have a variety of motivations to donate organs , one or more of which could be altruistic . presumably the authors would not endorse the claim that the presence of an altruistic motivation amongst a number of distinctly non - altruistic motivations ( such as to deliberately spite family / friends ) would render the donation altruistically motivated . although there may be an altruistic element to a donation decision in this instance , it would seem incorrect to deduce from this that the donation is altruistically motivated . although they acknowledge that real - life may present situations where motivations are mixed , the altruistic nature of a donation is secured only when the difficulty here is that establishing motivation is , as we have already discussed , notoriously difficult and it was perhaps beyond the scope of the nuffield report to provide a solution to this point . although the donor may be partly motivated to donate by his funeral expenses being paid , he could do so with others interests in mind he would receive no benefit himself from his funeral expenses being paid ( he would be dead ) but might be trying to lift this financial burden from his relatives . this , according to the nuffield report , is altruistic even though it displays a level of partiality ( towards next - of - kin and relatives ) that is not ordinarily acceptable under oda ( p.1754 ) . by suggesting that altruism in organ donation might be directed beyond the organ recipients , nuffield altruism could permit more direct payments in exchange for donation for instance , money given to next - of - kin to spend as they wish this would still be altruistic insofar as the donor would be motivated to donate organs in order to benefit other people ; it just happens that the intended beneficiaries would be his family . the only difference between the two cases is how the money provided is to be spent . a further complexity is the role of the next - of - kin in decisions about deceased donation . the nuffield report concedes that if the next - of - kin agree to the donation only because they will benefit from the funeral expenses being paid , the payment of funeral expenses in this case would be a non - altruist focused intervention ( p.1394 ) . oda acts as a guiding principle for determining and promoting ethical donations , but features inconsistencies that result in arbitrariness which , at the very least , border on unfairness to those who are deprived of the benefits of receiving an organ as a result . applying a philosophical definition of altruism would be a remedy for this inconsistency but would result in many fewer acceptable donations ( nagel ) , or additional principles might be needed to identify donations that were undesirable for reasons unrelated to altruism ( blum ) . nuffield altruism falls between the two ; it is consistent , and promotes organ donation , but fails to provide a single guiding principle for determining the acceptability of a donation . the nuffield report accepts however , that altruism can not be the only guiding principle ( ch 54 ) . this is a sensible way forward for transplantation ethics , since it allows unethical practice to be robustly and roundly rejected without distorting a single principle ( to the extent that it becomes inconsistent ) like altruism to suit a wide range of specifics . comparing accounts of altruism oda , organ donation altruism . nuffield altruism is controversial , however , insofar as it seems to permit practices that oda was deliberately trying to prevent , principally financial incentives . moreover , accepting that a decision to donate is unlikely to result from a single motivation , and by widening the recipient pool for the other - regarding beneficial motivation that characterises altruism , the concept is virtually redundant as a measure of whether a donation is acceptable . for example , it would permit racial direction as a condition of donation , as a person could still be motivated to donate by a concern for the welfare of others , although that concern could be conditional upon the recipients being of a certain race . racist conditions would only be discounted if it could be demonstrated that the donor 's primary motivation was to deprive a particular racial group of some benefit ( rather than to benefit a different group ) and the definition of altruism was tightened so that only primary motivations were assessed . the outstanding issue would then remain whether it is ever possible to be certain what a person 's motives are , let alone what their primary motives might be . the nuffield report provides us with one reason why we should continue to associate organ donation with altruism : it maintains the communal virtue of a general disposition to be moved to self - sacrifice by the health needs of others ( p.1444 ) . in this sense , people are urged to donate partly to fulfil their desire to help others , but also because this will promote the kind of community where others would do the same for them . while it may be important that people are moved to self - sacrifice ( p.1444 ) in order to help others , it is not clear that the health needs of others ought to be the only motivating factor . also , the degree of self - sacrifice in deceased organ donation is open to debate , and some people may not view it as being a sacrifice at all . it is unlikely that under the current system people are moved to self - sacrifice purely by the health needs of others ; as the nuffield report itself notes , people donate organs for a number of reasons , some of which may be selfless and other regarding , and others of which may be more self - interested ( ie , non - altruistic ) . the current nhs blood and transplant prove it campaign seems designed to promote reciprocity or fairness rather than altruism,23 which suggests something of a retreat from altruism as the only acceptable motivator . the 2011 nice guidelines for organ donation do not even mention altruism , and state that , similarly to other documents,24 25 organ donation should be considered as a usual part of end of life care planning ( p.626 ) . making organ donation usual may make it part and parcel ( p.10919 ) of the social role of dying people , and if this were the case , according to miller 's altruism , organ donation could not be altruistic yet nice 's aim does not appear undesirable as a result . we agree with the nuffield report that a society where there is a general disposition to altruism seems , on the face of it at least , to be preferable to one where we are each only concerned with our own interests . this does not , however , seem to justify insisting that that altruism is a necessary as opposed to desirable component of ethical donation . and , as we have shown , problems of arbitrary injustice seem to have arisen from an insistence that it should be . the charade that donation must be altruistic to be acceptable altruistic living donation and living - related donation. the latter has continued apace despite general recognition that there are usually self - interested reasons for wanting to save the life of a loved one . perhaps it is time to accept that whilst altruism might be a sign of an acceptable donation it is not the only sign . these other signs may be used to preclude donations that ( all but nagel 's definition of ) altruism may accommodate , but coherent , robust and consistent justifications are required every time an offer of donation is declined because of the cost to the recipients of rejecting offers of organs .

altruism has long been taken to be the guiding principle of ethical organ donation in the uk , and has been used as justification for rejecting or allowing certain types of donation . we argue that , despite this prominent role , altruism has been poorly defined in policy and position documents , and used confusingly and inconsistently . looking at how the term has been used over recent years allows us to define organ donation altruism , and comparing this with accounts in the philosophical literature highlights its theoretical shortcomings . the recent report from the nuffield council on bioethics reaffirmed the importance of altruism in organ donation , and offered a clearer definition . this definition is , however , more permissive than that of altruism previously seen in uk policy , and as a result allows some donations that previously have been considered unacceptable . we argue that while altruistic motivation may be desirable , it is not necessary .

Background Altruism and the gift of life Altruism and the department of health Philosophical accounts of altruism Altruism and the Nuffield report What is left? Is donor altruism really necessary?

not all potential organ donations are considered ethically acceptable , however , and the reasons for donation have been regarded as significant in determining the overall acceptability of particular donations ( p.129).1 suspect motivations in deceased organ donation include profit ( financial or otherwise)2 and seeking to prevent specific recipients from benefiting or , conversely , ensuring that they do.3 the apparent absence of altruistic motivation has been considered sufficient to exclude donations that have conditions or directions attached or involve any kind of transaction,3 and for an otherwise potentially lifesaving / enhancing donation to be rejected . the nuffield council on bioethics 2011 report ( henceforth nuffield report ) emphasises the central position of altruism in the uk : altruism , long promulgated as the only ethical basis for donation of bodily material , should continue to play a central role in ethical thinking in this field . while some of the claims made for altruism may be overblown , the notion of altruism as underpinning important communal values expresses something very significant about the kind of society in which we wish to live ( p.5 ) . altruism , long promulgated as the only ethical basis for donation of bodily material , should continue to play a central role in ethical thinking in this field . while some of the claims made for altruism may be overblown , the notion of altruism as underpinning important communal values expresses something very significant about the kind of society in which we wish to live ( p.5 ) . it is difficult to determine in advance whether a specific organ donation is an altruistic action because while most recipients will benefit from their transplantation , if an organ is rejected or fails the recipient may be worse off . since altruistic action / practice is judged on the basis of outcome it is a poor guide to determining in advance of surgery whether any particular offer of donation is acceptable . aside from this practical point , it is clearly motivational altruism that is currently considered important in organ donation . what are of concern to transplant authorities are the ( altruistic ) reasons that motivate a donation , rather than the contingent ( altruistic ) consequence ( it is reasonable to suppose that transplant surgery would not be offered unless it was thought on balance to be of benefit to the recipient ) . as we shall see , it may nonetheless be difficult to be certain of an individual 's motives . this paper , however , explores the way in which altruistic motivation has been , and could be , used as a guiding principle when attempting to determine whether individual offers of donation are acceptable . the nuffield report defines altruism as entailing a selfless gift to others without expectation of remuneration ( p.1204 ) and this definition is provided as though it were uncontroversial . there are , however , long - standing philosophical debates over whether altruism really exists , and if so , what it means and how it manifests itself . despite some superficial similarities , the term altruism as used in uk transplantation is quite different from the mainstream views in philosophy , and has given rise to a form of organ donation altruism ( oda ) that has specific purposes and more limited scope . oda is used throughout this paper to refer to the account of altruism seemingly endorsed by organ donation policy and guidance in the uk . the nuffield report has repositioned altruism at the forefront of organ donation and transplantation ethics , so it is timely to reconsider what it means to say organ donation must be altruistic. since the application of altruism results in the rejection of potentially life - saving ( and life - enhancing ) donations , a precise , rigorous and justifiable definition is required . although the nuffield report 's discussion of altruism is arguably a step forward , it will be maintained that even it offers a severely restricted assessment against which to measure potential donors motivations . organ donation has been described as the gift of life,7 and the gift relationship has long underpinned organ donation systems . titmuss 's analysis of the gift relationship focused on blood donation , and concluded that there were many advantages to having a voluntary system based upon altruistic giving . titmuss acknowledges , however , that no donor type can be characterised by complete , disinterested , spontaneous altruism ( p.898 ) . a problem with motivational altruism is that it is difficult ( if not impossible ) for transplant staff to establish the motivation of donors . it may be easier to establish that something is not altruistically motivated than to decisively conclude that it is ; for instance , demanding reward for donating suggests a non - altruistic motivation , but the mere absence of this sort of demand does not mean that the donation is altruistically motivated . given this , the suitability of altruistic motivation as a fundamental requirement for acceptable organ donation is immediately questionable . oda is sometimes taken to mean giving without expectation of reward ( ordinarily financial reward).9 for example , regardless of the legal position , trading in organs ( from living / deceased donors ) has been rejected on the grounds that it is non - altruistic ( p.7410 ) . oda is sometimes taken to mean simply the opposite of a commerce - based system ( p.1311 ) . although oda may have started as a means of prohibiting organ trade , it has since been used to determine the acceptability of other practices relating to organ donation . conditional and directed deceased donations are distinguished from one another by the human tissue authority , according to which : a condition would serve to exclude certain recipients ; a direction would identify one or more recipients.12 both sorts of donation are generally prohibited . this suggests that oda entails more than just giving without anticipation of financial or other rewards , since there were no clear benefits to the donor or donor family that could not also apply to most , if not all , deceased organ donation ( some comfort for the bereaved , sense of some good coming out of tragedy , etc ) . the relationship between conditions and altruism was further refined in guidance issued in march 2010 in relation to requests for directed allocation of organs.13 the dh report 2000 states that conditional donation offends against the fundamental principle that organs are donated altruistically and should go to patients in the greatest need ( p.253 ) , whereas the 2010 guidance states that conditional donation offends against the fundamental principle that organs are donated voluntarily and freely and should go to patients according to the agreed criteria ( p.413 ) . this suggests that for a deceased donation to be altruistic it must be donated without coercion or constraints on who receives the organ . the objection to conditional deceased donation must go deeper than the fact that conditional donations interfere with traditional allocation models based on medical need and clinical judgement , since the racial constraints in the 1998 case did not alter the allocation . the recipients who topped the waiting lists were all white and as such the conditions were met without disadvantaging any non - white recipients.14 even so , the dh concluded the donation was non - altruistic . the without constraint element of altruism employed here appears to be linked to impartial allocation ( where impartial means according to medical criteria ) . this suggests that to be altruistic , a donation must be motivated by a desire to help other people in general , rather than a specific person or group of people . this definition of altruism as impartial and undirected runs into trouble when the 2010 guidance13 is explored further . if , however , a decision to donate is strongly motivated by a desire to help a specific family member , this can not be considered impartial . this understanding of altruism is not consistently applied in living donation , where the vast majority of donations are contingent upon the donated organ being transplanted to a specific individual . for nagel altruism is not to be confused with a generalised affection for the human race , and is instead merely a willingness to act in consideration of the interests of other persons , without the need of ulterior motives ( p.116 ) . nagel is interested in the fundamental reasons for action and the possibility that others interests can give one reasons to act . for nagel , in order to act altruistically , the interests of others must provide the reason for action , and this requires that one must consider oneself to be just one person amongst many:[i]n any situation in which there is reason for one person to promote some end , we must be able to discover an end which there is reason for anyone to promote , should he be in a position to do so . accordingly , altruism must be objective and impartial , which means that acting in the interests of a friend because he is a friend is not altruistic . the sort of directed but unconditional donation permitted in the 2010 guidance would not be altruistic according to nagel , because it would express unacceptable partiality , as would directed living related donation . some partiality could , however , be accommodated in altruism according to blum , for whom these kinds of emotions are central to altruistic behaviour because it is good to be sympathetic , compassionate , concerned , and caring for other human beings ( p.717 ) . often these sorts of emotions will be most strongly felt towards family or friends , and blum 's account of altruism accommodates the partiality these relationships might entail . importantly , blum does not argue that favouring friends or family is always permissible and altruistic merely that it is acceptable to give some additional weight to their interests . accordingly , blum 's account would allow directed donations to count as altruistic in circumstances like those outlined in the 2010 guidance . the latter , sometimes referred to as non - directed altruistic living donation,18 refers to cases where living individuals donate an organ usually a kidney or part of an organ , ordinarily to be allocated according to the usual criteria applied to deceased donation that is , they are not donating to a specific known / loved individual . that altruism features in the name of non - directed living donation , but not living - related donation , suggests that organ donation is only considered altruistically motivated if the organs are available for allocation to recipients with whom the donor does not have a genetic or longstanding personal relationship , which is contrary to blum 's account . living - related donation could be considered with reference to miller 's account of altruism which distinguishes it from that which we have an interest in doing or which we are obliged for other reasons to do . for miller , altruism is behaviour that is intended to meet the needs of others , where there is no immediate self - interested reason to help , and where there is no institutional requirement that one should ( p.10819 ) . living - related donation would not , therefore , be considered altruistically motivated even though it is nonetheless acceptable . part of the problem for altruism in transplantation is that the link between altruism and the ethical acceptability of something is not always clear cut . depending upon which account of altruism one uses moral myopia ( p.1720 ) ; altruism can tell us whether an act is altruistic or not , but not always whether it is right or wrong , especially when motives may be mixed . for instance , a person may be motivated to donate partly by a desire to help people and partly because her family disapproves . the relationship between altruism and rightness is not a problem for nagel 's account which , owing to its strictly rational demands and kantian grounding , aligns altruistically motivated acts with right acts . blum 's account though , and the account apparently used in transplantation , are more vulnerable to this criticism . but suppose the rescuer , calculating that only three children could be saved in the time available , targeted her own children . it would also not be altruism according to miller , as one could argue that parents have a responsibility to save their children . if , however , she had not been prepared to save other children , and only ever intended to save her own , she would not be altruistically motivated according to the 2010 guidance . this shows us that even taking competing philosophical definitions into account , altruism as used to guide transplantation practice is confused . this prevents people from directing organs in cases like the racist donation in 1998 , or from directing organs in response to media - facilitated appeal by individuals.21 yet if one does have a relative on a transplant waiting list , then one 's altruism can be partial and directed , more like blum 's . unlike the 2010 guidance , however , blum does not insist that we must be willing to follow through with an action even if our family member can not after all benefit from it , but then neither does usual practice in relation to living - related donation . if one were to adopt any of these philosophical accounts of altruism as the basis for ethical organ donation , significantly more potential donations would have to be rejected . nagel 's altruism is particularly demanding , and would require the rejection of any organ not donated completely impartially . moreover , although blum 's altruism focuses on altruistic emotions , it does not overly concern itself with their origins ; if a racist person felt altruistic emotions for another person of his own race purely by virtue of their skin colour , this could be compatible with blum 's altruism . the nuffield report discusses altruism in detail , and presents a distinctive account that has elements in common with oda and accounts found in philosophical literature . in a departure from oda , however , the recipient of the beneficent behaviour need not necessarily be the eventual organ recipient . much media attention was given to the nuffield report 's proposal to pay a donor 's funeral expenses and the claim that this is compatible with altruism4 ( ch . presumably the authors would not endorse the claim that the presence of an altruistic motivation amongst a number of distinctly non - altruistic motivations ( such as to deliberately spite family / friends ) would render the donation altruistically motivated . although there may be an altruistic element to a donation decision in this instance , it would seem incorrect to deduce from this that the donation is altruistically motivated . although they acknowledge that real - life may present situations where motivations are mixed , the altruistic nature of a donation is secured only when the difficulty here is that establishing motivation is , as we have already discussed , notoriously difficult and it was perhaps beyond the scope of the nuffield report to provide a solution to this point . although the donor may be partly motivated to donate by his funeral expenses being paid , he could do so with others interests in mind he would receive no benefit himself from his funeral expenses being paid ( he would be dead ) but might be trying to lift this financial burden from his relatives . this , according to the nuffield report , is altruistic even though it displays a level of partiality ( towards next - of - kin and relatives ) that is not ordinarily acceptable under oda ( p.1754 ) . by suggesting that altruism in organ donation might be directed beyond the organ recipients , nuffield altruism could permit more direct payments in exchange for donation for instance , money given to next - of - kin to spend as they wish this would still be altruistic insofar as the donor would be motivated to donate organs in order to benefit other people ; it just happens that the intended beneficiaries would be his family . the only difference between the two cases is how the money provided is to be spent . the nuffield report concedes that if the next - of - kin agree to the donation only because they will benefit from the funeral expenses being paid , the payment of funeral expenses in this case would be a non - altruist focused intervention ( p.1394 ) . oda acts as a guiding principle for determining and promoting ethical donations , but features inconsistencies that result in arbitrariness which , at the very least , border on unfairness to those who are deprived of the benefits of receiving an organ as a result . applying a philosophical definition of altruism would be a remedy for this inconsistency but would result in many fewer acceptable donations ( nagel ) , or additional principles might be needed to identify donations that were undesirable for reasons unrelated to altruism ( blum ) . nuffield altruism falls between the two ; it is consistent , and promotes organ donation , but fails to provide a single guiding principle for determining the acceptability of a donation . the nuffield report accepts however , that altruism can not be the only guiding principle ( ch 54 ) . this is a sensible way forward for transplantation ethics , since it allows unethical practice to be robustly and roundly rejected without distorting a single principle ( to the extent that it becomes inconsistent ) like altruism to suit a wide range of specifics . comparing accounts of altruism oda , organ donation altruism . nuffield altruism is controversial , however , insofar as it seems to permit practices that oda was deliberately trying to prevent , principally financial incentives . moreover , accepting that a decision to donate is unlikely to result from a single motivation , and by widening the recipient pool for the other - regarding beneficial motivation that characterises altruism , the concept is virtually redundant as a measure of whether a donation is acceptable . for example , it would permit racial direction as a condition of donation , as a person could still be motivated to donate by a concern for the welfare of others , although that concern could be conditional upon the recipients being of a certain race . racist conditions would only be discounted if it could be demonstrated that the donor 's primary motivation was to deprive a particular racial group of some benefit ( rather than to benefit a different group ) and the definition of altruism was tightened so that only primary motivations were assessed . the outstanding issue would then remain whether it is ever possible to be certain what a person 's motives are , let alone what their primary motives might be . the nuffield report provides us with one reason why we should continue to associate organ donation with altruism : it maintains the communal virtue of a general disposition to be moved to self - sacrifice by the health needs of others ( p.1444 ) . while it may be important that people are moved to self - sacrifice ( p.1444 ) in order to help others , it is not clear that the health needs of others ought to be the only motivating factor . also , the degree of self - sacrifice in deceased organ donation is open to debate , and some people may not view it as being a sacrifice at all . it is unlikely that under the current system people are moved to self - sacrifice purely by the health needs of others ; as the nuffield report itself notes , people donate organs for a number of reasons , some of which may be selfless and other regarding , and others of which may be more self - interested ( ie , non - altruistic ) . the 2011 nice guidelines for organ donation do not even mention altruism , and state that , similarly to other documents,24 25 organ donation should be considered as a usual part of end of life care planning ( p.626 ) . making organ donation usual may make it part and parcel ( p.10919 ) of the social role of dying people , and if this were the case , according to miller 's altruism , organ donation could not be altruistic yet nice 's aim does not appear undesirable as a result . we agree with the nuffield report that a society where there is a general disposition to altruism seems , on the face of it at least , to be preferable to one where we are each only concerned with our own interests . this does not , however , seem to justify insisting that that altruism is a necessary as opposed to desirable component of ethical donation . the charade that donation must be altruistic to be acceptable altruistic living donation and living - related donation. perhaps it is time to accept that whilst altruism might be a sign of an acceptable donation it is not the only sign . these other signs may be used to preclude donations that ( all but nagel 's definition of ) altruism may accommodate , but coherent , robust and consistent justifications are required every time an offer of donation is declined because of the cost to the recipients of rejecting offers of organs .

current hiv prevention strategies ( condoms and abstinence ) force hiv - serodiscordant couples to choose between risking hiv transmission to a partner , or accepting childlessness [ 112 ] . behavioral strategies ( home artificial insemination , sex without condoms limited to peak fertility ) , male circumcision [ 1315 ] , antiretroviral therapy ( art ) for the infected partner [ 1618 ] , and preexposure antiretroviral prophylaxis ( prep ) for the negative partner [ 1922 ] create opportunities for hiv - serodiscordant couples to realize fertility goals and minimize periconception hiv transmission [ 2327 ] . prior to effective hiv treatment , the prevailing professional recommendation was for people living with hiv to avoid having children [ 28 , 29 ] . in 2001 , the u.s . centers for disease control recommended that healthcare providers support the fertility desires of people living with hiv . south africa 's constitution protects the right to reproductive choice for hiv - infected persons , and the most recent guidelines from the southern african hiv clinicians society offer risk - reduction strategies for hiv - serodiscordant couples who choose to conceive [ 31 , 32 ] . cross - sectional studies indicate that people living with hiv are receptive to safer conception advice from providers [ 3336 ] , but that healthcare workers ( hcws ) are not routinely engaging in conversations about fertility desires or plans , a crucial first piece of any reproductive health intervention [ 31 , 34 , 35 , 37 ] . in cape town , over 30% of hiv - infected women and 65% of hiv - infected men attending public sector clinics were interested in having additional children , yet only 19% and 6% , respectively , had discussed this with a hcw . among hiv - infected women in johannesburg , with plans to conceive in the next year , 40% had ever had a conversation about fertility plans with a provider . conversations with hcws about conception plans were also infrequent in argentina , brazil , and the united states . barriers to these conversations include a combination of patient ( e.g. , fear of judgment , lack of pregnancy planning ) , provider ( e.g. , limited experience , knowledge , or skills ) , and structural factors ( e.g. , competing healthcare demands , limited resources , poor integration of family planning and hiv services ) [ 31 , 3537 , 40 , 41 ] . while some conversations may be initiated with hiv lay counselors , they may not have the skills to address clinical issues beyond their focused training [ 42 , 43 ] . data demonstrating that arvs minimize hiv transmission suggest that periconception risk - reduction interventions will require hcw involvement [ 16 , 1922 , 27 ] . in our conceptual framework for periconception risk behavior , hcws have the potential to minimize risk behavior through providing information about hiv risk , offering prevention strategies , and promoting adherence to risk reduction strategies . there are no data on practices in kwazulu - natal , the most hiv - affected province in south africa , where 41% of pregnant women attending antenatal clinics are hiv positive . a better understanding of current hcw knowledge , attitudes , and practices will enhance the provision of reproductive counseling for hiv - infected individuals by allowing for development of interventions that capitalize on hcw strengths and address weaknesses . we present qualitative data resulting from interviews with 30 hiv - infected women and 20 hiv - infected men with serodiscordant sexual partners in durban , south africa . we previously reported on periconception risk behavior within this sample and here focus on participant reports of their experiences with hcw provision of reproductive counseling . these data offer early insight into current hcw practices and may contribute to development of feasible safer conception interventions . participants were recruited from the antiretroviral ( arv ) and preventing maternal - to - child - transmission ( pmtct ) clinics within a state - aided ( public / private partnership ) general hospital serving a predominantly urban population from the greater durban area where district antenatal clinic hiv prevalence is estimated at 41.5% . in 2011 , the arv clinic was providing care and treatment to 4734 patients on art . sixty percent of these patients are women ; the majority ( > 90% ) are black south africans . patients pay approximately 25 usd per month for comprehensive hiv services , which includes the cost of arvs . pregnant patients pay approximately 35 usd per visit to access antenatal care , which includes pmtct clinic services . in 2010 , 200 hiv - infected pregnant women enrolled in this program . male participants were recruited from the arv clinic and female participants were recruited from the arv and pmtct clinics . inclusion criteria were ( 1 ) age 1845 years ; ( 2 ) hiv - positive ; ( 3 ) pregnancy in the prior 12 months , including currently pregnant ( for women ) ; ( 4 ) partner of unknown or seronegative hiv status ( prior to referent pregnancy ) by participant report ( father of the referent pregnancy for women , current sexual partner for men ) ; ( 5 ) fluent in english or isizulu ; ( 6 ) able to give informed consent . initial attempts to recruit men with partner pregnancy in the past year were unsuccessful , men were subsequently recruited independent of recent partner pregnancy . it is not clear if initial recruitment challenges were due to sensitivities of reporting partner pregnancy ( in a setting where condoms are promoted strongly ) or a paucity of men with recent partner pregnancy . we conducted in - depth , qualitative , individual interviews to explore reproductive decision - making , sexual transmission risk understanding and practices , and periconception risk understanding and practices . here , we focus on data from the questions what have health care workers advised you about having children ( after knowing your status ) ? and what advice have you received around getting pregnant / having children safely ( since knowing your status ) ? participants were recruited from march through july 2010 via purposive sampling from patients awaiting clinical consultation . after obtaining informed consent , a gender - concordant research assistant trained in qualitative interviewing techniques interviewed participants in a private setting in isizulu or english . transcripts were independently reviewed and coded , and resultant conceptual categories and emergent themes were discussed by the research team using content analysis [ 46 , 47 ] . several authors reviewed coding categories and emergent themes with the research assistants in order to explore additional themes and confirm accuracy of interpretation . ethics approvals were obtained from the mccord hospital research ethics committee ( durban , south africa ) and from the partners healthcare institutional review board ( boston , usa ) . baseline demographic data , hiv history , reported partner hiv status , and reproductive history for 30 female and 20 male participants are shown in table 1 . women and men averaged 30 ( sd 4 ) and 34 ( sd 6 ) years of age , respectively , and had been diagnosed with hiv for a mean of 3 years ( sd 2 women , sd 5 men ) . seventy - three percent of women and 60% of men had completed secondary school , while 63% of women and 75% of men reported current employment . women had an average of 2 ( sd 1 ) pregnancies ( including current ) , 1.1 ( sd 0.7 ) prior live births , 0.9 ( sd 0.6 ) living children , and 18 ( 60% ) were pregnant at the time of interview . we have previously detailed the complexities of pregnancy intention in this sample , but about a third ( 11 ) of women described the referent pregnancy as explicitly planned . men had an average of 0.9 ( sd 1 ) living children ; three ( 16% ) reported partner pregnancy in the past year . structural , individual , and dyadic domains affect access to risk reduction information ( e.g. , knowledge that arvs can reduce sexual transmission risk ) , motivation ( e.g. , to adhere to prevention strategies ) , and ability to implement behavioral change ( e.g. , partnership dynamics and discussions of safer sex practices ) . within this framework , hcws have the potential to minimize risk behavior through providing hiv - affected couples with information about hiv transmission and conception , supporting disclosure , providing arvs as prevention , and promoting adherence to hiv risk reduction strategies . here we present themes suggesting that , in this sample , hiv - infected men and women in serodiscordant partnerships ( 1 ) are aware of and receptive to the idea that one should speak to an hcw prior to becoming pregnant , ( 2 ) seldom seek or receive conception advice from healthcare workers , and ( 3 ) when advice is shared , patients receive a range of information around safer conception . in addition , ( 4 ) men are eager for safer conception advice . most participants indicated that they had been informed by a hcw that they should seek advice when they were ready to conceive . in some cases , participants communicated that this advice might help minimize transmission to a child , and in some cases to a partner . this awareness was related to general information shared by counselors during routine arv adherence training sessions ( group adherence training sessions prior to art initiation are routine in south africa ) , regardless of the participant 's fertility goals at the time . they [ healthcare workers during the training session ] told me that when i have told her [ disclosed to partner ] , we should come here [ to the clinic ] together so that they will explain to both of us . they tell us that we should come back with our partners so that the doctor tells us what to do in order for the virus not to be transferred to the child . ( participant b03 , 26-year - old man)they [ counselors ] said that we should come with our partners so that you explain to the doctor that you want to have a child so that he tells you what to do , and that was it . no , they only said that we should consult a doctor with your partner if you want to have children . ( participant b15 , 29-year - old man)in addition to knowing that they should tell a hcw if they wanted to have children , participants were open to seeking this advice from healthcare workers . with us black people , if it happens that you get the virus and you die not having a child , your name just disappears and you are never mentioned . so if it happens that you do get hiv then you should go to the clinic and talk to them [ healthcare workers ] so that they help you to get a child in a safe way . ( participant b04 , 28-year - old man)we want to have a child together because we love each other and we are so close to each other i will probably go to special doctors when i am ready for that so that i hear from them what it is that i have to do in order to have a child since i have the virus and my partner does not . ( participant b16 , 36-year - old man)if you want to have children you need to consult the doctor then they will advise you what to do . ( participant a27 , 34-year - old woman ) they [ healthcare workers during the training session ] told me that when i have told her [ disclosed to partner ] , we should come here [ to the clinic ] together so that they will explain to both of us . they tell us that we should come back with our partners so that the doctor tells us what to do in order for the virus not to be transferred to the child . ( participant b03 , 26-year - old man ) they [ counselors ] said that we should come with our partners so that you explain to the doctor that you want to have a child so that he tells you what to do , and that was it . no , they only said that we should consult a doctor with your partner if you want to have children . ( participant b15 , 29-year - old man ) with us black people , if it happens that you get the virus and you die not having a child , your name just disappears and you are never mentioned so if it happens that you do get hiv then you should go to the clinic and talk to them [ healthcare workers ] so that they help you to get a child in a safe way . ( participant b04 , 28-year - old man ) we want to have a child together because we love each other and we are so close to each other i will probably go to special doctors when i am ready for that so that i hear from them what it is that i have to do in order to have a child since i have the virus and my partner does not . ( participant b16 , 36-year - old man ) if you want to have children you need to consult the doctor then they will advise you what to do . ( participant a27 , 34-year - old woman ) while most participants were aware that they should approach a healthcare worker prior to conception , few sought , or received safer conception advice . participants described several barriers to accessing reproductive counseling prior to conception including fear of judgment from nurses and financial challenges . they did not tell us that we should no longer have children , but you could see that is what they meant . the other day , one of the nurses made a comment and asked why do n't we go for family planning because they do not want to see us coming back to take the treatment [ arvs for pmtct ] again . ( participant a26 , 23-year - old woman ) i spoke to the doctor who said it is safe to have more children if i want to . he said i can come to him , but i did n't because he was expensive as he is a gynecologist . ( participant a13 , 32-year - old woman ) they did not tell us that we should no longer have children , but you could see that is what they meant . the other day , one of the nurses made a comment and asked why do n't we go for family planning because they do not want to see us coming back to take the treatment [ arvs for pmtct ] again . ( participant a26 , 23-year - old woman ) i spoke to the doctor who said it is safe to have more children if i want to . he said i can come to him , but i did n't because he was expensive as he is a gynecologist . ( participant a13 , 32-year - old woman ) additional barriers may be related to dyadic factors . for example , participants described that they were told to seek reproductive counseling at the arv clinic with their partners . however , about a third of participants had not disclosed his / her hiv status to their partner , making this scenario unlikely . further , about a third of the women in this sample described the referent pregnancy as unplanned . participants reported a range of information received from providers . at one end of the spectrum , some participants had not received advice about options for having children since their hiv diagnosis , including in the setting of expressing plans for having children . they [ healthcare workers ] haven't advised me : they have asked me about it but they have never given me any advice . they asked me if i am planning on having more children and i told them , ( participant a28 , 24-year - old woman)what advice have you received from health care workers around having children after knowing your status ? except that i have to practice safe sex , there is nothing ( participant a30 , 31-year - old woman ) they [ healthcare workers ] haven't advised me : they have asked me about it but they have never given me any advice . they asked me if i am planning on having more children and i told them , ( participant a28 , 24-year - old woman ) what advice have you received from health care workers around having children after knowing your status ? except that i have to practice safe sex , there is nothing ( participant a30 , 31-year - old woman ) others had received helpful safer conception information from doctors or nurses at pmtct , arv , and gynecology clinics . risk reduction strategies that participants had learned included artificial insemination , intercourse timed to peak fertility , manual insemination ( for male uninfected couples the uninfected male ejaculates into a condom or other container and the semen is inserted into the woman 's vaginal canal via a syringe or reversed condom ) , sperm washing , and intercourse with lubrication ( to avoid abrasions ) . they told me that i can go to one of those private hospitals to ask about sperm washing one of the times when i came into the clinic there was a poster on the wall saying if you want to have a child , speak to either a doctor or a nurse . so i asked the doctor , do you think is it possible for me to have a child even if i 'm positive ? but i had to go to one of those private hospitals to look into that thing [ sperm washing ] . ( participant b05 , 31-year - old man)a doctor gave us a [ syringe ] which we had to use after sex to withdraw sperm [ from the condom to insert into the woman ] . we are both aware that i am positive and so we would use a condom all the time and we received advice from the doctor on how to use the syringe that he gave us so that he [ partner ] does not get infected . ( participant a25 , 32-year - old woman)one participant suggested the importance of a lower plasma hiv viral load prior to conception , but it was not clear where she and her partner had learned this information . art as prevention was not a component of the south african department of health treatment guidelines at the time of this study ( or now ) .after finding out that my viral load was very low , even undetectable , he [ husband ] decided that let 's take a chance and try and see what is going to happen . he said he has taken that decision , it 's not that i am forcing him to take it . ( participant a04 , 28-year - old woman)some participants explained that the advice they had received about whether or not they , as hiv - infected individuals , could have children evolved over time or varied with different providers . but then they changed [ and said ] that i can have children , and that is what i am not sure of . ( participant b11 , 33-year - old man ) they told me that i can go to one of those private hospitals to ask about sperm washing one of the times when i came into the clinic there was a poster on the wall saying if you want to have a child , speak to either a doctor or a nurse . so i asked the doctor , do you think is it possible for me to have a child even if i 'm positive ? but i had to go to one of those private hospitals to look into that thing [ sperm washing ] . ( participant b05 , 31-year - old man ) a doctor gave us a [ syringe ] which we had to use after sex to withdraw sperm [ from the condom to insert into the woman ] . we are both aware that i am positive and so we would use a condom all the time and we received advice from the doctor on how to use the syringe that he gave us so that he [ partner ] does not get infected . ( participant a25 , 32-year - old woman ) after finding out that my viral load was very low , even undetectable , he [ husband ] decided that let 's take a chance and try and see what is going to happen . he said he has taken that decision , it 's not that i am forcing him to take it . ( participant a04 , 28-year - old woman ) so far they told me that i can not have children . but then they changed [ and said ] that i can have children , and that is what i am not sure of . ( participant b11 , 33-year - old man ) from our sample , many male participants had engaged with a hcw around discussions of safer conception or had sought out information from the internet , relatives , or other news sources . no [ i have not had a conversation with a hcw ] , but i 've done research and they said that one can do artificial insemination but i am not sure if it is done on humans . artificial insemination means that a person can be impregnated by a man , but technology can be used to make a baby without them having sex the child is yours because the semen is taken from you and can be injected into the women and a baby grows inside the woman , the genes of that child are yours . ( participant b13 , 28-year - old man)several male participants became quite interested in safer conception options during the interview and planned to seek out additional advice from a hcw . one participant ( b12 , 35-year - old man ) responded to a question about fertility desire by saying , so in the situation that i am in now i do not think i want to have children again , but by the end of the interview he asks : is it possible to have children if i am positive and my partner is negative ? even before i was diagnosed [ with hiv ] , we were not planning to have more two is enough . however , by the end of the interview , he asks : i want to know if i decide to have kids how is that possible ? maybe i 'll try to find more ways to get information about how to have kids when you 're hiv positive . however , this may simply reflect the study sample given that fewer women were planning a future pregnancy and many were still close to or in the midst of a current pregnancy . no [ i have not had a conversation with a hcw ] , but i 've done research and they said that one can do artificial insemination but i am not sure if it is done on humans . artificial insemination means that a person can be impregnated by a man , but technology can be used to make a baby without them having sex the child is yours because the semen is taken from you and can be injected into the women and a baby grows inside the woman , the genes of that child are yours . ( participant b13 , 28-year - old man ) is it possible to have children if i am positive and my partner is negative ? maybe i 'll try to find more ways to get information about how to have kids when you 're hiv positive . these qualitative data suggest that hiv - infected patients are increasingly aware of reproductive counseling opportunities . many participants understood that hcws may have valuable advice to offer to facilitate safer conception and were open to seeking this advice . while few participants had sought advice , some participants had received safer conception advice from healthcare encounters . prior studies suggest that the majority of hiv - infected patients are not receiving reproductive counseling and are reluctant to engage with healthcare workers to discuss reproductive plans [ 12 , 35 , 36 , 39 , 4951 ] . [ 34 , page 73 ] presented data collected during 2009 and reported that only 41% of hiv - infected women reported that an art healthcare provider had spoken with them about their options should they want to conceive in the future . in work by cooper et al . , collected in 2006 , 19% of women and 6% of men had consulted a doctor , nurse , or counselor in hiv care about fertility intentions [ 38 , page s44 ] . the fact that many participants , including men , in this small sample knew that hcws may have advice to offer captures data not mentioned in prior studies . these data suggest that clinical settings may be adapting to accommodate the fertility goals of people living with hiv . many participants reported the expectation that they could access detailed counseling if they returned to the clinic when they were ready to have a child . from work by our group and others , several facts of periconception practice waiting to talk to a provider until one is ready to have a child eliminates the opportunity to discuss the risks of having children in order to inform the decision to have biologic children [ 24 , 52 , 53 ] . many men and women living with hiv do not know their partner 's status and/or have not disclosed to their partners . prior to testing and disclosure , it is impossible for an individual or a couple to assess sexual transmission risk in the context of conception . continued efforts to promote couple - based testing and supported disclosure are critical . for those who decide to have children , a preconception conversation with a healthcare worker should include an assessment of the hiv - infected partner 's health ; the hiv status of the partner ; if the woman is positive , a discussion of the risks of pregnancy when hiv infected ; risks of transmission to a partner with various periconception risk reduction strategies ; fertility assessment ; the increased risk of transmission and acquisition during unprotected sex during pregnancy ; the risks of perinatal transmission . furthermore , asking individuals or couples to return when they are ready to have children presupposes conception planning . however , many pregnancies are not explicitly planned [ 55 , 56 ] . providing up - front information about the options for safer conception may communicate the importance of preconception planning to protect the future child and the partner . recent data from johannesburg ( south africa ) suggest high fertility intentions at art initiation , reflecting the importance of providing safer conception information at treatment initiation and in followup . a partner ( who is not attending clinic ) may make decisions about having children if his or her partner has not been educated it is impossible for them to have an informed conversation about the risks and options for safer conception . in addition , life transitions may be quick and there may be plans for pregnancy long before a next visit with a provider . expecting a patient to raise the issue of fertility plans on his or her own may be problematic . while not a theme in our data , published data suggest that women and men living with hiv hesitate to reveal fertility plans to hcws for fear of judgment [ 36 , 58 ] . it is not part of routine clinical practice to expect patients to tell providers when they are ready to discuss health behavior change ( e.g. , smoking , substance abuse , exercise , sexual behavior)the onus is on the provider to actively inquire about behaviors that compromise health . routine assessment of fertility goals and plans should be incorporated into clinical care for people living with hiv , with subsequent recommendations for safer conception or effective contraception options , depending on fertility goals . how to execute this in overburdened healthcare systems is a challenge but may require increased training in fertility intention assessment and comprehensive reproductive counseling for counselors . participants had learned of safer conception strategies from hcws including artificial insemination , intercourse timed to peak fertility , sperm washing , home manual insemination , and intercourse with lubrication to avoid abrasions . the frequency with which sperm washing and artificial insemination was raised is interesting since these are some of the least accessible ( geographically , economically ) strategies for reducing transmission risk . simpler risk reduction strategies such as delaying conception until the infected partner is on treatment with suppressed hiv rna viral load , timing sex without condoms to peak fertility , circumcision for the male partner if he is uninfected , and manual insemination are likely to be more feasible . our semistructured interview guide was not designed to probe specifically about particular techniques and it is possible that participants were more likely to recall discussions about and have faith in high - tech concepts such as sperm washing compared to behavioral modifications such as timing unprotected sex to peak fertility . in addition , patients may have received limited information , perhaps due to insufficient clinician training on this topic . the who guidelines for serodiscordant couples offer some safer conception recommendations , in addition , a more comprehensive guideline was recently published by the southern african hiv clinicians society and will be helpful for clinicians [ 32 , 54 ] . hcw training on the interpretation and application of these guidelines should be a priority for promoting comprehensive reproductive health counseling . we found that , in this sample , male participants were eager to engage with hcws in order to seek reproductive counseling . prior data suggests that providers may have less insight into male reproductive intentions and that men are less likely to seek advice from providers . we previously published on the important role of men in conception decisions from an earlier analysis of these data , an observation which has been reported by others [ 9 , 11 , 12 , 36 , 5961 ] . as reproductive counseling is integrated into hiv care , it will be crucial to increase male involvement . interventions to engage men in contraception and family planning have been effective in several sub - saharan african settings [ 6163 ] . in addition , the dyadic nature of conception decisions and periconception risk behavior emphasizes the importance of a couple - oriented counseling approach when feasible . findings from this small qualitative sample are meant to generate hypotheses to pursue in future larger scale research . in addition , our participants were attending clinical services at a semiprivate hospital and may not represent the broader population who access public sector care or those who do not access any healthcare . while this clinic does not have a formal program for safer conception counseling , several of the authors previously worked at this clinic which may have heightened some of the clinic providers ' awareness around reproductive counseling for people living with hiv . finally , patient perspectives of past experiences with provider counseling may not accurately reflect what occurred ; provider perspectives are also needed to understand current practices . these are the first data to explore patient experiences with provider provision of reproductive counseling in kwazulu natal , where 41% of women attending antenatal clinics are hiv infected . this work suggests that men and women living with hiv are aware that safer conception options may be available and are interested in accessing this information . further , there is a clear need to expand the quality and reach of periconception risk reduction information to both providers and patients . these data serve as important hypothesis generation to guide future research as reproductive counseling interventions are developed .

background . understanding hiv - infected patient experiences and perceptions of reproductive counseling in the health care context is critical to inform design of effective pharmaco - behavioral interventions that minimize periconception hiv risk and support hiv - affected couples to realize their fertility goals . methods . we conducted semistructured , in - depth interviews with 30 hiv - infected women ( with pregnancy in prior year ) and 20 hiv - infected men , all reporting serodiscordant partners and accessing care in durban , south africa . we investigated patient - reported experiences with safer conception counseling from health care workers ( hcws ) . interview transcripts were reviewed and coded using content analysis for conceptual categories and emergent themes . results . the study findings indicate that hiv - infected patients recognize hcws as a resource for periconception - related information and are receptive to speaking to a hcw prior to becoming pregnant , but seldom seek or receive conception advice in the clinic setting . hiv nondisclosure and unplanned pregnancy are important intervening factors . when advice is shared , patients reported receiving a range of information . male participants showed particular interest in accessing safer conception information . conclusions . hiv - infected men and women with serodiscordant partners are receptive to the idea of safer conception counseling . hcws need to be supported to routinely initiate accurate safer conception counseling with hiv - infected patients of reproductive age .

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

current hiv prevention strategies ( condoms and abstinence ) force hiv - serodiscordant couples to choose between risking hiv transmission to a partner , or accepting childlessness [ 112 ] . behavioral strategies ( home artificial insemination , sex without condoms limited to peak fertility ) , male circumcision [ 1315 ] , antiretroviral therapy ( art ) for the infected partner [ 1618 ] , and preexposure antiretroviral prophylaxis ( prep ) for the negative partner [ 1922 ] create opportunities for hiv - serodiscordant couples to realize fertility goals and minimize periconception hiv transmission [ 2327 ] . prior to effective hiv treatment , the prevailing professional recommendation was for people living with hiv to avoid having children [ 28 , 29 ] . centers for disease control recommended that healthcare providers support the fertility desires of people living with hiv . south africa 's constitution protects the right to reproductive choice for hiv - infected persons , and the most recent guidelines from the southern african hiv clinicians society offer risk - reduction strategies for hiv - serodiscordant couples who choose to conceive [ 31 , 32 ] . cross - sectional studies indicate that people living with hiv are receptive to safer conception advice from providers [ 3336 ] , but that healthcare workers ( hcws ) are not routinely engaging in conversations about fertility desires or plans , a crucial first piece of any reproductive health intervention [ 31 , 34 , 35 , 37 ] . in cape town , over 30% of hiv - infected women and 65% of hiv - infected men attending public sector clinics were interested in having additional children , yet only 19% and 6% , respectively , had discussed this with a hcw . among hiv - infected women in johannesburg , with plans to conceive in the next year , 40% had ever had a conversation about fertility plans with a provider . while some conversations may be initiated with hiv lay counselors , they may not have the skills to address clinical issues beyond their focused training [ 42 , 43 ] . in our conceptual framework for periconception risk behavior , hcws have the potential to minimize risk behavior through providing information about hiv risk , offering prevention strategies , and promoting adherence to risk reduction strategies . there are no data on practices in kwazulu - natal , the most hiv - affected province in south africa , where 41% of pregnant women attending antenatal clinics are hiv positive . a better understanding of current hcw knowledge , attitudes , and practices will enhance the provision of reproductive counseling for hiv - infected individuals by allowing for development of interventions that capitalize on hcw strengths and address weaknesses . we present qualitative data resulting from interviews with 30 hiv - infected women and 20 hiv - infected men with serodiscordant sexual partners in durban , south africa . we previously reported on periconception risk behavior within this sample and here focus on participant reports of their experiences with hcw provision of reproductive counseling . these data offer early insight into current hcw practices and may contribute to development of feasible safer conception interventions . participants were recruited from the antiretroviral ( arv ) and preventing maternal - to - child - transmission ( pmtct ) clinics within a state - aided ( public / private partnership ) general hospital serving a predominantly urban population from the greater durban area where district antenatal clinic hiv prevalence is estimated at 41.5% . in 2010 , 200 hiv - infected pregnant women enrolled in this program . male participants were recruited from the arv clinic and female participants were recruited from the arv and pmtct clinics . inclusion criteria were ( 1 ) age 1845 years ; ( 2 ) hiv - positive ; ( 3 ) pregnancy in the prior 12 months , including currently pregnant ( for women ) ; ( 4 ) partner of unknown or seronegative hiv status ( prior to referent pregnancy ) by participant report ( father of the referent pregnancy for women , current sexual partner for men ) ; ( 5 ) fluent in english or isizulu ; ( 6 ) able to give informed consent . initial attempts to recruit men with partner pregnancy in the past year were unsuccessful , men were subsequently recruited independent of recent partner pregnancy . we conducted in - depth , qualitative , individual interviews to explore reproductive decision - making , sexual transmission risk understanding and practices , and periconception risk understanding and practices . here , we focus on data from the questions what have health care workers advised you about having children ( after knowing your status ) ? transcripts were independently reviewed and coded , and resultant conceptual categories and emergent themes were discussed by the research team using content analysis [ 46 , 47 ] . several authors reviewed coding categories and emergent themes with the research assistants in order to explore additional themes and confirm accuracy of interpretation . ethics approvals were obtained from the mccord hospital research ethics committee ( durban , south africa ) and from the partners healthcare institutional review board ( boston , usa ) . baseline demographic data , hiv history , reported partner hiv status , and reproductive history for 30 female and 20 male participants are shown in table 1 . women and men averaged 30 ( sd 4 ) and 34 ( sd 6 ) years of age , respectively , and had been diagnosed with hiv for a mean of 3 years ( sd 2 women , sd 5 men ) . we have previously detailed the complexities of pregnancy intention in this sample , but about a third ( 11 ) of women described the referent pregnancy as explicitly planned . men had an average of 0.9 ( sd 1 ) living children ; three ( 16% ) reported partner pregnancy in the past year . , partnership dynamics and discussions of safer sex practices ) . within this framework , hcws have the potential to minimize risk behavior through providing hiv - affected couples with information about hiv transmission and conception , supporting disclosure , providing arvs as prevention , and promoting adherence to hiv risk reduction strategies . here we present themes suggesting that , in this sample , hiv - infected men and women in serodiscordant partnerships ( 1 ) are aware of and receptive to the idea that one should speak to an hcw prior to becoming pregnant , ( 2 ) seldom seek or receive conception advice from healthcare workers , and ( 3 ) when advice is shared , patients receive a range of information around safer conception . in addition , ( 4 ) men are eager for safer conception advice . most participants indicated that they had been informed by a hcw that they should seek advice when they were ready to conceive . in some cases , participants communicated that this advice might help minimize transmission to a child , and in some cases to a partner . this awareness was related to general information shared by counselors during routine arv adherence training sessions ( group adherence training sessions prior to art initiation are routine in south africa ) , regardless of the participant 's fertility goals at the time . they [ healthcare workers during the training session ] told me that when i have told her [ disclosed to partner ] , we should come here [ to the clinic ] together so that they will explain to both of us . they tell us that we should come back with our partners so that the doctor tells us what to do in order for the virus not to be transferred to the child . ( participant b03 , 26-year - old man)they [ counselors ] said that we should come with our partners so that you explain to the doctor that you want to have a child so that he tells you what to do , and that was it . ( participant b15 , 29-year - old man)in addition to knowing that they should tell a hcw if they wanted to have children , participants were open to seeking this advice from healthcare workers . so if it happens that you do get hiv then you should go to the clinic and talk to them [ healthcare workers ] so that they help you to get a child in a safe way . ( participant b16 , 36-year - old man)if you want to have children you need to consult the doctor then they will advise you what to do . ( participant a27 , 34-year - old woman ) they [ healthcare workers during the training session ] told me that when i have told her [ disclosed to partner ] , we should come here [ to the clinic ] together so that they will explain to both of us . they tell us that we should come back with our partners so that the doctor tells us what to do in order for the virus not to be transferred to the child . ( participant b03 , 26-year - old man ) they [ counselors ] said that we should come with our partners so that you explain to the doctor that you want to have a child so that he tells you what to do , and that was it . ( participant b15 , 29-year - old man ) with us black people , if it happens that you get the virus and you die not having a child , your name just disappears and you are never mentioned so if it happens that you do get hiv then you should go to the clinic and talk to them [ healthcare workers ] so that they help you to get a child in a safe way . ( participant b16 , 36-year - old man ) if you want to have children you need to consult the doctor then they will advise you what to do . ( participant a27 , 34-year - old woman ) while most participants were aware that they should approach a healthcare worker prior to conception , few sought , or received safer conception advice . participants described several barriers to accessing reproductive counseling prior to conception including fear of judgment from nurses and financial challenges . they did not tell us that we should no longer have children , but you could see that is what they meant . ( participant a26 , 23-year - old woman ) i spoke to the doctor who said it is safe to have more children if i want to . he said i can come to him , but i did n't because he was expensive as he is a gynecologist . ( participant a13 , 32-year - old woman ) they did not tell us that we should no longer have children , but you could see that is what they meant . ( participant a26 , 23-year - old woman ) i spoke to the doctor who said it is safe to have more children if i want to . he said i can come to him , but i did n't because he was expensive as he is a gynecologist . for example , participants described that they were told to seek reproductive counseling at the arv clinic with their partners . participants reported a range of information received from providers . at one end of the spectrum , some participants had not received advice about options for having children since their hiv diagnosis , including in the setting of expressing plans for having children . they asked me if i am planning on having more children and i told them , ( participant a28 , 24-year - old woman)what advice have you received from health care workers around having children after knowing your status ? they asked me if i am planning on having more children and i told them , ( participant a28 , 24-year - old woman ) what advice have you received from health care workers around having children after knowing your status ? except that i have to practice safe sex , there is nothing ( participant a30 , 31-year - old woman ) others had received helpful safer conception information from doctors or nurses at pmtct , arv , and gynecology clinics . they told me that i can go to one of those private hospitals to ask about sperm washing one of the times when i came into the clinic there was a poster on the wall saying if you want to have a child , speak to either a doctor or a nurse . ( participant a25 , 32-year - old woman)one participant suggested the importance of a lower plasma hiv viral load prior to conception , but it was not clear where she and her partner had learned this information . ( participant a04 , 28-year - old woman)some participants explained that the advice they had received about whether or not they , as hiv - infected individuals , could have children evolved over time or varied with different providers . ( participant b11 , 33-year - old man ) they told me that i can go to one of those private hospitals to ask about sperm washing one of the times when i came into the clinic there was a poster on the wall saying if you want to have a child , speak to either a doctor or a nurse . ( participant b11 , 33-year - old man ) from our sample , many male participants had engaged with a hcw around discussions of safer conception or had sought out information from the internet , relatives , or other news sources . no [ i have not had a conversation with a hcw ] , but i 've done research and they said that one can do artificial insemination but i am not sure if it is done on humans . artificial insemination means that a person can be impregnated by a man , but technology can be used to make a baby without them having sex the child is yours because the semen is taken from you and can be injected into the women and a baby grows inside the woman , the genes of that child are yours . ( participant b13 , 28-year - old man)several male participants became quite interested in safer conception options during the interview and planned to seek out additional advice from a hcw . one participant ( b12 , 35-year - old man ) responded to a question about fertility desire by saying , so in the situation that i am in now i do not think i want to have children again , but by the end of the interview he asks : is it possible to have children if i am positive and my partner is negative ? even before i was diagnosed [ with hiv ] , we were not planning to have more two is enough . however , this may simply reflect the study sample given that fewer women were planning a future pregnancy and many were still close to or in the midst of a current pregnancy . no [ i have not had a conversation with a hcw ] , but i 've done research and they said that one can do artificial insemination but i am not sure if it is done on humans . artificial insemination means that a person can be impregnated by a man , but technology can be used to make a baby without them having sex the child is yours because the semen is taken from you and can be injected into the women and a baby grows inside the woman , the genes of that child are yours . these qualitative data suggest that hiv - infected patients are increasingly aware of reproductive counseling opportunities . many participants understood that hcws may have valuable advice to offer to facilitate safer conception and were open to seeking this advice . while few participants had sought advice , some participants had received safer conception advice from healthcare encounters . prior studies suggest that the majority of hiv - infected patients are not receiving reproductive counseling and are reluctant to engage with healthcare workers to discuss reproductive plans [ 12 , 35 , 36 , 39 , 4951 ] . [ 34 , page 73 ] presented data collected during 2009 and reported that only 41% of hiv - infected women reported that an art healthcare provider had spoken with them about their options should they want to conceive in the future . the fact that many participants , including men , in this small sample knew that hcws may have advice to offer captures data not mentioned in prior studies . these data suggest that clinical settings may be adapting to accommodate the fertility goals of people living with hiv . many participants reported the expectation that they could access detailed counseling if they returned to the clinic when they were ready to have a child . from work by our group and others , several facts of periconception practice waiting to talk to a provider until one is ready to have a child eliminates the opportunity to discuss the risks of having children in order to inform the decision to have biologic children [ 24 , 52 , 53 ] . many men and women living with hiv do not know their partner 's status and/or have not disclosed to their partners . prior to testing and disclosure , it is impossible for an individual or a couple to assess sexual transmission risk in the context of conception . for those who decide to have children , a preconception conversation with a healthcare worker should include an assessment of the hiv - infected partner 's health ; the hiv status of the partner ; if the woman is positive , a discussion of the risks of pregnancy when hiv infected ; risks of transmission to a partner with various periconception risk reduction strategies ; fertility assessment ; the increased risk of transmission and acquisition during unprotected sex during pregnancy ; the risks of perinatal transmission . furthermore , asking individuals or couples to return when they are ready to have children presupposes conception planning . providing up - front information about the options for safer conception may communicate the importance of preconception planning to protect the future child and the partner . recent data from johannesburg ( south africa ) suggest high fertility intentions at art initiation , reflecting the importance of providing safer conception information at treatment initiation and in followup . a partner ( who is not attending clinic ) may make decisions about having children if his or her partner has not been educated it is impossible for them to have an informed conversation about the risks and options for safer conception . while not a theme in our data , published data suggest that women and men living with hiv hesitate to reveal fertility plans to hcws for fear of judgment [ 36 , 58 ] . routine assessment of fertility goals and plans should be incorporated into clinical care for people living with hiv , with subsequent recommendations for safer conception or effective contraception options , depending on fertility goals . how to execute this in overburdened healthcare systems is a challenge but may require increased training in fertility intention assessment and comprehensive reproductive counseling for counselors . participants had learned of safer conception strategies from hcws including artificial insemination , intercourse timed to peak fertility , sperm washing , home manual insemination , and intercourse with lubrication to avoid abrasions . simpler risk reduction strategies such as delaying conception until the infected partner is on treatment with suppressed hiv rna viral load , timing sex without condoms to peak fertility , circumcision for the male partner if he is uninfected , and manual insemination are likely to be more feasible . in addition , patients may have received limited information , perhaps due to insufficient clinician training on this topic . the who guidelines for serodiscordant couples offer some safer conception recommendations , in addition , a more comprehensive guideline was recently published by the southern african hiv clinicians society and will be helpful for clinicians [ 32 , 54 ] . we found that , in this sample , male participants were eager to engage with hcws in order to seek reproductive counseling . as reproductive counseling is integrated into hiv care , it will be crucial to increase male involvement . while this clinic does not have a formal program for safer conception counseling , several of the authors previously worked at this clinic which may have heightened some of the clinic providers ' awareness around reproductive counseling for people living with hiv . finally , patient perspectives of past experiences with provider counseling may not accurately reflect what occurred ; provider perspectives are also needed to understand current practices . these are the first data to explore patient experiences with provider provision of reproductive counseling in kwazulu natal , where 41% of women attending antenatal clinics are hiv infected . this work suggests that men and women living with hiv are aware that safer conception options may be available and are interested in accessing this information . further , there is a clear need to expand the quality and reach of periconception risk reduction information to both providers and patients . these data serve as important hypothesis generation to guide future research as reproductive counseling interventions are developed .

the last decade has seen an exponential rise in the availability and accessibility of assisted reproductive treatment opportunities in the indian subcontinent . couple 's desires to achieve maximum chances of conceiving as well as the option of fetal reduction are important reasons for multiple embryo transfer in an assisted reproductive technique ( art ) cycle . balancing benefit ( increased pregnancy rate ) against the disadvantage of multiple embryo transfer is not easy as both the clinician and the couple very easily appreciate the benefit , whereas the disadvantages ( pregnancy loss , preterm delivery , maternal and/or neonatal morbidity / mortality ) are more distant consequences . therefore , in the absence of any standardized legislature to guide the number of embryos transferred after an art procedure , multiple pregnancies have become one of the most important outcomes to be considered in an art conception . the risk of pregnancy loss and preterm delivery leading to neonatal morbidity and mortality increases in the presence of multiple pregnancy , especially higher order gestation like triplet or more . maternal complications for women carrying high - order multiple gestations include increased incidence of preeclampsia , cesarean delivery , postpartum hemorrhage , and fatty liver . many studies in the past have evaluated the risk of continuation of triplet conception versus reduction to twin or singleton pregnancy to improve perinatal outcomes . while some studies have found an increase in gestational age at delivery in triplet pregnancy after fetal reduction , some have not found any significant advantage . the role of multifetal pregnancy reduction ( mfpr ) is more clearly advantageous in quadruplets or higher order gestation . a fetal loss of 8%16% has been reported in literature after mfpr although this incidence depends not only on operator 's experience but also on gestational age at reduction , number of fetuses reduced , patient factors , etc . in many cases , spontaneous fetal reduction ( sfr ) is seen , wherein one or several embryos naturally disappear . pregnancies undergoing sfr have found to be at higher risk of preterm delivery and poorer neonatal outcomes , especially when they were reduced to singleton from twin and more so when reduced to a twin from triplets . this retrospective analysis was performed in the department of reproductive medicine and surgery from january 2012 to december 2015 . details of any pregnancy conceived after art in vitro fertilization ( ivf ) or intracytoplasmic sperm injection ( icsi ) was recorded in the departmental record in the form of an individual patient file which is periodically updated either at the time of patient follow - up visit or telephonic enquiry . records of patients who had conceived triplet gestation after ivf / icsi and had delivered were evaluated for the purpose of this study , and age , parity , number of embryo transferred , calculated last menstrual period , number of gestational sacs , antenatal course , pregnancy outcome , date of delivery , birth weight , and neonatal course were collected from them . the patients with three gestational sacs ( trichorionic triamniotic ) with cardiac activity seen at 6 weeks of gestation on transvaginal scan were considered for the study . patients , who were lost to follow - up , had heterotopic pregnancies , had previous pregnancy losses , or continued with triplet or higher order gestation beyond 12 weeks and/or whose records were incomplete were excluded from the study . as per departmental protocol , ultrasonography ( usg ) was initially performed to determine the location and number of gestational sacs when quantitative beta - human chorionic gonadotropin levels were expected to be 2000 miu / ml or more between 5.5 and 6.5 gestational weeks ( 3.54.5 weeks after embryo transfer ) . pregnant women were followed up in the department under a fetal medicine specialist until 12 weeks . patients not under regular care were periodically followed contacted via telephone . for the purpose of the study , sfr was defined as disappearance of gestational sac or loss of cardiac activity in one or two gestational sacs ( after its identification ) out of the triplet pregnancies . a single operator performed mfpr at 1113 weeks in cases where cardiac activity was seen in all three gestational sacs between 10 and 12 weeks gestation . the reduction was done abdominally under ultrasound guidance with intracardiac kcl injection such that single fetus was reduced in triplet gestation . fetuses were selected for reduction by : ( 1 ) discordant fetal growth , ( 2 ) the presence of increased nuchal translucency , ( 3 ) distance of the amniotic sac from the internal os , and ( 4 ) accessibility to transabdominal reduction . no cases of quadruplet or higher order gestation were noted / considered in the study population . in the mfpr group , triplet gestation reduced to singleton was not considered for the study to reduce confounding factors . abortion was defined as disappearance of cardiac activity in utero after sfr / mfpr or delivery before 28 completed weeks of gestation . preterm delivery was defined as the birth of a viable baby ( after 28 weeks ) at or before 37 completed weeks of gestation . neonatal death was defined as the death of a live baby within 4 weeks of delivery . restricted fetal growth or intrauterine growth restriction ( iugr ) was defined as a birth weight less than the 10 percentile for gestational age on the basis of national singleton birth weights . eighty pregnancies with triplet gestation at 6 weeks scan fulfilled the inclusion criteria and underwent spontaneous or mfpr before 1213 weeks of gestation . the patients were divided into two groups sfr and mfpr according to the reduction method . secondary outcome measures were live birth rate , average birth weight , and preterm birth rate . the outcome variables studied in the present study were pregnancy loss , weeks of gestation at delivery , birth weight of the baby , incidence of iugr , incidence of preterm labor , and incidence of single and twin delivery . the maternal and fetal parameters of the two groups were compared using chi - square test and t - test wherever applicable to determine statistical significance . association of the type of fetal reduction with fetal outcome was evaluated using chi - square test . the average age of patients in the present study was 32.76 years . only 12 ( 15% ) had a history of previous conception . of 80 patients considered for the study , 34 had sfr , whereas 46 underwent mfpr as per protocol . of eighty triplet pregnancies considered in the present study , a total of 15 patients aborted before the period of viability ( 28 weeks ) . the difference in probability of abortion in both groups was not statistically significant ( p = 0.3942 ) , even if only early abortions are considered ( gestational age < 14 weeks ) . the sfr group had higher risk ( odds ratio [ or ] = 3.600 , 95% confidence interval [ ci ] = 0.279446.388 ) of loss of all the fetuses over the next 2 weeks ( < 14 weeks ) than mfpr groups ( 37.5% vs. 14.3% , respectively ) . twenty - six patients out of 34 ( 76.5% ) in the sfr group had live births in comparison to 84.78% ( 39/46 ) in the mfpr group . although the percentage of live birth was higher in patients who had mfpr , it was not statistically significant [ table 1 ] . perinatal outcome in two groups there were more chances of loss of extra fetus in sfr ( 23.5% ) group than mfpr group ( 8.7% ) ( or = 3.889 , 95% ci = 1.03014.680 , p = 0.0781 ) . the statistical probability of having a term delivery , preterm or very preterm delivery did not differ in both groups statistically ( p = 0.297 ) . after sfr or mfpr , a total of 118 babies were born to eighty patients either as singleton or twins ( 106 ) . 58.33% ( 7/12 ) singleton babies had a birth weight of more than 2.6 kg , while in twin gestation , only 6.6% ( 7/106 ) babies had a birth weight of more than 2.5 kg ( p < 0.0001 ) . however , no association of birth weight was observed with the type of fetal reduction ( sfr and mfpr group ) ( p = 0.364 ) [ table 1 ] . the average gestational age in the sfr group was lower in comparison to the mfpr group , but this does not reach clinical significance ( p = 0.2025 ) [ table 1 ] . the incidence of pregnancy - induced hypertension was more in sfr than mfpr group ; however , the difference was statistically insignificant . mfpr group had 74 babies with an average birth weight of 2.03 0.49 kg . although birth weight was higher than sfr group ( 1.98 0.61 kg ) , the difference does not reach clinical significance . no significant difference was observed in terms of incidence of iugr , congenital abnormalities , and neonatal death in two groups [ table 1 ] . of the three congenital anomalies , there were two fetuses with cardiac anomalies ( one in each group ) and one fetus had an absence of bilateral toe in mfpr group . of the ten neonatal deaths , seven were early neonatal death , one was late neonatal death , and two were immediate neonatal deaths . in art pregnancies , the first scan is done at around 6 weeks of gestation . on visualization of triplet sacs , the patient is counseled regarding the pros and cons of continuation of triplet pregnancies as against fetal reduction . however , at times before the reduction is scheduled ( generally after 1112 weeks ) , spontaneous loss of one or more fetuses is seen . this study aims to compare the perinatal outcome in cases where fetal reduction occurred spontaneously as against wherein mpfr was performed . most of the studies done in the past have compared the perinatal outcomes in reduced and nonreduced pregnancies . this study is relevant not only to understand the implications of sfr for counseling patients but also to decide whether it is prudent to wait for 12 weeks or more before mfpr was done , so as to allow for sfr . this study is also needed because knowing the probability of preterm delivery or growth restriction will aid not only in counseling the couple but also in planning the place of delivery and measures thereof . it is reported that sfr rate is 12%30% in art cycles and even as high as 50%80% in triplet or higher order gestation . association of sfr with increasing maternal age was found in these studies as well . in the present study , although the percentage of sfr in previous studies is similar to our own , we did not find any difference or association with maternal age in both groups because average maternal age in our study was less ( 32.76 years ) . in a large study by zhang et al . therefore , it can be postulated that multiple gestational sacs in it predispose to the loss of one or more fetuses in utero . the possible reasons may be small uterine space and the relative lack of blood supply of the gestation sac caused by multiple pregnancies . zhang et al . have found that sfr occurred within 8 weeks in 78.4% of cases . in patients who underwent sfr later in their gestation , they found an increasing trend of low birth weight and very low birth weight rates . therefore , we believe that although our study does not show any significant difference in the perinatal outcome between sfr and mfpr groups , waiting for sfr in advanced gestation ( > 12 weeks ) may not be beneficial to the patient because patients in mfpr group were selected for reduction by discordant growth , increased nuchal translucency as already mentioned in the materials and methods . in an observational study by papageorghiou et al . , the rate of miscarriage ( pregnancy loss before 24 weeks ) was 8.3% , while the rate of early preterm delivery was 9.7% . the rate of abortion is lower to our own ( 15.2% ) as the period of viability considered by us is 28 weeks as against 24 weeks in the above - mentioned study . our percentage of abortion is higher than some previous studies as well . as ours is a retrospective analysis and the follow - up of patients beyond 12 weeks was not done at our own center in all cases , the reasons for such high abortion rates are not clear . it is possible that some of our patients were not under expert medical care after the second trimester and therefore were not as closely monitored as was required . our study shows that loss of fetus occurred after mfpr occurred in 1 patient within 2 weeks . however , in over 80% cases , interval between mfpr and miscarriage was more than 2 weeks . it is , therefore , important to counsel the couple who choose to have a fetal reduction that most of the excess loss with mfpr occurs several weeks after the procedure and is likely to be the consequence of the resorbing dead fetoplacental tissue , rather than the technique itself . when we look into the pattern of miscarriage in the sfr group , we found that miscarriage of the remaining fetuses in 37.5% ( 3/8 ) occurred with 2 weeks of sfr . this shows that counseling needs to be done in weeks following sfr in comparison to those who have undergone mfpr . in the mfpr group , only 2 ( 4.35% ) patients out of 46 underwent loss of remaining fetuses within 2 weeks of reduction as against 8.8% ( 3/34 ) in the sfr group . , 14.4% ( 13/90 ) patients undergoing mfpr from triplet to twins had fetal losses within 1 week ( 7.7% ) and rest within 4 weeks . in the study by dickey et al . , the average gestation of patients with sfr was shortened by 4 days in comparison to unreduced twin gestation . although the comparison of gestational age of delivery of reduced and unreduced pregnancies is beyond the scope of the present study , it is expected that in twin gestations delivered after sfr , the patients delivered earlier than unreduced twin pregnancies . the average gestational age at delivery in our study is about 30 weeks , which is much lower than that of dickey et al . ( 35 weeks ) . this may be secondary to patient factors and lower degree of maternal care in some of our cases who did not have access to higher centers . , in their study , showed that triplet gestation undergoing sfr in the first trimester had more propensity of delivering earlier than their twin or singleton counterparts . our study has compared the risk of preterm birth in spontaneous versus reduced multifetal gestation . this study shows that in comparison to sfr , mfpr offers a higher chance of delivering at later gestation . it is important to point out that the average gestation in both cases remains in the preterm range and therefore practically neither of the two procedures offers a chance to prevent preterm birth . the sfr group had earlier delivery probably because the sfr occurred due to certain maternal and/or uterine factors , which eventually led to preterm delivery too . however , determination of the reason for lower average gestation in sfr group in comparison to mfpr requires further studies . in an indian prospective study , this is very similar to our own study , wherein 44.2 ( 29/65 ) patients delivered at term even though the average gestational age of the entire cohort was 32.14 weeks . of the two groups even though the average gestational age of sfr group at delivery is lesser , 53.84% ( 14/26 ) delivered at or after 37 weeks in comparison to 38.46% ( 14/39 ) in the mfpr group . however , this difference in the term delivery rate in the two groups is not statistically different . in the above - mentioned study , the mean gestational age at delivery was 35.4 weeks which is a little higher than our own ( 32 weeks ) . the sample size in the former is much lesser than our own and could lead to a difference in the result . some studies have reported that sfr improved neonatal prognosis while others have found conflicting results . in these studies , the authors have compared the perinatal outcomes after reduction with pregnancies where no reduction was required ( singleton ) or was not done ( twins ) . it depends on the gestational age of fetal reduction , maternal factors influencing pregnancy , as well as number of gestational sacs , to begin with . our study shows that while these factors indeed may alter neonatal prognosis , effect of nature of fetal reduction ( sfr or mfpr ) is not a clinically significant factor , especially if triplet to twin reduction is considered . in our study , triplets ' pregnancy underwent mfpr to twins if sfr did not occur by 12 weeks . therefore , the two groups formed ( sfr and mfpr ) were mutually exclusive . in the study by zhang et al . this study shows that in patients with only mfpr , the perinatal outcome was significantly better than those in sfr group . this may be because loss of fetuses in sfr group was secondary to mfpr itself or some other confounding factor in this group . these results may be difficult to compare with our own study due to the difference in design ; however , as neonatal prognosis is inversely related to gestational age at reduction , waiting for sfr in advanced gestation ( > 12 weeks ) may not be beneficial to the patient . at the same time , as most sfr occurred within 810 weeks of gestation , it is appropriate to wait for sfr before planning for mfpr for the patient . it is reported that sfr rate is 12%30% in art cycles and even as high as 50%80% in triplet or higher order gestation . association of sfr with increasing maternal age was found in these studies as well . in the present study , 34 out of eighty triplet pregnancies ( 42.5% ) underwent sfr . although the percentage of sfr in previous studies is similar to our own , we did not find any difference or association with maternal age in both groups because average maternal age in our study was less ( 32.76 years ) . in a large study by zhang et al . therefore , it can be postulated that multiple gestational sacs in it predispose to the loss of one or more fetuses in utero . the possible reasons may be small uterine space and the relative lack of blood supply of the gestation sac caused by multiple pregnancies . zhang et al . have found that sfr occurred within 8 weeks in 78.4% of cases . in patients who underwent sfr later in their gestation , they found an increasing trend of low birth weight and very low birth weight rates . therefore , we believe that although our study does not show any significant difference in the perinatal outcome between sfr and mfpr groups , waiting for sfr in advanced gestation ( > 12 weeks ) may not be beneficial to the patient because patients in mfpr group were selected for reduction by discordant growth , increased nuchal translucency as already mentioned in the materials and methods . in an observational study by papageorghiou et al . , the rate of miscarriage ( pregnancy loss before 24 weeks ) was 8.3% , while the rate of early preterm delivery was 9.7% . the rate of abortion is lower to our own ( 15.2% ) as the period of viability considered by us is 28 weeks as against 24 weeks in the above - mentioned study . our percentage of abortion is higher than some previous studies as well . as ours is a retrospective analysis and the follow - up of patients beyond 12 weeks was not done at our own center in all cases , the reasons for such high abortion rates are not clear . it is possible that some of our patients were not under expert medical care after the second trimester and therefore were not as closely monitored as was required . our study shows that loss of fetus occurred after mfpr occurred in 1 patient within 2 weeks . however , in over 80% cases , interval between mfpr and miscarriage was more than 2 weeks . it is , therefore , important to counsel the couple who choose to have a fetal reduction that most of the excess loss with mfpr occurs several weeks after the procedure and is likely to be the consequence of the resorbing dead fetoplacental tissue , rather than the technique itself . when we look into the pattern of miscarriage in the sfr group , we found that miscarriage of the remaining fetuses in 37.5% ( 3/8 ) occurred with 2 weeks of sfr . this shows that counseling needs to be done in weeks following sfr in comparison to those who have undergone mfpr . in the mfpr group , only 2 ( 4.35% ) patients out of 46 underwent loss of remaining fetuses within 2 weeks of reduction as against 8.8% ( 3/34 ) in the sfr group . in the study by zhang et al . , 14.4% ( 13/90 ) patients undergoing mfpr from triplet to twins had fetal losses within 1 week ( 7.7% ) and rest within 4 weeks . in the study by dickey et al . , the average gestation of patients with sfr was shortened by 4 days in comparison to unreduced twin gestation . although the comparison of gestational age of delivery of reduced and unreduced pregnancies is beyond the scope of the present study , it is expected that in twin gestations delivered after sfr , the patients delivered earlier than unreduced twin pregnancies . the average gestational age at delivery in our study is about 30 weeks , which is much lower than that of dickey et al . ( 35 weeks ) . this may be secondary to patient factors and lower degree of maternal care in some of our cases who did not have access to higher centers . dickey et al . , in their study , showed that triplet gestation undergoing sfr in the first trimester had more propensity of delivering earlier than their twin or singleton counterparts . our study has compared the risk of preterm birth in spontaneous versus reduced multifetal gestation . this study shows that in comparison to sfr , mfpr offers a higher chance of delivering at later gestation . it is important to point out that the average gestation in both cases remains in the preterm range and therefore practically neither of the two procedures offers a chance to prevent preterm birth . the sfr group had earlier delivery probably because the sfr occurred due to certain maternal and/or uterine factors , which eventually led to preterm delivery too . however , determination of the reason for lower average gestation in sfr group in comparison to mfpr requires further studies . in an indian prospective study , 45% patients ( 5/12 ) carried the pregnancy to term . this is very similar to our own study , wherein 44.2 ( 29/65 ) patients delivered at term even though the average gestational age of the entire cohort was 32.14 weeks . of the two groups even though the average gestational age of sfr group at delivery is lesser , 53.84% ( 14/26 ) delivered at or after 37 weeks in comparison to 38.46% ( 14/39 ) in the mfpr group . however , this difference in the term delivery rate in the two groups is not statistically different . in the above - mentioned study , the mean gestational age at delivery was 35.4 weeks which is a little higher than our own ( 32 weeks ) . the sample size in the former is much lesser than our own and could lead to a difference in the result . some studies have reported that sfr improved neonatal prognosis while others have found conflicting results . in these studies , the authors have compared the perinatal outcomes after reduction with pregnancies where no reduction was required ( singleton ) or was not done ( twins ) . it depends on the gestational age of fetal reduction , maternal factors influencing pregnancy , as well as number of gestational sacs , to begin with . our study shows that while these factors indeed may alter neonatal prognosis , effect of nature of fetal reduction ( sfr or mfpr ) is not a clinically significant factor , especially if triplet to twin reduction is considered . in our study , triplets ' pregnancy underwent mfpr to twins if sfr did not occur by 12 weeks . therefore , the two groups formed ( sfr and mfpr ) were mutually exclusive . in the study by zhang et al . this study shows that in patients with only mfpr , the perinatal outcome was significantly better than those in sfr group . this may be because loss of fetuses in sfr group was secondary to mfpr itself or some other confounding factor in this group . these results may be difficult to compare with our own study due to the difference in design ; however , as neonatal prognosis is inversely related to gestational age at reduction , waiting for sfr in advanced gestation ( > 12 weeks ) may not be beneficial to the patient . at the same time , as most sfr occurred within 810 weeks of gestation , it is appropriate to wait for sfr before planning for mfpr for the patient . mfppr have a better prognosis than sfr in terms of gestational age at delivery , fetal birth weight as well as abortion rates although the differences are not clinically significant . as neither strategy offers a benefit when compared to singleton pregnancies or unreduced pregnancies as per previous studies , the primary aim of any art should be to reduce multiple embryo transfer in the first place .

introduction : with the advent of assisted reproductive treatment options , the incidence of multiple pregnancies has increased . although the need for elective single embryo transfer is emphasized time and again , its uniform applicability in practice is yet a distant goal . in view of the fact that triplet and higher order pregnancies are associated with significant fetomaternal complications , the fetal reduction is a commonly used option in such cases . this retrospective study aims to compare the perinatal outcome in patients with triplet gestation who have undergone spontaneous fetal reduction ( sfr ) as against those in whom multifetal pregnancy reduction ( mfpr ) was done.materials and methods : in the present study , eighty patients with triplet gestation at 6 weeks were considered . the patients underwent sfr or mfpr at or before 1213 weeks and were divided into two groups ( 34 and 46 ) , respectively.results:our study found no statistical difference in perinatal outcome between the sfr and mfpr groups in terms of average gestational age at delivery , abortion rate , preterm delivery rate , and birth weight . the study shows that the risk of aborting all fetuses after sfr is three times ( odds ratio [ or ] = 3.600 , 95% confidence interval [ ci ] = 0.279446.388 ) that of mfpr in subsequent 2 weeks . there were more chances of loss of extra fetus in sfr ( 23.5% ) group than mfpr group ( 8.7% ) ( or = 3.889 , 95% ci = 1.03014.680 ) . as neither group offers any significant benefit from preterm delivery , multiple pregnancies continue to be responsible for preterm delivery despite fetal reduction.conclusion:there appears to be some advantages of mfpr in perinatal outcome when compared to sfr , especially if the latter happens at advanced gestation . therefore , although it is advisable to wait for sfr to occur , in patients with triplet gestation at 1112 weeks , mfpr is a viable option to be considered .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION Spontaneous fetal reduction and multifetal pregnancy reduction Risk of abortion after spontaneous fetal reduction and multifetal pregnancy reduction Chronology of abortion in spontaneous fetal reduction and multifetal pregnancy reduction Gestational age at delivery Preterm delivery in spontaneous fetal reduction and multifetal pregnancy reduction Term delivery in spontaneous fetal reduction and multifetal pregnancy reduction Effect of fetal reduction on neonatal outcomes Can multifetal pregnancy reduction be avoided in favor of spontaneous fetal reduction? CONCLUSION Financial support and sponsorship Conflicts of interest

the last decade has seen an exponential rise in the availability and accessibility of assisted reproductive treatment opportunities in the indian subcontinent . couple 's desires to achieve maximum chances of conceiving as well as the option of fetal reduction are important reasons for multiple embryo transfer in an assisted reproductive technique ( art ) cycle . balancing benefit ( increased pregnancy rate ) against the disadvantage of multiple embryo transfer is not easy as both the clinician and the couple very easily appreciate the benefit , whereas the disadvantages ( pregnancy loss , preterm delivery , maternal and/or neonatal morbidity / mortality ) are more distant consequences . therefore , in the absence of any standardized legislature to guide the number of embryos transferred after an art procedure , multiple pregnancies have become one of the most important outcomes to be considered in an art conception . the risk of pregnancy loss and preterm delivery leading to neonatal morbidity and mortality increases in the presence of multiple pregnancy , especially higher order gestation like triplet or more . maternal complications for women carrying high - order multiple gestations include increased incidence of preeclampsia , cesarean delivery , postpartum hemorrhage , and fatty liver . many studies in the past have evaluated the risk of continuation of triplet conception versus reduction to twin or singleton pregnancy to improve perinatal outcomes . while some studies have found an increase in gestational age at delivery in triplet pregnancy after fetal reduction , some have not found any significant advantage . the role of multifetal pregnancy reduction ( mfpr ) is more clearly advantageous in quadruplets or higher order gestation . a fetal loss of 8%16% has been reported in literature after mfpr although this incidence depends not only on operator 's experience but also on gestational age at reduction , number of fetuses reduced , patient factors , etc . in many cases , spontaneous fetal reduction ( sfr ) is seen , wherein one or several embryos naturally disappear . pregnancies undergoing sfr have found to be at higher risk of preterm delivery and poorer neonatal outcomes , especially when they were reduced to singleton from twin and more so when reduced to a twin from triplets . this retrospective analysis was performed in the department of reproductive medicine and surgery from january 2012 to december 2015 . details of any pregnancy conceived after art in vitro fertilization ( ivf ) or intracytoplasmic sperm injection ( icsi ) was recorded in the departmental record in the form of an individual patient file which is periodically updated either at the time of patient follow - up visit or telephonic enquiry . records of patients who had conceived triplet gestation after ivf / icsi and had delivered were evaluated for the purpose of this study , and age , parity , number of embryo transferred , calculated last menstrual period , number of gestational sacs , antenatal course , pregnancy outcome , date of delivery , birth weight , and neonatal course were collected from them . the patients with three gestational sacs ( trichorionic triamniotic ) with cardiac activity seen at 6 weeks of gestation on transvaginal scan were considered for the study . patients , who were lost to follow - up , had heterotopic pregnancies , had previous pregnancy losses , or continued with triplet or higher order gestation beyond 12 weeks and/or whose records were incomplete were excluded from the study . as per departmental protocol , ultrasonography ( usg ) was initially performed to determine the location and number of gestational sacs when quantitative beta - human chorionic gonadotropin levels were expected to be 2000 miu / ml or more between 5.5 and 6.5 gestational weeks ( 3.54.5 weeks after embryo transfer ) . for the purpose of the study , sfr was defined as disappearance of gestational sac or loss of cardiac activity in one or two gestational sacs ( after its identification ) out of the triplet pregnancies . no cases of quadruplet or higher order gestation were noted / considered in the study population . in the mfpr group , triplet gestation reduced to singleton was not considered for the study to reduce confounding factors . preterm delivery was defined as the birth of a viable baby ( after 28 weeks ) at or before 37 completed weeks of gestation . restricted fetal growth or intrauterine growth restriction ( iugr ) was defined as a birth weight less than the 10 percentile for gestational age on the basis of national singleton birth weights . eighty pregnancies with triplet gestation at 6 weeks scan fulfilled the inclusion criteria and underwent spontaneous or mfpr before 1213 weeks of gestation . the patients were divided into two groups sfr and mfpr according to the reduction method . secondary outcome measures were live birth rate , average birth weight , and preterm birth rate . the outcome variables studied in the present study were pregnancy loss , weeks of gestation at delivery , birth weight of the baby , incidence of iugr , incidence of preterm labor , and incidence of single and twin delivery . the average age of patients in the present study was 32.76 years . of 80 patients considered for the study , 34 had sfr , whereas 46 underwent mfpr as per protocol . of eighty triplet pregnancies considered in the present study , a total of 15 patients aborted before the period of viability ( 28 weeks ) . the difference in probability of abortion in both groups was not statistically significant ( p = 0.3942 ) , even if only early abortions are considered ( gestational age < 14 weeks ) . the sfr group had higher risk ( odds ratio [ or ] = 3.600 , 95% confidence interval [ ci ] = 0.279446.388 ) of loss of all the fetuses over the next 2 weeks ( < 14 weeks ) than mfpr groups ( 37.5% vs. 14.3% , respectively ) . twenty - six patients out of 34 ( 76.5% ) in the sfr group had live births in comparison to 84.78% ( 39/46 ) in the mfpr group . although the percentage of live birth was higher in patients who had mfpr , it was not statistically significant [ table 1 ] . perinatal outcome in two groups there were more chances of loss of extra fetus in sfr ( 23.5% ) group than mfpr group ( 8.7% ) ( or = 3.889 , 95% ci = 1.03014.680 , p = 0.0781 ) . the statistical probability of having a term delivery , preterm or very preterm delivery did not differ in both groups statistically ( p = 0.297 ) . after sfr or mfpr , a total of 118 babies were born to eighty patients either as singleton or twins ( 106 ) . however , no association of birth weight was observed with the type of fetal reduction ( sfr and mfpr group ) ( p = 0.364 ) [ table 1 ] . the average gestational age in the sfr group was lower in comparison to the mfpr group , but this does not reach clinical significance ( p = 0.2025 ) [ table 1 ] . the incidence of pregnancy - induced hypertension was more in sfr than mfpr group ; however , the difference was statistically insignificant . mfpr group had 74 babies with an average birth weight of 2.03 0.49 kg . although birth weight was higher than sfr group ( 1.98 0.61 kg ) , the difference does not reach clinical significance . no significant difference was observed in terms of incidence of iugr , congenital abnormalities , and neonatal death in two groups [ table 1 ] . of the three congenital anomalies , there were two fetuses with cardiac anomalies ( one in each group ) and one fetus had an absence of bilateral toe in mfpr group . of the ten neonatal deaths , seven were early neonatal death , one was late neonatal death , and two were immediate neonatal deaths . on visualization of triplet sacs , the patient is counseled regarding the pros and cons of continuation of triplet pregnancies as against fetal reduction . however , at times before the reduction is scheduled ( generally after 1112 weeks ) , spontaneous loss of one or more fetuses is seen . this study aims to compare the perinatal outcome in cases where fetal reduction occurred spontaneously as against wherein mpfr was performed . most of the studies done in the past have compared the perinatal outcomes in reduced and nonreduced pregnancies . this study is relevant not only to understand the implications of sfr for counseling patients but also to decide whether it is prudent to wait for 12 weeks or more before mfpr was done , so as to allow for sfr . it is reported that sfr rate is 12%30% in art cycles and even as high as 50%80% in triplet or higher order gestation . in the present study , although the percentage of sfr in previous studies is similar to our own , we did not find any difference or association with maternal age in both groups because average maternal age in our study was less ( 32.76 years ) . the possible reasons may be small uterine space and the relative lack of blood supply of the gestation sac caused by multiple pregnancies . in patients who underwent sfr later in their gestation , they found an increasing trend of low birth weight and very low birth weight rates . therefore , we believe that although our study does not show any significant difference in the perinatal outcome between sfr and mfpr groups , waiting for sfr in advanced gestation ( > 12 weeks ) may not be beneficial to the patient because patients in mfpr group were selected for reduction by discordant growth , increased nuchal translucency as already mentioned in the materials and methods . , the rate of miscarriage ( pregnancy loss before 24 weeks ) was 8.3% , while the rate of early preterm delivery was 9.7% . the rate of abortion is lower to our own ( 15.2% ) as the period of viability considered by us is 28 weeks as against 24 weeks in the above - mentioned study . our study shows that loss of fetus occurred after mfpr occurred in 1 patient within 2 weeks . however , in over 80% cases , interval between mfpr and miscarriage was more than 2 weeks . it is , therefore , important to counsel the couple who choose to have a fetal reduction that most of the excess loss with mfpr occurs several weeks after the procedure and is likely to be the consequence of the resorbing dead fetoplacental tissue , rather than the technique itself . when we look into the pattern of miscarriage in the sfr group , we found that miscarriage of the remaining fetuses in 37.5% ( 3/8 ) occurred with 2 weeks of sfr . this shows that counseling needs to be done in weeks following sfr in comparison to those who have undergone mfpr . in the mfpr group , only 2 ( 4.35% ) patients out of 46 underwent loss of remaining fetuses within 2 weeks of reduction as against 8.8% ( 3/34 ) in the sfr group . in the study by dickey et al . , the average gestation of patients with sfr was shortened by 4 days in comparison to unreduced twin gestation . although the comparison of gestational age of delivery of reduced and unreduced pregnancies is beyond the scope of the present study , it is expected that in twin gestations delivered after sfr , the patients delivered earlier than unreduced twin pregnancies . the average gestational age at delivery in our study is about 30 weeks , which is much lower than that of dickey et al . , in their study , showed that triplet gestation undergoing sfr in the first trimester had more propensity of delivering earlier than their twin or singleton counterparts . our study has compared the risk of preterm birth in spontaneous versus reduced multifetal gestation . this study shows that in comparison to sfr , mfpr offers a higher chance of delivering at later gestation . it is important to point out that the average gestation in both cases remains in the preterm range and therefore practically neither of the two procedures offers a chance to prevent preterm birth . in an indian prospective study , this is very similar to our own study , wherein 44.2 ( 29/65 ) patients delivered at term even though the average gestational age of the entire cohort was 32.14 weeks . of the two groups even though the average gestational age of sfr group at delivery is lesser , 53.84% ( 14/26 ) delivered at or after 37 weeks in comparison to 38.46% ( 14/39 ) in the mfpr group . however , this difference in the term delivery rate in the two groups is not statistically different . in the above - mentioned study , the mean gestational age at delivery was 35.4 weeks which is a little higher than our own ( 32 weeks ) . the sample size in the former is much lesser than our own and could lead to a difference in the result . it depends on the gestational age of fetal reduction , maternal factors influencing pregnancy , as well as number of gestational sacs , to begin with . our study shows that while these factors indeed may alter neonatal prognosis , effect of nature of fetal reduction ( sfr or mfpr ) is not a clinically significant factor , especially if triplet to twin reduction is considered . therefore , the two groups formed ( sfr and mfpr ) were mutually exclusive . this study shows that in patients with only mfpr , the perinatal outcome was significantly better than those in sfr group . this may be because loss of fetuses in sfr group was secondary to mfpr itself or some other confounding factor in this group . these results may be difficult to compare with our own study due to the difference in design ; however , as neonatal prognosis is inversely related to gestational age at reduction , waiting for sfr in advanced gestation ( > 12 weeks ) may not be beneficial to the patient . at the same time , as most sfr occurred within 810 weeks of gestation , it is appropriate to wait for sfr before planning for mfpr for the patient . it is reported that sfr rate is 12%30% in art cycles and even as high as 50%80% in triplet or higher order gestation . in the present study , 34 out of eighty triplet pregnancies ( 42.5% ) underwent sfr . the possible reasons may be small uterine space and the relative lack of blood supply of the gestation sac caused by multiple pregnancies . in patients who underwent sfr later in their gestation , they found an increasing trend of low birth weight and very low birth weight rates . therefore , we believe that although our study does not show any significant difference in the perinatal outcome between sfr and mfpr groups , waiting for sfr in advanced gestation ( > 12 weeks ) may not be beneficial to the patient because patients in mfpr group were selected for reduction by discordant growth , increased nuchal translucency as already mentioned in the materials and methods . , the rate of miscarriage ( pregnancy loss before 24 weeks ) was 8.3% , while the rate of early preterm delivery was 9.7% . the rate of abortion is lower to our own ( 15.2% ) as the period of viability considered by us is 28 weeks as against 24 weeks in the above - mentioned study . our study shows that loss of fetus occurred after mfpr occurred in 1 patient within 2 weeks . it is , therefore , important to counsel the couple who choose to have a fetal reduction that most of the excess loss with mfpr occurs several weeks after the procedure and is likely to be the consequence of the resorbing dead fetoplacental tissue , rather than the technique itself . when we look into the pattern of miscarriage in the sfr group , we found that miscarriage of the remaining fetuses in 37.5% ( 3/8 ) occurred with 2 weeks of sfr . this shows that counseling needs to be done in weeks following sfr in comparison to those who have undergone mfpr . in the mfpr group , only 2 ( 4.35% ) patients out of 46 underwent loss of remaining fetuses within 2 weeks of reduction as against 8.8% ( 3/34 ) in the sfr group . in the study by zhang et al . , the average gestation of patients with sfr was shortened by 4 days in comparison to unreduced twin gestation . although the comparison of gestational age of delivery of reduced and unreduced pregnancies is beyond the scope of the present study , it is expected that in twin gestations delivered after sfr , the patients delivered earlier than unreduced twin pregnancies . the average gestational age at delivery in our study is about 30 weeks , which is much lower than that of dickey et al . , in their study , showed that triplet gestation undergoing sfr in the first trimester had more propensity of delivering earlier than their twin or singleton counterparts . this study shows that in comparison to sfr , mfpr offers a higher chance of delivering at later gestation . it is important to point out that the average gestation in both cases remains in the preterm range and therefore practically neither of the two procedures offers a chance to prevent preterm birth . however , determination of the reason for lower average gestation in sfr group in comparison to mfpr requires further studies . this is very similar to our own study , wherein 44.2 ( 29/65 ) patients delivered at term even though the average gestational age of the entire cohort was 32.14 weeks . of the two groups even though the average gestational age of sfr group at delivery is lesser , 53.84% ( 14/26 ) delivered at or after 37 weeks in comparison to 38.46% ( 14/39 ) in the mfpr group . however , this difference in the term delivery rate in the two groups is not statistically different . in the above - mentioned study , the mean gestational age at delivery was 35.4 weeks which is a little higher than our own ( 32 weeks ) . the sample size in the former is much lesser than our own and could lead to a difference in the result . in these studies , the authors have compared the perinatal outcomes after reduction with pregnancies where no reduction was required ( singleton ) or was not done ( twins ) . it depends on the gestational age of fetal reduction , maternal factors influencing pregnancy , as well as number of gestational sacs , to begin with . our study shows that while these factors indeed may alter neonatal prognosis , effect of nature of fetal reduction ( sfr or mfpr ) is not a clinically significant factor , especially if triplet to twin reduction is considered . therefore , the two groups formed ( sfr and mfpr ) were mutually exclusive . in the study by zhang et al . this study shows that in patients with only mfpr , the perinatal outcome was significantly better than those in sfr group . this may be because loss of fetuses in sfr group was secondary to mfpr itself or some other confounding factor in this group . these results may be difficult to compare with our own study due to the difference in design ; however , as neonatal prognosis is inversely related to gestational age at reduction , waiting for sfr in advanced gestation ( > 12 weeks ) may not be beneficial to the patient . at the same time , as most sfr occurred within 810 weeks of gestation , it is appropriate to wait for sfr before planning for mfpr for the patient . mfppr have a better prognosis than sfr in terms of gestational age at delivery , fetal birth weight as well as abortion rates although the differences are not clinically significant . as neither strategy offers a benefit when compared to singleton pregnancies or unreduced pregnancies as per previous studies , the primary aim of any art should be to reduce multiple embryo transfer in the first place .

determining whether an instrument is operating within acceptable performance metrics is an essential step during scientific investigation . a variety of methods are commonly used in liquid chromatography mass spectrometry ( lc ms ) , with many groups routinely utilizing a common sample which is regularly analyzed , for example , weekly . samples range from simple proteins and mixtures to whole cell - lysates . in addition to regularly running a quality control ( qc ) sample , recent research has investigated analysis methods and metrics for assessing data quality . rudnick et al . defined 46 nist metrics to quantify the performance of various lc ms aspects , such as chromatography , ion source , dynamic sampling , and peptide identifications . these metrics are largely dependent on ms / ms data and their subsequent peptide identifications . the quameter software package proposed an additional set of metrics , which are not dependent on ms / ms identifications . the nist and quameter efforts focused on generating a comprehensive set of metrics for monitoring the various aspects of system performance . when employing the metrics to monitor the day - to - day operation of an lc ms system , it is important to note that some metrics are heavily dependent on the specific instrument and configuration . what is an acceptable value for one setup may be wholly unacceptable for another . thus , a critical shortcoming of current metric sets is that they produce an array of values and leave interpretation to the user . specifically , there is no guidance as to when the performance of the lc given the large number of metrics available , routine interpretation of the metrics and quality assurance is typically determined via a subjective weighting of a subset of the metrics . the specific subset of metrics , and associated acceptable values , varies from lab to lab and among individual operators . as pointed out by tabb decision support tools for the interpretation of metrics derived from data . a variety of statistical methods may be used to explore and model how the multivariate metrics predict data quality , including principal component analysis , classification and regression and model selection techniques . leveraging a large corpus of manually curated qc data , we developed a lasso logistic regression classifier ( llrc ) that predicts , with high sensitivity and specificity , whether a qc data set is in or out of control . this signature , which is a composite of lc ms performance metrics , is more robust than any single metric itself . as performance can be viewed as a continuum , the llrc model natively computes a quality score within the range 01 . the trained model then identifies a cutoff for the dichotomous classification that achieves the highest sensitivity and specificity . an important feature of the llrc is that it differentiates between false positive and false negative errors . therefore , the penalties associated with these errors are separately defined , which allows the classifier to be more responsive in the balance between sensitivity and specificity . finally , we present the software used to create the classifier , so that other laboratories can create a classifier specific to their qc regimen , instrumentation , and needs . a shewanella oneidensis mr-1 lysate digest is used as a quality control sample in our laboratory to provide sufficient proteomic complexity to assess both lc and ms performance . the cultures ( 7 l ) were grown in fed - batch mode using a bioflow 3000 model fermentor ( new brunswick , inc . ) and allowed to achieve steady state before sampling and harvesting . the medium was hba mr-1 with 0.5 ml / l of 100 mm ferric nta , 1 ml / l of 1 mm na2seo4 , and 1 ml / l of 3 m mgcl26h2o as well as vitamins , minerals , and amino acids . the cultures used o2 as the terminal electron acceptor from a house air source ( 5 l / min flow rate ) and were maintained at 20% dissolved oxygen ( do ) . other monitored parameters included maintaining a ph of 7.00 ( 0.03 ) , a constant agitation of the culture at 5000 rpm , and temperature of 30 c . these samples were pelleted ( 11900 g for 8 min at 4 c ) and frozen at 80 c until processing for proteomic analysis . enough material was prepared in this manner to provide qc samples for the last four years . the majority of the culture is still waiting to be used . cells were then lysed by homogenizing the cells with 0.1 mm zirconia / silica beads in the bullet blender ( next advance , averill park ny ) speed 8 for 3 min . samples were then immediately placed on ice to inhibit proteolysis and then transferred , and the beads were rinsed with 100 l of 100 mm na4hco3 and 1 ml of 100 mm ammonium bicarbonate . a bca protein assay ( thermo scientific , san jose ca ) was performed to determine concentration . samples were denatured by adding urea to 7 m and reduced by dithiothreitol ( dtt ) to a concentration of 5 mm . the samples were then incubated for 30 min at 60 c , and then diluted 10-fold with 100 mm na4hco3 . next , trypsin ( affymetrix , santa clara ca ) was added in a ratio 1:50 trypsin / protein and incubated at 37 c for 3 h. samples were desalted by 100 mg dsc-18 columns ( sigma aldrich ) . each column was conditioned with 3 ml of meoh and rinsed with 2 ml of 0.1% tfa in h2o . peptides were then loaded on the resin and washed with 4 ml of 95:5 h2o / acn with 0.1% tfa . collected sample was concentrated via speedvac ( thermo scientific , san jose ca ) , and then the samples were transferred to ultracentrifuge tubes and ultracentrifuged at 100 000 rpm for 10 min at 4 c , and the supernatant was drawn off and pooled . final concentration was determined by peptide bca ( thermo scientific , san jose ca ) . samples were brought to a concentration of 0.5 g/l with h2o ( millipore , billerica ma ) and divided into aliquots for injections on the hplc . the hplcs used to run the samples were built in - house utilizing various commercial pumps , valves , and auto samplers , all of which were coordinated by a custom software packaged called lcmsnet . the data sets analyzed for this paper were run using lc columns that were 75 m inner diameter , and either 30 or 65 cm in length . these lc columns were packed in house with phenomenex jupiter c18 3 m porous beads . both 60 and 100 min acquisitions were used . mobile phase a is 0.1% formic acid in h2o and mobile phase b is 0.1% formic acid in acetonitrile . the 100 min gradient was delivered by starting at 5% mobile phase b and advancing to 8% , 12% , 35% , 60% , and 75% at times ( in minutes ) 2 , 20 , 75 , 97 , 100 respectively . typically 2.5 g of shewanella digest was loaded to the head of the column or to a trapping column . although operating conditions varied by capabilities of each instrument , typical conditions for each are as follows . the ltq was run in data - dependent ms - ms mode , selecting the top 10 parent ions from each survey scan . the exactive runs were high resolution ms only with the target resolution set to 100 000 . the ltq - orbitrap and the velos - orbitrap instruments were typically set to have a high resolution survey scan of 60 000 resolution followed by the top 6 or 10 data - dependent ms ms scans , respectively . because of the diversity of data sets used in this study , this is not a comprehensive list of conditions . data are presented from four classes of instruments from thermo scientific : the ltq linear ion trap , exactive , ltq - orbitrap , and velos orbitrap platforms . there are 1150 testing and training data sets : 224 on ltq instruments , 85 on exactive instruments , 380 on ltq - orbitrap instruments , and 461 on velos - orbitrap instruments ( see table 1 ) . for each qc data set , the nist and quameter metrics were calculated exactly as described in the original publications . the quameter metrics were calculated using the software from the tabb group ( http://fenchurch.mc.vanderbilt.edu/software.php ) ; the nist metrics were calculated using in - house software , smaqc , which is posted on our github repository ( https://github.com/pnnl-comp-mass-spec ) . for data from the exactive instrument class , no ms - ms data were collected , and therefore any metrics relating to ms / ms data were omitted . peptide identifications were performed with msgf+ ( version v9593 , 05/06/2013 ) . the protein sequence database was the shewanella oneidensis mr1 proteome supplemented with trypsin and keratin sequences . relevant search parameters were tryptic specificity ( semi tryptic allowed ) , no static modifications , dynamic methionine oxidation . high resolution instruments used a 20 ppm tolerance , low resolution data used 2.5 da . all of the data has been deposited to the proteomexchange consortium ( http://proteomecentral.proteomexchange.org ) via the pride partner repository with the data set identifiers pxd000320 , pxd000321 , pxd000322 , pxd000323 , and pxd000324 . this includes the instrument .raw files , the ms - gf+ peptide identifications in .mzidentml format , .mgf converted spectra files , and the spreadsheet containing the metrics for every data set . the data sets were manually reviewed by three expert instrument operators ( 30 + years of combined lc this viewer contained the base peak chromatogram , total ion current chromatogram , plots of both the top 50 000 and top 500 000 lc ms detected features , and the number of peptides identified . in the first round , 1150 data sets were manually curated as good , okay , or poor and used to develop the classifier . in cases where the assessors disagreed ( 510% ) , moreover , the okay value was used to denote the wide range of performance , which , although not optimal , was still acceptable . for the validation , an additional 1321 data sets classified with the statistical model from which a subset of 100 data sets were chosen for manual curation ( supplemental files 1 and 2 , supporting information ) . data quality assessment requires knowledge of the conditions under which the qc was run to properly account for the variety of run parameters , for example , high resolution ms only , high resolution ms low resolution ms / ms , high resolution ms high resolution ms / ms , and low resolution ms low resolution ms / ms . data - dependent acquisition regimes varied from ms / ms of top 3 , top 6 , and top 10 most intense peaks from the survey ms scan . fragmentation methods included high energy collisional dissociation ( hcd ) and collision induced dissociation ( cid ) . resolution ranged from 1000 to 100 000 based on instrument capabilities and settings to meet the needs of ongoing experiments . run times were either 60 or 100 min , and lc column lengths varied accordingly . each of these criteria is considered to determine what constitutes an acceptable ( in control ) data set . for example , under identical mass spectrometer conditions , an acceptable 60 min hplc run would provide results that would be unacceptable ( out of control ) for a 100 min run . multivariate statistical techniques were applied to the data to identify which metrics might be useful to understand the quality of a given data set and to develop a model to predict the quality of future data sets . principal component analysis ( pca ) was applied on the nist and quameter metrics , 87 in all , using the pca package in the r statistical programming language . we applied a few of them , including classification and regression tree algorithms ( cart ) , linear discriminant analysis ( lda ) , and logistic regression augmented by a classification threshold . the most effective was logistic regression , an approach for predicting a binary response using a linear combination of continuous and/or categorical predictor variables . the goal for the classifier is to identify data sets that are out of control . to that end , the binary response was coded as a 0 for a data set that was in control ( annotated as good or 1 for a data set that was out of control ( annotated as poor ) . the linear component of the logistic regression model can be expressed aswhere i , ( i = 1 , ... , n ) indexes the data sets , 0 is the intercept , 1 is a vector of coefficients ( one for each of the nist and quameter quality metrics included in the model ) , and xi is a vector representing the quality metrics included in the model . the probability of the binomial response is modeled by the logistic function : where ( xi ) estimates the probability of data set i being out of control . an important step in any regression model is determining the set of variables that best predict the response . we used the lasso approach , a model selection technique that accounts for collinearity among the predictors while selecting a subset of the predictor variables that results in the best regression model . in this case , best is defined as the subset of variables whose coefficients maximize a penalized log - likelihood function ( see appendix ) . the inputs to the lasso logistic regression model include the complete set of possible quality metrics , the binary expert annotations ( 0 = good or , 1 = poor ) , and , a regularization parameter that simultaneously restricts the size of model coefficients and the number of quality metrics included in the model . we developed a separate lasso logistic regression model for each of the four instruments types . we used a threshold parameter , , to make a definitive prediction as to whether a data set was good or poor . specifically , we classify data set i as out of control if (xi ) > and in control otherwise . we will refer to the combination of the logistic regression and the threshold , , as the lasso logistic regression classifier ( llrc ) . when predicting the quality of a data set , the llrc will either predict a data set correctly , or will make either a false positive or false negative error . a false positive error occurs when the llrc predicts the data set to be out of control , when it was annotated as in control . the false negative error occurs when the llrc predicts the data set as being in control when it was annotated as out of control . the rates of these errors depend on the threshold criteria and are inversely related ( as one goes up , the other generally goes down ) . to this end , a loss function is constructed that reflects the consequences we attribute to the two types of errors . when the llrc predicts a data set correctly , zero loss occurs . when the llrc produces a false positive , a loss of 1 occurs , and a false negative receives a loss of 1 . hence , the consequences ( or cost ) of a false negative are times greater than those of a false positive . cross validation was employed to determine the optimal and that minimize the expected loss . cross validation was performed on the 1150 testing / training data sets by randomly dividing the data set into five equal parts . four parts were then combined to train the llrc which was subsequently used to predict the held - out part . this continues such that each part was predicted from a model trained by the other four parts . the r package used in the creation of the llrc , and a tutorial for its use is available for download at http://omics.pnl.gov/software/msdataqualitysignatures.php . one critical setting where subjective or time - consuming manual data analysis can lead to systemic problems is in the evaluation of quality control ( qc ) data sets . in an effort to automate the assessment of qc data sets , we selected a testing / training sample of 1150 data sets ( see table 1 ) and manually annotated them as being these data sets originated from four types of mass spectrometers and are all replicate analyses of the same whole - cell lysate of s. oneidensis ( see experimental procedures ) . in addition to the manual rating , all computational metrics from nist and quameter were calculated for each data set . initial analyses of the 1150 data sets using pca showed a separation by quality rating and also by instrument type ( figure 1 ) . the separation by quality rating indicates that statistical classification approaches that rely on multiple variables are likely to be successful , to some degree , in predicting data quality ( supplemental figure 1 , supporting information ) . each data point on the plot represents a single qc data set and is identified by its instrument class ( the symbol ) and its manually curated quality ( the color ) . the first two principal components explain about 40% of the variability in the original data . only the quameter data quality metrics could be calculated for exactive data , as it lacks ms2 fragmentation scans . the primary goal was to build a classifier that accurately predicts the data set quality , specifically when a data set is out of control ( poor ) . we utilized a lasso logistic regression classifier ( llrc ) which simultaneously restricts the size of model coefficients and the number of quality metrics included the model ( see experimental procedures ) . importantly , the model does not treat false negatives and false positives as equally deleterious events . the different impact of each error type is reflected in the practical implication of day - to - day instrument operation . while false positives incur extra work and inconvenience for operators to review and overturn the decision , the false - negative error is of much greater consequence ; if the classifier fails to predict an out of control data set , a problem in the lc ms system may not be discovered and poor quality data will continue to be produced . optimal parameter values were obtained by using cross validation to minimize a loss function that treated false negatives as five times worse than false positives . the resulting llrcs for each of the four instrument types are shown in table 2 . this reflects the continuum of performance of real lc ms systems in day - to - day operation . converting this value into the binary classification is accomplished by applying a threshold cutoff . figure 2 shows the sensitivity and specificity as the threshold value varies for each instrument platform . the dashed gray line on each plot in figure 2 indicates the optimal threshold that minimizes the loss function . mathematical and algorithmic details for obtaining the llrc are provided in the appendix . sensitivity and specificity trade - off . the 1150 curated data sets are shown according to their classification from the cross - validation results . we define sensitivity as the probability of correctly classifying an out of control ( or poor ) data set , equal to 1 minus the false negative rate . specificity is the proportion of good data sets that are correctly classified and is equal to 1 minus the false positive rate ( see experimental procedures ) . the dotted vertical line indicates the optimal threshold , , which balances the cost of a false positive or false negative classification error . the final step was to develop an llrc for predicting the quality of future data sets on each platform . these classifiers were created by training on all the available data using the optimal parameter values previously obtained . in developing the llrc , the lasso method selected between 2 and 12 quality metrics to be included in the llrc for each of the four instrument types . the llrc utilized a different number of metrics to classify data from each of the different instrument types . the number of metrics used seems to correlate with how well separated the data sets appeared in the pca analysis ( supplemental figure 1 , supporting information ) . exactive data , which required only two metrics , was the most distinctly separated by the first two principle components . in contrast , orbitrap data required 12 metrics and was visibly less clearly separated in the pca analysis . it is important to note that many of the nist and quameter quality metrics are highly correlated with each other . although lasso selected a certain subset of these metrics , it is likely there are other subsets that would have performed similarly had they been selected . consequently , one can not directly compare the subset of metrics chosen for one instrument class to another , and we do not claim that the metrics in table 3 constitute the best set . table 2 shows the sensitivity and specificity at the optimal threshold for each instrument platform . the sensitivity for each instrument category ranged from 100% for exactive to 89.4% for ltq - orbitrap . the specificity ranged from 97% for exactive to 84.6% for ltq ion trap ( which had a very high sensitivity of 97% ) . a value of that performs well with both sensitivity and specificity is preferable . however , if one measure is more important in a specific cost / benefit scenario , the threshold will adjust to reflect the trade - offs captured in the loss function . sensitivity was more desirable , and , therefore , resulted in a threshold with a larger discrepancy between sensitivity and specificity . moreover , the expected rates of false positives and false negative are implicitly considered as part of the optimization . the llrc is a composite classifier , in that it relies on multiple metrics to determine the quality of each data set . some instrument operators may rely only on a single metric to assess data quality . commonly used single metrics include : total tryptic peptide count ( p_2a ) , distinct tryptic peptide count ( p_2c ) , and the absolute intensities of peaks in the tic ( ms1_2b ) . in table 4 , we present a comparison of the llrc to these single measures of quality for velos - orbitrap data sets . the table illustrates the proportion of false positives and false negatives that would occur when using the llrc , as well as the four aforementioned metrics , each applied individually with their own threshold . as before , the loss function used to optimize the llrc parameters assumed that false negatives are 5 times worse than false positives , resulting in only 7.1% ( 24 ) false positives and 6.6% ( 8) false negatives ( see table 4 ) . to create an equitable comparison , the threshold for each individual metric was selected to achieve a false negative rate of 0.066 ( equal to that of the llrc ) . the llrc had a false positive rate of 7.1% , while the single metrics resulted in considerably worse false positive rates ranging between 32.3% and 73.5% ( see table 4 ) . the llrc kept the false negative rate low ( i.e. , high sensitivity to detecting out - of - control data sets ) while also controlling the false positive rate . to frame this comparison in real - world terms , let us compare llrc to the p_2c single metric classifier in terms of the total number of data sets that would require validation . our comparison depends on two assumptions : both classifiers have the same sensitivity ( 0.934 ) , and if a classifier predicts a data set is poor it will be curated . consequently , if we use the p_2c classifier instead of the llrc , we would expect to have to validate an additional ( 0.3230.071 ) 100% of the data sets . here , is the unknown fraction of data sets that truly are out of control . so , in a group of 1000 data sets , if were 0.15 , we would expect to have to curate 38 additional data sets . although the time spent in curating a single data set is typically small ( 12 min ) , there is a distinct advantage for automating curation and avoiding manual revalidation . individually , the 38 additional manual analyses may not take much time , but the repetitive monotony increases the propensity for operator error and drifting subjectivity . the sensitivity is held constant at 0.934 to show the sensitivity for any single metric compared to the llrc . from rudnick et al . p_2c is the total unique tryptic peptide identifications ; p_2a is the total spectrum identifications ; ms1_2b is the median tic . to further validate the llrc , we predicted the data quality of an additional 1321 noncurated velos - orbitrap data sets using the optimal llrc trained on all the data ( denoted f * in the appendix ) . we randomly ( but systematically ) selected 100 of these classified data sets such that the probabilities estimated by the logistic regression were evenly spaced across the range of predicted probabilities . the sensitivity was quite similar to the cross validation estimates ( shown in table 2 ) , but the specificity was 11% lower than what was observed in cross validation . notwithstanding this reduction in specificity , it is noteworthy that sensitivity was preserved , especially because sensitivity is more important than specificity ; that is , a false negative error is considerably worse than the false positive error . the primary purpose for which we use the data is to build a classifier for automatically detecting out - of - control data sets and therefore poor instrument performance . we envision , however , that this corpus could be used for a myriad of other bioinformatics and biostatistics applications . with data presented on four different classes of instruments , an obvious application would be the development of ms / ms scoring functions , using this data to understand how fragmentation differs between instrument types . a second type of analysis could be to understand the qualitative presence / absence of various peptides observed across a large number of replicates . the missing data problem is an important issue for quantitative proteomics , and this corpus would allow for rigorous understanding of the scope of the problem and potentially causes for sporadic peptide observation . another application that we envision is the investigation of unidentified or unattributed spectra . with thousands of lc ms / ms data sets , there are a very large number of fragmentation spectra for which there is not a confident identification . of those unidentified species , many are fragmented in multiple data sets ; spectrum averaging or other methods could be utilized to obtain a confident identification . for these and doubtless many other bioinformatics explorations , we have posted the data to public repositories and encourage researchers to use them as a resource . we note that the original intent of our research ( classifying bad data sets ) necessitated the publication of lc we openly acknowledge that some of the data are of low quality , as was required for our goals . therefore , in secondary analyses of these data , we encourage users to carefully consider whether such data are appropriate for their application , and consult supplementary files 1 and 2 , supporting information for a list of data that is considered out - of - control . the presented approach demonstrates how the lasso logistic regression classifier ( llrc ) approach can leverage a collection lc we note that our classifier models may not be directly usable by those whose lc however , the described methodology and accompanying software can be applied by others who want to develop an automated qc analysis . given the diverse qc samples , instruments and even goals of various research laboratories and core facilities , we strongly recommend that each group train their own classifier . our research shows that a statistically trained composite metric is dramatically more effective than any single metric . thus , this work describes an automated process that will greatly improve the use of quality metrics . while the approach produces a dichotomous prediction ( i.e. , in or out of control ) for each data set , operators may find it more useful to omit the final classification step and base their assessments on the probability scores alone . these scores , which range in the unit interval , may be useful in making decisions about borderline cases . similarly , adding an explicit borderline class would be a valuable future direction . the llrc may also provide the underpinnings for a more comprehensive statistical model designed to identify the onset of operational drift . such a methodology would alert the instrument operator to more closely monitor the system . a final extension understanding the particular subsets of the quality metrics that correspond to out - of - control conditions in lc or ms would assist operators in diagnosing malfunctions . moreover , new and more specific metrics could be created to specifically target different types of malfunction . such classifications would provide immense utility for day - to - day instrument operation . all data used for this project are available at http://omics.pnl.gov and have been deposited to the proteomexchange consortium ( http://proteomecentral.proteomexchange.org ) via the pride partner repository with the data set identifiers pxd000320 , pxd000321 , pxd000322 , pxd000323 , and pxd000324 . this includes representative instrument raw files and the complete list of qc metrics for all data sets ( supplemental files 1 and 2 , supporting information ) .

ensuring data quality and proper instrument functionality is a prerequisite for scientific investigation . manual quality assurance is time - consuming and subjective . metrics for describing liquid chromatography mass spectrometry ( lc ms ) data have been developed ; however , the wide variety of lc ms instruments and configurations precludes applying a simple cutoff . using 1150 manually classified quality control ( qc ) data sets , we trained logistic regression classification models to predict whether a data set is in or out of control . model parameters were optimized by minimizing a loss function that accounts for the trade - off between false positive and false negative errors . the classifier models detected bad data sets with high sensitivity while maintaining high specificity . moreover , the composite classifier was dramatically more specific than single metrics . finally , we evaluated the performance of the classifier on a separate validation set where it performed comparably to the results for the testing / training data sets . by presenting the methods and software used to create the classifier , other groups can create a classifier for their specific qc regimen , which is highly variable lab - to - lab . in total , this manuscript presents 3400 lc ms data sets for the same qc sample ( whole cell lysate of shewanella oneidensis ) , deposited to the proteomexchange with identifiers pxd000320pxd000324 .

Introduction Experimental Procedures Results Discussion

a variety of methods are commonly used in liquid chromatography mass spectrometry ( lc ms ) , with many groups routinely utilizing a common sample which is regularly analyzed , for example , weekly . in addition to regularly running a quality control ( qc ) sample , recent research has investigated analysis methods and metrics for assessing data quality . defined 46 nist metrics to quantify the performance of various lc ms aspects , such as chromatography , ion source , dynamic sampling , and peptide identifications . when employing the metrics to monitor the day - to - day operation of an lc ms system , it is important to note that some metrics are heavily dependent on the specific instrument and configuration . specifically , there is no guidance as to when the performance of the lc given the large number of metrics available , routine interpretation of the metrics and quality assurance is typically determined via a subjective weighting of a subset of the metrics . a variety of statistical methods may be used to explore and model how the multivariate metrics predict data quality , including principal component analysis , classification and regression and model selection techniques . leveraging a large corpus of manually curated qc data , we developed a lasso logistic regression classifier ( llrc ) that predicts , with high sensitivity and specificity , whether a qc data set is in or out of control . this signature , which is a composite of lc ms performance metrics , is more robust than any single metric itself . as performance can be viewed as a continuum , the llrc model natively computes a quality score within the range 01 . an important feature of the llrc is that it differentiates between false positive and false negative errors . therefore , the penalties associated with these errors are separately defined , which allows the classifier to be more responsive in the balance between sensitivity and specificity . finally , we present the software used to create the classifier , so that other laboratories can create a classifier specific to their qc regimen , instrumentation , and needs . a shewanella oneidensis mr-1 lysate digest is used as a quality control sample in our laboratory to provide sufficient proteomic complexity to assess both lc and ms performance . other monitored parameters included maintaining a ph of 7.00 ( 0.03 ) , a constant agitation of the culture at 5000 rpm , and temperature of 30 c . typically 2.5 g of shewanella digest was loaded to the head of the column or to a trapping column . because of the diversity of data sets used in this study , this is not a comprehensive list of conditions . there are 1150 testing and training data sets : 224 on ltq instruments , 85 on exactive instruments , 380 on ltq - orbitrap instruments , and 461 on velos - orbitrap instruments ( see table 1 ) . for each qc data set , the nist and quameter metrics were calculated exactly as described in the original publications . the quameter metrics were calculated using the software from the tabb group ( http://fenchurch.mc.vanderbilt.edu/software.php ) ; the nist metrics were calculated using in - house software , smaqc , which is posted on our github repository ( https://github.com/pnnl-comp-mass-spec ) . relevant search parameters were tryptic specificity ( semi tryptic allowed ) , no static modifications , dynamic methionine oxidation . all of the data has been deposited to the proteomexchange consortium ( http://proteomecentral.proteomexchange.org ) via the pride partner repository with the data set identifiers pxd000320 , pxd000321 , pxd000322 , pxd000323 , and pxd000324 . this includes the instrument .raw files , the ms - gf+ peptide identifications in .mzidentml format , .mgf converted spectra files , and the spreadsheet containing the metrics for every data set . the data sets were manually reviewed by three expert instrument operators ( 30 + years of combined lc this viewer contained the base peak chromatogram , total ion current chromatogram , plots of both the top 50 000 and top 500 000 lc ms detected features , and the number of peptides identified . in the first round , 1150 data sets were manually curated as good , okay , or poor and used to develop the classifier . in cases where the assessors disagreed ( 510% ) , moreover , the okay value was used to denote the wide range of performance , which , although not optimal , was still acceptable . for the validation , an additional 1321 data sets classified with the statistical model from which a subset of 100 data sets were chosen for manual curation ( supplemental files 1 and 2 , supporting information ) . data quality assessment requires knowledge of the conditions under which the qc was run to properly account for the variety of run parameters , for example , high resolution ms only , high resolution ms low resolution ms / ms , high resolution ms high resolution ms / ms , and low resolution ms low resolution ms / ms . each of these criteria is considered to determine what constitutes an acceptable ( in control ) data set . for example , under identical mass spectrometer conditions , an acceptable 60 min hplc run would provide results that would be unacceptable ( out of control ) for a 100 min run . multivariate statistical techniques were applied to the data to identify which metrics might be useful to understand the quality of a given data set and to develop a model to predict the quality of future data sets . we applied a few of them , including classification and regression tree algorithms ( cart ) , linear discriminant analysis ( lda ) , and logistic regression augmented by a classification threshold . the most effective was logistic regression , an approach for predicting a binary response using a linear combination of continuous and/or categorical predictor variables . the goal for the classifier is to identify data sets that are out of control . to that end , the binary response was coded as a 0 for a data set that was in control ( annotated as good or 1 for a data set that was out of control ( annotated as poor ) . the linear component of the logistic regression model can be expressed aswhere i , ( i = 1 , ... , n ) indexes the data sets , 0 is the intercept , 1 is a vector of coefficients ( one for each of the nist and quameter quality metrics included in the model ) , and xi is a vector representing the quality metrics included in the model . the probability of the binomial response is modeled by the logistic function : where ( xi ) estimates the probability of data set i being out of control . we used the lasso approach , a model selection technique that accounts for collinearity among the predictors while selecting a subset of the predictor variables that results in the best regression model . the inputs to the lasso logistic regression model include the complete set of possible quality metrics , the binary expert annotations ( 0 = good or , 1 = poor ) , and , a regularization parameter that simultaneously restricts the size of model coefficients and the number of quality metrics included in the model . we developed a separate lasso logistic regression model for each of the four instruments types . we used a threshold parameter , , to make a definitive prediction as to whether a data set was good or poor . specifically , we classify data set i as out of control if (xi ) > and in control otherwise . we will refer to the combination of the logistic regression and the threshold , , as the lasso logistic regression classifier ( llrc ) . when predicting the quality of a data set , the llrc will either predict a data set correctly , or will make either a false positive or false negative error . a false positive error occurs when the llrc predicts the data set to be out of control , when it was annotated as in control . the false negative error occurs when the llrc predicts the data set as being in control when it was annotated as out of control . to this end , a loss function is constructed that reflects the consequences we attribute to the two types of errors . when the llrc predicts a data set correctly , zero loss occurs . when the llrc produces a false positive , a loss of 1 occurs , and a false negative receives a loss of 1 . hence , the consequences ( or cost ) of a false negative are times greater than those of a false positive . cross validation was performed on the 1150 testing / training data sets by randomly dividing the data set into five equal parts . four parts were then combined to train the llrc which was subsequently used to predict the held - out part . the r package used in the creation of the llrc , and a tutorial for its use is available for download at http://omics.pnl.gov/software/msdataqualitysignatures.php . one critical setting where subjective or time - consuming manual data analysis can lead to systemic problems is in the evaluation of quality control ( qc ) data sets . in an effort to automate the assessment of qc data sets , we selected a testing / training sample of 1150 data sets ( see table 1 ) and manually annotated them as being these data sets originated from four types of mass spectrometers and are all replicate analyses of the same whole - cell lysate of s. oneidensis ( see experimental procedures ) . in addition to the manual rating , all computational metrics from nist and quameter were calculated for each data set . initial analyses of the 1150 data sets using pca showed a separation by quality rating and also by instrument type ( figure 1 ) . each data point on the plot represents a single qc data set and is identified by its instrument class ( the symbol ) and its manually curated quality ( the color ) . the primary goal was to build a classifier that accurately predicts the data set quality , specifically when a data set is out of control ( poor ) . importantly , the model does not treat false negatives and false positives as equally deleterious events . the different impact of each error type is reflected in the practical implication of day - to - day instrument operation . while false positives incur extra work and inconvenience for operators to review and overturn the decision , the false - negative error is of much greater consequence ; if the classifier fails to predict an out of control data set , a problem in the lc ms system may not be discovered and poor quality data will continue to be produced . optimal parameter values were obtained by using cross validation to minimize a loss function that treated false negatives as five times worse than false positives . the resulting llrcs for each of the four instrument types are shown in table 2 . this reflects the continuum of performance of real lc ms systems in day - to - day operation . sensitivity and specificity trade - off . we define sensitivity as the probability of correctly classifying an out of control ( or poor ) data set , equal to 1 minus the false negative rate . specificity is the proportion of good data sets that are correctly classified and is equal to 1 minus the false positive rate ( see experimental procedures ) . the dotted vertical line indicates the optimal threshold , , which balances the cost of a false positive or false negative classification error . in developing the llrc , the lasso method selected between 2 and 12 quality metrics to be included in the llrc for each of the four instrument types . the number of metrics used seems to correlate with how well separated the data sets appeared in the pca analysis ( supplemental figure 1 , supporting information ) . however , if one measure is more important in a specific cost / benefit scenario , the threshold will adjust to reflect the trade - offs captured in the loss function . moreover , the expected rates of false positives and false negative are implicitly considered as part of the optimization . the llrc is a composite classifier , in that it relies on multiple metrics to determine the quality of each data set . some instrument operators may rely only on a single metric to assess data quality . commonly used single metrics include : total tryptic peptide count ( p_2a ) , distinct tryptic peptide count ( p_2c ) , and the absolute intensities of peaks in the tic ( ms1_2b ) . in table 4 , we present a comparison of the llrc to these single measures of quality for velos - orbitrap data sets . as before , the loss function used to optimize the llrc parameters assumed that false negatives are 5 times worse than false positives , resulting in only 7.1% ( 24 ) false positives and 6.6% ( 8) false negatives ( see table 4 ) . to create an equitable comparison , the threshold for each individual metric was selected to achieve a false negative rate of 0.066 ( equal to that of the llrc ) . the llrc had a false positive rate of 7.1% , while the single metrics resulted in considerably worse false positive rates ranging between 32.3% and 73.5% ( see table 4 ) . the llrc kept the false negative rate low ( i.e. , high sensitivity to detecting out - of - control data sets ) while also controlling the false positive rate . to frame this comparison in real - world terms , let us compare llrc to the p_2c single metric classifier in terms of the total number of data sets that would require validation . our comparison depends on two assumptions : both classifiers have the same sensitivity ( 0.934 ) , and if a classifier predicts a data set is poor it will be curated . consequently , if we use the p_2c classifier instead of the llrc , we would expect to have to validate an additional ( 0.3230.071 ) 100% of the data sets . here , is the unknown fraction of data sets that truly are out of control . so , in a group of 1000 data sets , if were 0.15 , we would expect to have to curate 38 additional data sets . although the time spent in curating a single data set is typically small ( 12 min ) , there is a distinct advantage for automating curation and avoiding manual revalidation . the sensitivity is held constant at 0.934 to show the sensitivity for any single metric compared to the llrc . to further validate the llrc , we predicted the data quality of an additional 1321 noncurated velos - orbitrap data sets using the optimal llrc trained on all the data ( denoted f * in the appendix ) . we randomly ( but systematically ) selected 100 of these classified data sets such that the probabilities estimated by the logistic regression were evenly spaced across the range of predicted probabilities . the sensitivity was quite similar to the cross validation estimates ( shown in table 2 ) , but the specificity was 11% lower than what was observed in cross validation . notwithstanding this reduction in specificity , it is noteworthy that sensitivity was preserved , especially because sensitivity is more important than specificity ; that is , a false negative error is considerably worse than the false positive error . the primary purpose for which we use the data is to build a classifier for automatically detecting out - of - control data sets and therefore poor instrument performance . the missing data problem is an important issue for quantitative proteomics , and this corpus would allow for rigorous understanding of the scope of the problem and potentially causes for sporadic peptide observation . with thousands of lc ms / ms data sets , there are a very large number of fragmentation spectra for which there is not a confident identification . for these and doubtless many other bioinformatics explorations , we have posted the data to public repositories and encourage researchers to use them as a resource . we note that the original intent of our research ( classifying bad data sets ) necessitated the publication of lc we openly acknowledge that some of the data are of low quality , as was required for our goals . therefore , in secondary analyses of these data , we encourage users to carefully consider whether such data are appropriate for their application , and consult supplementary files 1 and 2 , supporting information for a list of data that is considered out - of - control . the presented approach demonstrates how the lasso logistic regression classifier ( llrc ) approach can leverage a collection lc we note that our classifier models may not be directly usable by those whose lc however , the described methodology and accompanying software can be applied by others who want to develop an automated qc analysis . given the diverse qc samples , instruments and even goals of various research laboratories and core facilities , we strongly recommend that each group train their own classifier . , in or out of control ) for each data set , operators may find it more useful to omit the final classification step and base their assessments on the probability scores alone . moreover , new and more specific metrics could be created to specifically target different types of malfunction . such classifications would provide immense utility for day - to - day instrument operation . all data used for this project are available at http://omics.pnl.gov and have been deposited to the proteomexchange consortium ( http://proteomecentral.proteomexchange.org ) via the pride partner repository with the data set identifiers pxd000320 , pxd000321 , pxd000322 , pxd000323 , and pxd000324 . this includes representative instrument raw files and the complete list of qc metrics for all data sets ( supplemental files 1 and 2 , supporting information ) .

the inclusion criteria were : patients with chronic renal failure undergoing stable maintenance haemodialysis three times weekly , for 12 weeks or more , before the initiation of the washout period ( week3 ) , and with planned continued haemodialysis during the treatment period ; patients with no change in their phosphate binder agent dose , for 4 weeks or more before the initiation of the washout period ; patients whose predialysis serum phosphorus concentration was > 1.94 mmol / l and 3.23 mmol / l at week 1 ; patients with no change , at least 4 weeks before the initiation of the washout period , in the dose of any vitamin d receptor activator , calcimimetic , or osteoporosis drug that they may have been receiving ; patients considered able to discontinue their current therapy for hyperphosphataemia for the 3week washout period ; and patients aged 20 or older , at the time their consent was obtained , regardless of sex . mmol / l , at week 1 ; patients whose intactparathyroid hormone ( pth ) concentration was > 800 ng / l at the beginning of the washout period ; patients with a history of haemochromatosis , or any other iron accumulation disorder , or patients whose serum ferritin was > 1797.60 pmol / l or whose transferrin saturation ( tsat ) was > 50% at the beginning of the washout period ; patients with severe gastrointestinal disorders based on the investigator 's diagnosis ; and patients with a history of a severe digestive tract procedure based on the investigator 's diagnosis . this was a phase iii , parallelgroup , randomized , openlabel , activecontrolled , multicentre study performed in haemodialysis patients with hyperphosphataemia . the washout period consisted of 3 weeks for discontinuing any phosphate binders that were taken before the study start , and the treatment period lasted 12 weeks . the randomization of subjects was carried out by an interactive web response system with subjects randomized in a 1:1 ratio to the pa21 group or the sevelamer group . the initiation dosage and administration methods for each drug were as follows : pa21 was orally administered at 250 mg / dose three times per day ( 750 mg / day ) immediately before every meal ; sevelamer was orally administered at 1000 mg / dose or 2000 mg / dose if the serum phosphorus concentration before dialysis at week 1 was < 2.58 mmol / l or 2.58 mmol / l , respectively . sevelamer was administered three times per day , immediately before every meal . during week 2 to week 8 , based on the serum phosphorus concentration from the previous week , the investigator decided to maintain , increase , or decrease the dose of each drug using the following criteria for dose adjustment . if the serum phosphorus concentration at the beginning of the previous week was > 1.94 mmol / l , the dose of pa21 was increased by 750 mg / day and that of sevelamer was increased by 750 or 1500 mg / day ; if it was 1.131.94 mmol / l , both pa21 and sevelamer doses were maintained ; and if it was < 1.13 mmol / l , the dose of pa21 was reduced by 750 mg / day and that of sevelamer was reduced by 750 or 1500 mg / day . the maximum allowed dose of pa21 was 1000 mg / dose three times per day ( 3000 mg / day ) and that of sevelamer was 3000 mg / dose three times per day ( 9000 mg / day ) . each pa21 tablet contained 250 mg of iron and each sevelamer tablet contained 250 mg of sevelamer hydrochloride . patients recorded in a diary the number of tablets taken , and compliance was checked by the investigator before each week 's first dialysis session ( at weeks 1 to 12 , or at discontinuation ) during the treatment period . concomitant use of the following drugs was prohibited during the study period : other phosphate binders , drugs with a phosphate binding action , oral iron agents , drugs having an effect on serum phosphorus concentrations , and any study drugs other than pa21 . the discontinuation criteria for individual subjects were as follows : the development of any adverse event ( ae ) that would make study continuation difficult ; any serum phosphorus concentration < 0.97 mmol / l or > 3.23 mmol / l , twice consecutively ; and any corrected serum calcium concentration or serum ferritin concentration 1.88 mmol / l or > 1797.60 the use of vitamin d receptor activators and calcimimetics was allowed as long as the subjects were receiving it for 4 weeks or more before the start of the observation period , and the dose was not to be changed during the study period . any patient not using vitamin d receptor activators or calcimimetics at the study start was not allowed to begin using them during the study period . were not to be changed during the study ( observation and treatment ) periods . measurements of parameters were standardized at a central laboratory . laboratory tests were processed collectively at an assay facility with the same measurement methods throughout the study period . tests at each evaluation point were conducted on the week 's first dialysis day . because the visiting time was different for each patient , laboratory tests were not standardized to the same time of day among all patients ; however , each individual patient visited the participating centre at the same time and under the same fasting condition throughout their individual treatment . ethical approval was obtained from the institutional review boards of each participating centre , and the study was performed in accordance with the ethical standards laid down in the declaration of helsinki as revised in 2013 . the primary efficacy outcome was the adjusted serum phosphorus concentration at the end of treatment . additional evaluations included serum phosphorus concentration at each time point , the change in serum phosphorus concentration from baseline to end of treatment , and the achievement rates for target serum phosphorus concentrations of both 1.131.94 mmol / l and 1.131.78 mmol / l . the target serum phosphorus concentrations of 1.13 mmol / l and 1.94 mmol / l were based on the target range of the japanese society for dialysis therapy ( jsdt ) guidelines,3 whereas the target serum phosphorus concentrations of 1.13 mmol / l and 1.78 mmol / l were based on the target range from the national kidney foundation kidney disease outcomes quality initiative guidelines.4 to assess the adequacy of dialysis treatment , the protein catabolic rate and urea clearance ( kt / v ) were calculated . safety outcomes were aes , adrs , and laboratory tests including tsat , ferritin , haemoglobin , and bicarbonate levels . from phase iii studies of sevelamer and similar drugs , a standard deviation of 0.480.65 mmol / l for serum phosphorus concentration range , and a noninferiority margin of 0.32 mmol / l were assumed . in a doseresponse study in japanese haemodialysis patients with hyperphosphataemia , standard deviations in a pa21 group were 0.370.51 mmol / l . thus , the standard deviation for pa21 and sevelamer in the present study was set at 0.55 mmol / l . the difference between pa21 and sevelamer was set at 0 mmol / l , and the noninferiority margin was established at 0.32 sixtytwo subjects would be needed per group , for a twosided significance level of 5% , and a detection power of 90% . therefore , a target number of 100 subjects per group was established . to compare demographic and baseline characteristics between the two treatment groups , twosample ttest , fisher 's exact test , and twosample wilcoxon test for the adjusted serum phosphorus concentration at the end of treatment ( primary efficacy outcome ) , analyses of covariance ( ancova ) were performed with the groups as fixed effects , and serum phosphorus concentrations at week 0 as covariates , and the noninferiority of pa21 versus sevelamer was confirmed . adjusted intergroup means and their twosided 95% confidence intervals were calculated . if the upper limit of the twosided 95% confidence interval did not exceed 0.32 mmol / l , the noninferiority of pa21 was defined as confirmed . serum phosphorus concentrations at each timepoint , summary statistics for each of the secondary endpoint measures by group at each evaluation time , and twosided 95% confidence intervals for means were calculated . the achievement rates of target serum phosphorus concentrations at each timepoint per group were calculated . for safety outcomes , the aes and adrs did not include discolouration events , such as discoloured faeces and discoloured tongue , caused by the iron contained in pa21 . the number of events , the number of patients with events , and event incidence were calculated in each group . aes and adrs were classified by meddra primary system organ class and preferred term . for laboratory test parameters , all statistical analyses were performed using sas 9.3 software ( sas institute inc . , cary , nc , usa ) . the inclusion criteria were : patients with chronic renal failure undergoing stable maintenance haemodialysis three times weekly , for 12 weeks or more , before the initiation of the washout period ( week3 ) , and with planned continued haemodialysis during the treatment period ; patients with no change in their phosphate binder agent dose , for 4 weeks or more before the initiation of the washout period ; patients whose predialysis serum phosphorus concentration was > 1.94 mmol / l and 3.23 mmol / l at week 1 ; patients with no change , at least 4 weeks before the initiation of the washout period , in the dose of any vitamin d receptor activator , calcimimetic , or osteoporosis drug that they may have been receiving ; patients considered able to discontinue their current therapy for hyperphosphataemia for the 3week washout period ; and patients aged 20 or older , at the time their consent was obtained , regardless of sex . mmol / l , at week 1 ; patients whose intactparathyroid hormone ( pth ) concentration was > 800 ng / l at the beginning of the washout period ; patients with a history of haemochromatosis , or any other iron accumulation disorder , or patients whose serum ferritin was > 1797.60 pmol / l or whose transferrin saturation ( tsat ) was > 50% at the beginning of the washout period ; patients with severe gastrointestinal disorders based on the investigator 's diagnosis ; and patients with a history of a severe digestive tract procedure based on the investigator 's diagnosis . this was a phase iii , parallelgroup , randomized , openlabel , activecontrolled , multicentre study performed in haemodialysis patients with hyperphosphataemia . the washout period consisted of 3 weeks for discontinuing any phosphate binders that were taken before the study start , and the treatment period lasted 12 weeks . the randomization of subjects was carried out by an interactive web response system with subjects randomized in a 1:1 ratio to the pa21 group or the sevelamer group . the initiation dosage and administration methods for each drug were as follows : pa21 was orally administered at 250 mg / dose three times per day ( 750 mg / day ) immediately before every meal ; sevelamer was orally administered at 1000 mg / dose or 2000 mg / dose if the serum phosphorus concentration before dialysis at week 1 was < 2.58 mmol / l or 2.58 mmol / l , respectively . sevelamer was administered three times per day , immediately before every meal . during week 2 to week 8 , based on the serum phosphorus concentration from the previous week , the investigator decided to maintain , increase , or decrease the dose of each drug using the following criteria for dose adjustment . if the serum phosphorus concentration at the beginning of the previous week was > 1.94 mmol / l , the dose of pa21 was increased by 750 mg / day and that of sevelamer was increased by 750 or 1500 mg / day ; if it was 1.131.94 mmol / l , both pa21 and sevelamer doses were maintained ; and if it was < 1.13 mmol / l , the dose of pa21 was reduced by 750 mg / day and that of sevelamer was reduced by 750 or 1500 mg / day . the maximum allowed dose of pa21 was 1000 mg / dose three times per day ( 3000 mg / day ) and that of sevelamer was 3000 mg / dose three times per day ( 9000 mg / day ) . each pa21 tablet contained 250 mg of iron and each sevelamer tablet contained 250 mg of sevelamer hydrochloride . patients recorded in a diary the number of tablets taken , and compliance was checked by the investigator before each week 's first dialysis session ( at weeks 1 to 12 , or at discontinuation ) during the treatment period . concomitant use of the following drugs was prohibited during the study period : other phosphate binders , drugs with a phosphate binding action , oral iron agents , drugs having an effect on serum phosphorus concentrations , and any study drugs other than pa21 . the discontinuation criteria for individual subjects were as follows : the development of any adverse event ( ae ) that would make study continuation difficult ; any serum phosphorus concentration < 0.97 mmol / l or > 3.23 mmol / l , twice consecutively ; and any corrected serum calcium concentration or serum ferritin concentration 1.88 mmol / l or > 1797.60 pmol / l , respectively . the use of vitamin d receptor activators and calcimimetics was allowed as long as the subjects were receiving it for 4 weeks or more before the start of the observation period , and the dose was not to be changed during the study period . any patient not using vitamin d receptor activators or calcimimetics at the study start was not allowed to begin using them during the study period . were not to be changed during the study ( observation and treatment ) periods . measurements of parameters were standardized at a central laboratory . laboratory tests were processed collectively at an assay facility with the same measurement methods throughout the study period . tests at each evaluation point were conducted on the week 's first dialysis day . because the visiting time was different for each patient , laboratory tests were not standardized to the same time of day among all patients ; however , each individual patient visited the participating centre at the same time and under the same fasting condition throughout their individual treatment . ethical approval was obtained from the institutional review boards of each participating centre , and the study was performed in accordance with the ethical standards laid down in the declaration of helsinki as revised in 2013 . the primary efficacy outcome was the adjusted serum phosphorus concentration at the end of treatment . additional evaluations included serum phosphorus concentration at each time point , the change in serum phosphorus concentration from baseline to end of treatment , and the achievement rates for target serum phosphorus concentrations of both 1.131.94 mmol / l and 1.131.78 mmol / l . the target serum phosphorus concentrations of 1.13 mmol / l and 1.94 mmol / l were based on the target range of the japanese society for dialysis therapy ( jsdt ) guidelines,3 whereas the target serum phosphorus concentrations of 1.13 mmol / l and 1.78 mmol / l were based on the target range from the national kidney foundation kidney disease outcomes quality initiative guidelines.4 to assess the adequacy of dialysis treatment , the protein catabolic rate and urea clearance ( kt / v ) were calculated . safety outcomes were aes , adrs , and laboratory tests including tsat , ferritin , haemoglobin , and bicarbonate levels . from phase iii studies of sevelamer and similar drugs , a standard deviation of 0.480.65 mmol / l for serum phosphorus concentration range , and a noninferiority margin of 0.32 mmol / l were assumed . in a doseresponse study in japanese haemodialysis patients with hyperphosphataemia , standard deviations in a pa21 group were 0.370.51 mmol / l . thus , the standard deviation for pa21 and sevelamer in the present study was set at 0.55 mmol / l . the difference between pa21 and sevelamer was set at 0 mmol / l , and the noninferiority margin was established at 0.32 sixtytwo subjects would be needed per group , for a twosided significance level of 5% , and a detection power of 90% . therefore , a target number of 100 subjects per group was established . to compare demographic and baseline characteristics between the two treatment groups , twosample ttest , fisher 's exact test , and twosample wilcoxon test for the adjusted serum phosphorus concentration at the end of treatment ( primary efficacy outcome ) , analyses of covariance ( ancova ) were performed with the groups as fixed effects , and serum phosphorus concentrations at week 0 as covariates , and the noninferiority of pa21 versus sevelamer was confirmed . adjusted intergroup means and their twosided 95% confidence intervals were calculated . if the upper limit of the twosided 95% confidence interval did not exceed 0.32 serum phosphorus concentrations at each timepoint , summary statistics for each of the secondary endpoint measures by group at each evaluation time , and twosided 95% confidence intervals for means were calculated . the achievement rates of target serum phosphorus concentrations at each timepoint per group were calculated . for safety outcomes , the aes and adrs did not include discolouration events , such as discoloured faeces and discoloured tongue , caused by the iron contained in pa21 . the number of events , the number of patients with events , and event incidence were calculated in each group . aes and adrs were classified by meddra primary system organ class and preferred term . for laboratory test parameters , all statistical analyses were performed using sas 9.3 software ( sas institute inc . , cary , nc , usa ) . a total of 321 patients were screened in this study , and 213 eligible patients were randomised to treatment with pa21 ( n = 108 ) or sevelamer ( n = 105 ) . a total of 209 ( pa21 group , 106 ; sevelamer group , 103 ) , 192 ( pa21 group , 100 ; sevelamer group , 92 ) , and 213 ( pa21 group , 108 ; sevelamer group , 105 ) patients were included in the full analysis set , per protocol set , and safety set , respectively . fas , full analysis set ; pps , per protocol set ; ss , safety set . in the pa21 and sevelamer groups , respectively , 14 patients ( 13.0% ) and 18 patients ( 17.1% ) discontinued the study . a total of 94 ( 87.0% ) and 87 ( 82.9% ) of the patients in the pa21 and sevelamer group completed the study , respectively . there were no statistically significant differences between the two groups except for sex and intactpth . adjusted analyses for sex and intactpth were performed , and the analysis indicated that the study results were unaffected by these imbalanced baseline characteristics . patient demographic and clinical characteristics ( per protocol set ) fisher 's exact test ; twosample wilcoxon test ; ns , not significant the adjusted mean serum phosphorus concentration at the end of treatment was 1.62 mmol / l in the pa21 group and 1.72 mmol / l in the sevelamer group , with a difference of 0.11 mmol / l ( 95% confidence interval [ ci ] , 0.20 to 0.02 mmol / l ) in the per protocol set ( fig . 2 ) . furthermore , the mean serum phosphorus concentration at the end of treatment was significantly lower in the pa21 group compared with that in the sevelamer group ( ancova , p = 0.020 ) . furthermore , in both groups , no subject showed deviation from the dose adjustment criteria . serum phosphorus concentrations ( mean + sd ) ( per protocol set ) . the adjusted mean serum phosphorus concentration at the end of treatment confirmed the noninferiority of pa21 ( sucroferric oxyhydroxide ) to sevelamer ( 1.62 vs 1.72 mmol / l ; 95% ci , 0.20 to 0.02 mmol / l ; ancova , p = 0.020 ) . a notable decrease in serum phosphorus concentrations the horizontal lines indicate the target range of serum phosphorus concentration in the jsdt guideline . ancova , analysis of covariance ; ci , confidence interval ; ep , endpoint ; sd , standard deviation . a notable decrease in serum phosphorus concentrations was shown at week 1 in both groups and was maintained until week 12 ( fig . 2 ) . the mean daily tablet intake at the end of treatment was 5.6 2.6 tablets ( mean daily dose , 1403 654 mg ) in the pa21 group and 18.7 7.1 tablets ( mean daily dose , 4671 1776 mg ) in the sevelamer group . the mean number of tablets for daily dose in each group at the end of treatment is shown in figure 3 . mean daily number of tablets for daily dose of pa21 ( sucroferric oxyhydroxide ) and sevelamer ( per protocol set ) . the mean number of tablets for daily dose of pa21 was notably lower than that of sevelamer . at the end of treatment , the achievement rate of target serum phosphorus concentration ( 1.131.94 mmol / the rates were over 80% in the pa21 group after week 6 ; meanwhile , the rates were significantly higher in the pa21 group than in the sevelamer group after week 8 ( fig . mmol / l ) was 63.0% in the pa21 group and 52.2% in the sevelamer group at the end of treatment . the achievement rates of target serum phosphorus concentration ( 1.131.94 mmol / l ) at the end of treatment were 82.0% and 67.4% in the pa21 ( sucroferric oxyhydroxide ) and sevelamer groups , respectively . meanwhile , the rates were significantly higher in the pa21 group than in the sevelamer group after week 8 . * p < 0.05 . no change was seen in protein catabolic rate and kt / v from baseline to end of treatment in both groups ( table 2 ) , which indicated that dietary protein intake and dialysis treatment were adequate in both groups . changes in serum phosphorus concentrations , corrected calcium levels , intactpth levels , protein catabolic rate , and kt / v ( per protocol set ) kt / v , urea clearance ; pth , parathyroid hormone . the changes in corrected serum calcium levels and intactpth levels are shown in table 2 . although the intactpth levels increased during the phosphate binder washout period , the levels decreased after treatment with pa21 or sevelamer . the incidence of aes ( excluding discolouration events caused by the iron contained in pa21 ) was 75.0% ( 81/108 ) and 66.7% ( 70/105 ) , and the incidence of adrs was 26.9% ( 29/108 ) and 26.7% ( 28/105 ) in the pa21 group and the sevelamer group , respectively ( table 3 ) . no significant difference in the incidence of either aes or adrs was seen between the two groups . the most frequently reported adr was diarrhoea ( pa21 , 21.3% ; sevelamer , 1.0% ) in the pa21 group and constipation ( pa21 , 0.0% ; sevelamer , 18.1% ) , abdominal discomfort ( pa21 , 0.0% ; sevelamer , 2.9% ) , and abdominal distension ( pa21 , 0.0% ; sevelamer , 2.9% ) in the sevelamer group . most cases of diarrhoea were mild , transient , and developed early in the treatment . there were two cases of serious adrs in the pa21 group ( congestive cardiac failure and acute pulmonary oedema ) and one case in the sevelamer group ( diverticulitis ) . adverse events that occurred at an incidence of 5% , adverse drug reactions that occurred at an incidence of 2% and adverse events that led to withdrawal at an incidence of 2% ( excluding discolouration events caused by the iron contained in pa21 ) ( safety set ) iron parameters , such as serum ferritin , transferrin saturation , and haemoglobin , were prone to increase in the pa21 group . no bicarbonate decrease was seen in the pa21 group ( table 4 ) . laboratory parameters ( ferritin levels , transferrin saturation , haemoglobin , and bicarbonate ) ( safety set ) a total of 321 patients were screened in this study , and 213 eligible patients were randomised to treatment with pa21 ( n = 108 ) or sevelamer ( n = 105 ) . a total of 209 ( pa21 group , 106 ; sevelamer group , 103 ) , 192 ( pa21 group , 100 ; sevelamer group , 92 ) , and 213 ( pa21 group , 108 ; sevelamer group , 105 ) patients were included in the full analysis set , per protocol set , and safety set , respectively . fas , full analysis set ; pps , per protocol set ; ss , safety set . in the pa21 and sevelamer groups , respectively , 14 patients ( 13.0% ) and 18 patients ( 17.1% ) discontinued the study . a total of 94 ( 87.0% ) and 87 ( 82.9% ) of the patients in the pa21 and sevelamer group completed the study , respectively . there were no statistically significant differences between the two groups except for sex and intactpth . adjusted analyses for sex and intactpth were performed , and the analysis indicated that the study results were unaffected by these imbalanced baseline characteristics . patient demographic and clinical characteristics ( per protocol set ) fisher 's exact test ; twosample wilcoxon test ; ns , not significant mmol / l in the pa21 group and 1.72 mmol / l in the sevelamer group , with a difference of 0.11 mmol / l ( 95% confidence interval [ ci ] , 0.20 to 0.02 mmol / l ) in the per protocol set ( fig . 2 ) . furthermore , the mean serum phosphorus concentration at the end of treatment was significantly lower in the pa21 group compared with that in the sevelamer group ( ancova , p = 0.020 ) . furthermore , in both groups , no subject showed deviation from the dose adjustment criteria . serum phosphorus concentrations ( mean + sd ) ( per protocol set ) . the adjusted mean serum phosphorus concentration at the end of treatment confirmed the noninferiority of pa21 ( sucroferric oxyhydroxide ) to sevelamer ( 1.62 vs 1.72 mmol / l ; difference , 0.11 mmol / l ; 95% ci , 0.20 to 0.02 mmol / l ; ancova , p = 0.020 ) . a notable decrease in serum phosphorus concentrations the horizontal lines indicate the target range of serum phosphorus concentration in the jsdt guideline . ancova , analysis of covariance ; ci , confidence interval ; ep , endpoint ; sd , standard deviation . a notable decrease in serum phosphorus concentrations was shown at week 1 in both groups and was maintained until week 12 ( fig . 2 ) . the mean daily tablet intake at the end of treatment was 5.6 2.6 tablets ( mean daily dose , 1403 654 mg ) in the pa21 group and 18.7 7.1 tablets ( mean daily dose , 4671 1776 mg ) in the sevelamer group . the mean number of tablets for daily dose in each group at the end of treatment is shown in figure 3 . mean daily number of tablets for daily dose of pa21 ( sucroferric oxyhydroxide ) and sevelamer ( per protocol set ) . the mean number of tablets for daily dose of pa21 was notably lower than that of sevelamer . at the end of treatment , the achievement rate of target serum phosphorus concentration ( 1.131.94 mmol / l ) was 82.0% in the pa21 group and 67.4% in the sevelamer group . the rates were over 80% in the pa21 group after week 6 ; meanwhile , the rates were significantly higher in the pa21 group than in the sevelamer group after week 8 ( fig . mmol / l ) was 63.0% in the pa21 group and 52.2% in the sevelamer group at the end of treatment . the achievement rates of target serum phosphorus concentration ( 1.131.94 mmol / l ) at the end of treatment were 82.0% and 67.4% in the pa21 ( sucroferric oxyhydroxide ) and sevelamer groups , respectively . meanwhile , the rates were significantly higher in the pa21 group than in the sevelamer group after week 8 . p < 0.05 . no change was seen in protein catabolic rate and kt / v from baseline to end of treatment in both groups ( table 2 ) , which indicated that dietary protein intake and dialysis treatment were adequate in both groups . changes in serum phosphorus concentrations , corrected calcium levels , intactpth levels , protein catabolic rate , and kt / v ( per protocol set ) kt / v , urea clearance ; pth , parathyroid hormone . the changes in corrected serum calcium levels and intactpth levels are shown in table 2 . although the intactpth levels increased during the phosphate binder washout period , the levels decreased after treatment with pa21 or sevelamer . the incidence of aes ( excluding discolouration events caused by the iron contained in pa21 ) was 75.0% ( 81/108 ) and 66.7% ( 70/105 ) , and the incidence of adrs was 26.9% ( 29/108 ) and 26.7% ( 28/105 ) in the pa21 group and the sevelamer group , respectively ( table 3 ) . no significant difference in the incidence of either aes or adrs was seen between the two groups . the most frequently reported adr was diarrhoea ( pa21 , 21.3% ; sevelamer , 1.0% ) in the pa21 group and constipation ( pa21 , 0.0% ; sevelamer , 18.1% ) , abdominal discomfort ( pa21 , 0.0% ; sevelamer , 2.9% ) , and abdominal distension ( pa21 , 0.0% ; sevelamer , 2.9% ) in the sevelamer group . most cases of diarrhoea were mild , transient , and developed early in the treatment . there were two cases of serious adrs in the pa21 group ( congestive cardiac failure and acute pulmonary oedema ) and one case in the sevelamer group ( diverticulitis ) . adverse events that occurred at an incidence of 5% , adverse drug reactions that occurred at an incidence of 2% and adverse events that led to withdrawal at an incidence of 2% ( excluding discolouration events caused by the iron contained in pa21 ) ( safety set ) iron parameters , such as serum ferritin , transferrin saturation , and haemoglobin , were prone to increase in the pa21 group . laboratory parameters ( ferritin levels , transferrin saturation , haemoglobin , and bicarbonate ) ( safety set ) the adjusted mean serum phosphorus concentrations at the end of treatment confirmed the noninferiority of pa21 to sevelamer , and a significant decrease was observed in the pa21 group compared with the sevelamer group . this significant difference in serum phosphorus concentration between both groups was not caused by differences in dietary protein intake or the adequacy of dialysis treatment . our findings are consistent with those of the phase iii study by floege et al . that showed the noninferiority of pa21 to sevelamer.10 the efficacy of both pa21 and sevelamer in decreasing the serum phosphorus concentrations was evident at week 1 of treatment and this was maintained until the end of the treatment period . the phosphoruslowering effect in the present study was comparable to that of the previous study.10 the changes in serum phosphorus concentration from baseline to the end of treatment were comparable in both treatment groups and similar to the findings in the previous studies.9 , 10 in both groups , the serum phosphorus concentrations at the end of treatment were within the range of the control target values ( between 1.13 and 1.94 mmol / l ) . furthermore , high achievement rates within the target range were maintained in the pa21 group . because a ushaped curve association between serum phosphorus and mortality in haemodialysis patients was reported,2 this result suggests that pa21 , which can maintain serum phosphorus concentration within the target range in more patients , demonstrates a clinical benefit . the mean daily tablet intake was markedly lower in the pa21 group than in the sevelamer group . pa21 showed an equivalent efficacy to sevelamer in lowering serum phosphorus concentration with a notably smaller number of tablets . the necessary number of tablets and the daily dose in the pa21 group ( 5.6 tablets and 1403 mg / day ) was approximately onethird that in the sevelamer group ( 18.7 tablets and 4671 mg / day ) . a similar result was shown in the previous phase iii study of pa2110 in which the number of tablets for daily dose for the first 12 weeks was 2.8 tablets in the pa21 group and 7.6 tablets in sevelamer group ( ratio , 1:4.3 when converted to daily dose ) . a high pill burden in patients undergoing dialysis was affirmed in a survey reporting that haemodialysis patients take a median of 19 tablets per day,11 and it has also been reported that a higher pill burden in dialysis patients is associated with poorer healthrelated quality of life.11 in the meantime , higher adherence and good control of phosphorus concentrations are achieved with a lower pill burden.12 pa21 is considered a clinically useful drug in ckd patients because it can control phosphorus concentrations with a smaller number of tablets than sevelamer , which is considered to have a high pill burden . the changes in corrected serum calcium levels and serum intactpth levels in the pa21 group were comparable to those of the sevelamer group . no increasing trend was seen during treatment in either group . in terms of safety , although diarrhoea was frequently observed with the pa21 group , most cases were mild and transient . only four patients withdrew from the treatment because of diarrhoea ; however , other patients who developed diarrhoea were able to continue the treatment . the onset and course , such as the incidence , severity , duration , and withdrawal , of diarrhoea were comparable to those of the previous phase iii study.10 the incidence of constipation in the pa21 group was low ; therefore , dialysis patients could benefit from pa21 treatment because it is known that many ckd patients undergoing dialysis suffer from constipation due to water restriction.13 the incidence of serious adr was similar between the pa21 and sevelamer groups . regarding the clinical laboratory tests , increasing trends in serum ferritin , tsat , and haemoglobin levels were observed in the pa21 group as well as the previous phase iii study.10 however , the previous nonclinical14 and clinical studies15 of pa21 confirmed that the iron absorption level was consistently low and no iron accumulation was indicated in the previous longterm study.16 when comparing the current study and the previous phase iii study , the absolute ferritin values and fluctuation range of ferritin were higher in the previous study . these differences may have been caused by the more frequent use of intravenous iron in the us than in japan . in fact , the dialysis outcomes and practice patterns study surveys indicated that among several countries , japan used the lowest monthly intravenous iron dose and subsequently showed the lowest mean serum ferritin.17 , 18 it is known that dialysis patients often develop metabolic acidosis resulting from a low bicarbonate condition due to renal dysfunction.19 in this study , the bicarbonate ion concentration decreased in the sevelamer group ; meanwhile , no notable change was seen in the pa21 group . regarding the study limitations , although a 12week study period would not be long enough to evaluate a therapy for chronic diseases , we have conducted a 52week longterm study to evaluate the safety and efficacy of pa21 in japanese haemodialysis patients , the results of which will be published separately . another limitation is that , because not all the patients visited the site at the same time of day and under the same fasting conditions , laboratory tests could not be standardized to the time of day or fasting vs nonfasting . finally , although the study protocol did not allow diet therapies to be changed throughout the observation and treatment periods , dietary phosphate intake was not standardized across all study centres . in conclusion , treatment with pa21 was noninferior to that with sevelamer in japanese haemodialysis patients with hyperphosphataemia . treatment with pa21 was effective , safe , and well tolerated in this patient population , while having a considerably lower pill burden than sevelamer . the effectiveness of pa21 in this current japanese study is comparable to that of previous overseas studies ; therefore , pa21 represents a new treatment alternative for haemodialysis patients with hyperphosphataemia .

abstractaimwe aimed to investigate the noninferiority of pa21 ( sucroferric oxyhydroxide ) to sevelamer hydrochloride ( sevelamer ) in terms of efficacy and safety in japanese haemodialysis patients with hyperphosphataemia.methodsin this phase iii , openlabel , multicentre study , 213 haemodialysis patients with hyperphosphataemia were randomized to pa21 or sevelamer treatment for 12 weeks . the primary outcome was adjusted serum phosphorus concentration at the end of treatment ; the noninferiority of pa21 was confirmed if the upper limit of the twosided 95% confidence interval ( ci ) is 0.32 mmol / l . secondary outcomes were corrected serum calcium and intactparathyroid hormone concentrations . adverse events ( aes ) and adverse drug reactions ( adrs ) were evaluated.resultsthe adjusted mean serum phosphorus concentration at the end of treatment confirmed the noninferiority of pa21 for lowering serum phosphorus compared with sevelamer ( 1.62 vs 1.72 mmol / l ; difference , 0.11 mmol / l ; 95% ci , 0.20 to 0.02 mmol / l ) . the mean daily tablet intake was 5.6 2.6 and 18.7 7.1 tablets in the pa21 and sevelamer groups , respectively . the incidences of aes and adrs were not significantly different between the two groups.conclusionthe noninferiority of pa21 to sevelamer was confirmed for the treatment of japanese haemodialysis patients with hyperphosphataemia . pa21 was effective , safe , and well tolerated , while having a considerably lower pill burden than sevelamer .

Methods Patients Study design Efficacy outcomes and additional evaluations Safety and tolerability Statistical analyses Results Patients Efficacy outcomes and additional evaluations Safety and tolerability Discussion

the inclusion criteria were : patients with chronic renal failure undergoing stable maintenance haemodialysis three times weekly , for 12 weeks or more , before the initiation of the washout period ( week3 ) , and with planned continued haemodialysis during the treatment period ; patients with no change in their phosphate binder agent dose , for 4 weeks or more before the initiation of the washout period ; patients whose predialysis serum phosphorus concentration was > 1.94 mmol / l and 3.23 mmol / l at week 1 ; patients with no change , at least 4 weeks before the initiation of the washout period , in the dose of any vitamin d receptor activator , calcimimetic , or osteoporosis drug that they may have been receiving ; patients considered able to discontinue their current therapy for hyperphosphataemia for the 3week washout period ; and patients aged 20 or older , at the time their consent was obtained , regardless of sex . mmol / l , at week 1 ; patients whose intactparathyroid hormone ( pth ) concentration was > 800 ng / l at the beginning of the washout period ; patients with a history of haemochromatosis , or any other iron accumulation disorder , or patients whose serum ferritin was > 1797.60 pmol / l or whose transferrin saturation ( tsat ) was > 50% at the beginning of the washout period ; patients with severe gastrointestinal disorders based on the investigator 's diagnosis ; and patients with a history of a severe digestive tract procedure based on the investigator 's diagnosis . this was a phase iii , parallelgroup , randomized , openlabel , activecontrolled , multicentre study performed in haemodialysis patients with hyperphosphataemia . the initiation dosage and administration methods for each drug were as follows : pa21 was orally administered at 250 mg / dose three times per day ( 750 mg / day ) immediately before every meal ; sevelamer was orally administered at 1000 mg / dose or 2000 mg / dose if the serum phosphorus concentration before dialysis at week 1 was < 2.58 mmol / l or 2.58 mmol / l , respectively . if the serum phosphorus concentration at the beginning of the previous week was > 1.94 mmol / l , the dose of pa21 was increased by 750 mg / day and that of sevelamer was increased by 750 or 1500 mg / day ; if it was 1.131.94 mmol / l , both pa21 and sevelamer doses were maintained ; and if it was < 1.13 mmol / l , the dose of pa21 was reduced by 750 mg / day and that of sevelamer was reduced by 750 or 1500 mg / day . the discontinuation criteria for individual subjects were as follows : the development of any adverse event ( ae ) that would make study continuation difficult ; any serum phosphorus concentration < 0.97 mmol / l or > 3.23 mmol / l , twice consecutively ; and any corrected serum calcium concentration or serum ferritin concentration 1.88 mmol / l or > 1797.60 the use of vitamin d receptor activators and calcimimetics was allowed as long as the subjects were receiving it for 4 weeks or more before the start of the observation period , and the dose was not to be changed during the study period . the primary efficacy outcome was the adjusted serum phosphorus concentration at the end of treatment . additional evaluations included serum phosphorus concentration at each time point , the change in serum phosphorus concentration from baseline to end of treatment , and the achievement rates for target serum phosphorus concentrations of both 1.131.94 mmol / l and 1.131.78 mmol / l . from phase iii studies of sevelamer and similar drugs , a standard deviation of 0.480.65 mmol / l for serum phosphorus concentration range , and a noninferiority margin of 0.32 mmol / l were assumed . in a doseresponse study in japanese haemodialysis patients with hyperphosphataemia , standard deviations in a pa21 group were 0.370.51 mmol / l . the difference between pa21 and sevelamer was set at 0 mmol / l , and the noninferiority margin was established at 0.32 sixtytwo subjects would be needed per group , for a twosided significance level of 5% , and a detection power of 90% . to compare demographic and baseline characteristics between the two treatment groups , twosample ttest , fisher 's exact test , and twosample wilcoxon test for the adjusted serum phosphorus concentration at the end of treatment ( primary efficacy outcome ) , analyses of covariance ( ancova ) were performed with the groups as fixed effects , and serum phosphorus concentrations at week 0 as covariates , and the noninferiority of pa21 versus sevelamer was confirmed . if the upper limit of the twosided 95% confidence interval did not exceed 0.32 mmol / l , the noninferiority of pa21 was defined as confirmed . the inclusion criteria were : patients with chronic renal failure undergoing stable maintenance haemodialysis three times weekly , for 12 weeks or more , before the initiation of the washout period ( week3 ) , and with planned continued haemodialysis during the treatment period ; patients with no change in their phosphate binder agent dose , for 4 weeks or more before the initiation of the washout period ; patients whose predialysis serum phosphorus concentration was > 1.94 mmol / l and 3.23 mmol / l at week 1 ; patients with no change , at least 4 weeks before the initiation of the washout period , in the dose of any vitamin d receptor activator , calcimimetic , or osteoporosis drug that they may have been receiving ; patients considered able to discontinue their current therapy for hyperphosphataemia for the 3week washout period ; and patients aged 20 or older , at the time their consent was obtained , regardless of sex . mmol / l , at week 1 ; patients whose intactparathyroid hormone ( pth ) concentration was > 800 ng / l at the beginning of the washout period ; patients with a history of haemochromatosis , or any other iron accumulation disorder , or patients whose serum ferritin was > 1797.60 pmol / l or whose transferrin saturation ( tsat ) was > 50% at the beginning of the washout period ; patients with severe gastrointestinal disorders based on the investigator 's diagnosis ; and patients with a history of a severe digestive tract procedure based on the investigator 's diagnosis . this was a phase iii , parallelgroup , randomized , openlabel , activecontrolled , multicentre study performed in haemodialysis patients with hyperphosphataemia . the initiation dosage and administration methods for each drug were as follows : pa21 was orally administered at 250 mg / dose three times per day ( 750 mg / day ) immediately before every meal ; sevelamer was orally administered at 1000 mg / dose or 2000 mg / dose if the serum phosphorus concentration before dialysis at week 1 was < 2.58 mmol / l or 2.58 mmol / l , respectively . if the serum phosphorus concentration at the beginning of the previous week was > 1.94 mmol / l , the dose of pa21 was increased by 750 mg / day and that of sevelamer was increased by 750 or 1500 mg / day ; if it was 1.131.94 mmol / l , both pa21 and sevelamer doses were maintained ; and if it was < 1.13 mmol / l , the dose of pa21 was reduced by 750 mg / day and that of sevelamer was reduced by 750 or 1500 mg / day . the discontinuation criteria for individual subjects were as follows : the development of any adverse event ( ae ) that would make study continuation difficult ; any serum phosphorus concentration < 0.97 mmol / l or > 3.23 mmol / l , twice consecutively ; and any corrected serum calcium concentration or serum ferritin concentration 1.88 mmol / l or > 1797.60 pmol / l , respectively . the primary efficacy outcome was the adjusted serum phosphorus concentration at the end of treatment . additional evaluations included serum phosphorus concentration at each time point , the change in serum phosphorus concentration from baseline to end of treatment , and the achievement rates for target serum phosphorus concentrations of both 1.131.94 mmol / l and 1.131.78 mmol / l . from phase iii studies of sevelamer and similar drugs , a standard deviation of 0.480.65 mmol / l for serum phosphorus concentration range , and a noninferiority margin of 0.32 mmol / l were assumed . in a doseresponse study in japanese haemodialysis patients with hyperphosphataemia , standard deviations in a pa21 group were 0.370.51 mmol / l . the difference between pa21 and sevelamer was set at 0 mmol / l , and the noninferiority margin was established at 0.32 sixtytwo subjects would be needed per group , for a twosided significance level of 5% , and a detection power of 90% . to compare demographic and baseline characteristics between the two treatment groups , twosample ttest , fisher 's exact test , and twosample wilcoxon test for the adjusted serum phosphorus concentration at the end of treatment ( primary efficacy outcome ) , analyses of covariance ( ancova ) were performed with the groups as fixed effects , and serum phosphorus concentrations at week 0 as covariates , and the noninferiority of pa21 versus sevelamer was confirmed . if the upper limit of the twosided 95% confidence interval did not exceed 0.32 serum phosphorus concentrations at each timepoint , summary statistics for each of the secondary endpoint measures by group at each evaluation time , and twosided 95% confidence intervals for means were calculated . in the pa21 and sevelamer groups , respectively , 14 patients ( 13.0% ) and 18 patients ( 17.1% ) discontinued the study . a total of 94 ( 87.0% ) and 87 ( 82.9% ) of the patients in the pa21 and sevelamer group completed the study , respectively . patient demographic and clinical characteristics ( per protocol set ) fisher 's exact test ; twosample wilcoxon test ; ns , not significant the adjusted mean serum phosphorus concentration at the end of treatment was 1.62 mmol / l in the pa21 group and 1.72 mmol / l in the sevelamer group , with a difference of 0.11 mmol / l ( 95% confidence interval [ ci ] , 0.20 to 0.02 mmol / l ) in the per protocol set ( fig . furthermore , the mean serum phosphorus concentration at the end of treatment was significantly lower in the pa21 group compared with that in the sevelamer group ( ancova , p = 0.020 ) . the adjusted mean serum phosphorus concentration at the end of treatment confirmed the noninferiority of pa21 ( sucroferric oxyhydroxide ) to sevelamer ( 1.62 vs 1.72 mmol / l ; 95% ci , 0.20 to 0.02 mmol / l ; ancova , p = 0.020 ) . the mean daily tablet intake at the end of treatment was 5.6 2.6 tablets ( mean daily dose , 1403 654 mg ) in the pa21 group and 18.7 7.1 tablets ( mean daily dose , 4671 1776 mg ) in the sevelamer group . mean daily number of tablets for daily dose of pa21 ( sucroferric oxyhydroxide ) and sevelamer ( per protocol set ) . at the end of treatment , the achievement rate of target serum phosphorus concentration ( 1.131.94 mmol / the rates were over 80% in the pa21 group after week 6 ; meanwhile , the rates were significantly higher in the pa21 group than in the sevelamer group after week 8 ( fig . mmol / l ) was 63.0% in the pa21 group and 52.2% in the sevelamer group at the end of treatment . the achievement rates of target serum phosphorus concentration ( 1.131.94 mmol / l ) at the end of treatment were 82.0% and 67.4% in the pa21 ( sucroferric oxyhydroxide ) and sevelamer groups , respectively . the incidence of aes ( excluding discolouration events caused by the iron contained in pa21 ) was 75.0% ( 81/108 ) and 66.7% ( 70/105 ) , and the incidence of adrs was 26.9% ( 29/108 ) and 26.7% ( 28/105 ) in the pa21 group and the sevelamer group , respectively ( table 3 ) . adverse events that occurred at an incidence of 5% , adverse drug reactions that occurred at an incidence of 2% and adverse events that led to withdrawal at an incidence of 2% ( excluding discolouration events caused by the iron contained in pa21 ) ( safety set ) iron parameters , such as serum ferritin , transferrin saturation , and haemoglobin , were prone to increase in the pa21 group . in the pa21 and sevelamer groups , respectively , 14 patients ( 13.0% ) and 18 patients ( 17.1% ) discontinued the study . a total of 94 ( 87.0% ) and 87 ( 82.9% ) of the patients in the pa21 and sevelamer group completed the study , respectively . patient demographic and clinical characteristics ( per protocol set ) fisher 's exact test ; twosample wilcoxon test ; ns , not significant mmol / l in the pa21 group and 1.72 mmol / l in the sevelamer group , with a difference of 0.11 mmol / l ( 95% confidence interval [ ci ] , 0.20 to 0.02 mmol / l ) in the per protocol set ( fig . furthermore , the mean serum phosphorus concentration at the end of treatment was significantly lower in the pa21 group compared with that in the sevelamer group ( ancova , p = 0.020 ) . the adjusted mean serum phosphorus concentration at the end of treatment confirmed the noninferiority of pa21 ( sucroferric oxyhydroxide ) to sevelamer ( 1.62 vs 1.72 mmol / l ; difference , 0.11 mmol / l ; 95% ci , 0.20 to 0.02 mmol / l ; ancova , p = 0.020 ) . the mean daily tablet intake at the end of treatment was 5.6 2.6 tablets ( mean daily dose , 1403 654 mg ) in the pa21 group and 18.7 7.1 tablets ( mean daily dose , 4671 1776 mg ) in the sevelamer group . mean daily number of tablets for daily dose of pa21 ( sucroferric oxyhydroxide ) and sevelamer ( per protocol set ) . at the end of treatment , the achievement rate of target serum phosphorus concentration ( 1.131.94 mmol / l ) was 82.0% in the pa21 group and 67.4% in the sevelamer group . mmol / l ) was 63.0% in the pa21 group and 52.2% in the sevelamer group at the end of treatment . the achievement rates of target serum phosphorus concentration ( 1.131.94 mmol / l ) at the end of treatment were 82.0% and 67.4% in the pa21 ( sucroferric oxyhydroxide ) and sevelamer groups , respectively . the incidence of aes ( excluding discolouration events caused by the iron contained in pa21 ) was 75.0% ( 81/108 ) and 66.7% ( 70/105 ) , and the incidence of adrs was 26.9% ( 29/108 ) and 26.7% ( 28/105 ) in the pa21 group and the sevelamer group , respectively ( table 3 ) . adverse events that occurred at an incidence of 5% , adverse drug reactions that occurred at an incidence of 2% and adverse events that led to withdrawal at an incidence of 2% ( excluding discolouration events caused by the iron contained in pa21 ) ( safety set ) iron parameters , such as serum ferritin , transferrin saturation , and haemoglobin , were prone to increase in the pa21 group . laboratory parameters ( ferritin levels , transferrin saturation , haemoglobin , and bicarbonate ) ( safety set ) the adjusted mean serum phosphorus concentrations at the end of treatment confirmed the noninferiority of pa21 to sevelamer , and a significant decrease was observed in the pa21 group compared with the sevelamer group . that showed the noninferiority of pa21 to sevelamer.10 the efficacy of both pa21 and sevelamer in decreasing the serum phosphorus concentrations was evident at week 1 of treatment and this was maintained until the end of the treatment period . the phosphoruslowering effect in the present study was comparable to that of the previous study.10 the changes in serum phosphorus concentration from baseline to the end of treatment were comparable in both treatment groups and similar to the findings in the previous studies.9 , 10 in both groups , the serum phosphorus concentrations at the end of treatment were within the range of the control target values ( between 1.13 and 1.94 mmol / l ) . the mean daily tablet intake was markedly lower in the pa21 group than in the sevelamer group . a similar result was shown in the previous phase iii study of pa2110 in which the number of tablets for daily dose for the first 12 weeks was 2.8 tablets in the pa21 group and 7.6 tablets in sevelamer group ( ratio , 1:4.3 when converted to daily dose ) . the onset and course , such as the incidence , severity , duration , and withdrawal , of diarrhoea were comparable to those of the previous phase iii study.10 the incidence of constipation in the pa21 group was low ; therefore , dialysis patients could benefit from pa21 treatment because it is known that many ckd patients undergoing dialysis suffer from constipation due to water restriction.13 the incidence of serious adr was similar between the pa21 and sevelamer groups . regarding the clinical laboratory tests , increasing trends in serum ferritin , tsat , and haemoglobin levels were observed in the pa21 group as well as the previous phase iii study.10 however , the previous nonclinical14 and clinical studies15 of pa21 confirmed that the iron absorption level was consistently low and no iron accumulation was indicated in the previous longterm study.16 when comparing the current study and the previous phase iii study , the absolute ferritin values and fluctuation range of ferritin were higher in the previous study . in conclusion , treatment with pa21 was noninferior to that with sevelamer in japanese haemodialysis patients with hyperphosphataemia . treatment with pa21 was effective , safe , and well tolerated in this patient population , while having a considerably lower pill burden than sevelamer .

treatment with fixed orthodontic devices such as brackets and bands creates numerous plaque accumulation sites impeding oral hygiene procedures and thus potentially leading to develop white spot lesions , caries , and periodontitis . it is recognized that microbial dental plaque is the main etiologic factor in the development of dental caries and periodontal disease . plaque accumulation can favor the transition of the microbial biofilm to a more aggressive periodontopathogenic flora in subgingival periodontal pockets and the production of proinflammatory cytokines . during fixed orthodontic treatment inflammation occurs and pathologic phenomena such as gingivitis , gingival bleeding , gingival enlargement , and increased gingival pocket depth are observed . furthermore , microbiological studies revealed significant changes in the bacterial composition of the subgingival dental plaque , so orthodontic treatment may affect the equilibrium of oral microflora and increase bacteria retention . it has been shown that treatment with fixed orthodontic appliances stimulates the growth of a subgingival plaque where some periodontopathogenic bacterial strains are prevalent such as porphyromonas gingivalis , prevotella intermedia , bacteroides forsythus , actinobacillus actinomycetemcomitans , fusobacterium nucleatum , and treponema denticola . in contrast , the use of removable orthodontic appliance is able to allow an adequate oral hygiene and reduce the risk for such negative dental and periodontal complication . orthodontic treatment should be able to expose the patients to none or limited side effects . along with the risk of root resorption periodontal complications are the most reported to occur . periodontal health should be regarded as one of the success criteria in orthodontic treatment . in 1999 , a new orthodontic system based on a polymer composed by a chain of organic units joined with urethane links was introduced ( invisalign , align technology , santa clara , california ) as a removable appliance able to gradually move the teeth to a treatment plan , which was formerly computer designed . few studies evaluated the subgingival pathogenic microflora via real - time polymerase chain reaction ( pcr ) analyses in fixed orthodontic therapy , and there is only one preliminary report evaluating microbiological and periodontal data related to periodontal disease risk development in patient treated with fixed and removable appliances . to date , the literature is scarce about scientific trials that are committed to elucidate the relationship between orthodontic treatment and periodontal health status . to the authors ' knowledge , there are only clinical trials evaluating the periodontal health of clear aligners compared to traditional orthodontic appliances but none compared the microbiological aspect of the invisalign treatment to the fixed orthodontic appliances . the hypothesis that we investigated was that patients treated with invisalign aligners had a better periodontal health compared to patients who were treated with fixed appliances . thus , the aim of this study was to evaluate the total microbiological biofilm mass and the presence of selected bacteria , via real - time pcr , in adults undergoing fixed or removable orthodontic therapy with the invisalign system . the modified plaque index ( pi ) , pocket probing depth ( pd ) , and the bleeding on probing ( bop ) were also evaluated during the entire period of treatment by clinical assessment . seventy - seven patients , 52 females and 25 males with a mean age of 24.3 years ( range from 16 to 30 ) were included in this study referring to the department of orthodontics of the university of insubria . sixty - seven patients referred to our clinic for orthodontic treatment and were randomly selected to the test invisalign treatment group and the fixed appliance treatment group . a group of ten patients who did not need any treatment was used as control group . composition of the groups smoking habitpresence of extensive dental restorations in proximity to the gingival marginpresence of fixed bridges / crowns or partial denturesprevious periodontal nonsurgical treatment ( such as full mouth disinfection , quadrant - by - quadrant therapy , full mouth debridement ) within the past yearmedications such as antibiotics , steroids , or nonsteroidal anti - inflammatory drugs within the past 6 months . presence of extensive dental restorations in proximity to the gingival margin presence of fixed bridges / crowns or partial dentures previous periodontal nonsurgical treatment ( such as full mouth disinfection , quadrant - by - quadrant therapy , full mouth debridement ) within the past year medications such as antibiotics , steroids , or nonsteroidal anti - inflammatory drugs within the past 6 months . moreover , the patients used no oral antiseptic solutions or mouthwash during the entire investigation , but who used dietary supplements with antioxidant properties were not excluded . class i skeletal relationshipnormo - divergent frankfort mandibular - plane angleage > 16class i molar relationshipminimal mandibular crowding in a range from 1 to 3 according to little 's index . class i skeletal relationship normo - divergent frankfort mandibular - plane angle class i molar relationship minimal mandibular crowding in a range from 1 to 3 according to little 's index . all patients were informed of the nature of the study to be carried out on an individual basis and read and signed a written consent form . the study protocol was conducted in accordance with the helsinki declaration of 1975 , as revised in 2007 . one - month before orthodontic therapy , professional oral hygiene was performed , and patients were instructed on a standardized oral hygiene protocol . oral hygiene instructions were specified by an experienced dental hygienist before the treatment and recapitulated during all the scheduled check - up . all patients had to use an orthodontic brush ( bass technique for 2 min ) and dental floss three times a day . the fixed orthodontic treatment was performed in all patients by treating the upper and lower arch simultaneously . mini sprint brackets ( forestadent , pforzheim , germany ) and standard elastic ligatures were used on incisors , canines , and premolars ; orthodontic bonded tubes were used for the first molars ( forestadent ) . the bonding procedure was performed with a direct technique using transbond xt ( 3 m , st . the patients in the invisalign group were instructed to wear the aligners 20 h a day . the invisalign aligners were replaced every 2 weeks with a new set , which had been previously developed according to the treatment plan of each single patient . the clinical assessment of the periodontal health status was achieved using the periodontal index , according to the criteria of the modified pi of loe and silness , pocket pd and bop . the pocket pd was measured to the nearest millimeter on the scale of the periodontal probe ( goldman - fox , hu - friedy mfg co. , inc . , chicago , il , usa ) and bop tendency was registered 20 s after probing ( absent = 0 , present = 1 ) . the pi was assessed by observing the plaque accumulation in the gingival area and was classified into one of four grades . scoring criteria were : 0 = no plaque / debris on inspection and probing1 = thin film of plaque only visible after probing2 = ribbon - like layer of plaque covering the gingival sulcus with no involvement of interproximal dental space3 = thick layer of plaque clearly visible at inspection and involving an interproximal dental space . 0 = no plaque / debris on inspection and probing 1 = thin film of plaque only visible after probing 2 = ribbon - like layer of plaque covering the gingival sulcus with no involvement of interproximal dental space 3 = thick layer of plaque clearly visible at inspection and involving an interproximal dental space . these clinical parameters were assessed on the mesio - vestibular surface of the examined teeth : upper right first molar ( site 0 ) and upper left central incisor ( site 1 ) , according to the ramfjord system . this periodontal assessment was performed at the beginning of the orthodontic treatment ( t0 ) , after 1-month ( t1 ) and after 3 months , corresponding to the end of the treatment ( t2 ) . the scoring registrations were executed by a single calibrated examiner while all reviews according to the protocol were carried out by two operators who were unaware of the experimental protocol . the microbiological samples were obtained from the same sites ( site 0 and 1 ) at t0 , t1 , and t3 as previously described in the periodontal assessment . in order to evaluate the biofilm present in the experimental sites , the microbiological investigation was performed to confirm the presence or absence of four periodontopathic anaerobes species : p. intermedia , a. actinomycetemcomitans , p. gingivalis , tannerella forsythia . these samples were collected in dry field conditions by inserting one sterile paper point into the deepest part of the gingival sulcus for 30 s. after insertion , paper points were closed into a test tube , refrigerated at 20c and sent to the dna sequencing service , university of cagliari , italy , where the microbiological analysis was performed . each paper point was suspended in 50 l of pure dimethyl sulfoxide ( dmso ) and centrifuged for 30 s. two microliter was used as dna suspension for real - time pcr reactions . periodontal pathogen and total bacteria enumeration ( biofilm mass ) were detected by real - time pcr procedure and molecular analysis protocols used in this paper has been described in previously published papers . real - time pcr was performed using a lightcycler instrument and a lightcycler dna master sybr green i kit ( roche diagnostics mannheim germany ) , according to the manufacturer 's instructions . among 10 fold serial dilutions of each bacterium in dmso ranging from 10 to 10 cells pcr mixture contained ( 20 l final volume ) : 4 mm mgcl2 , 1 m of each primer , and 2 l of dmso suspension . the pcr program was the following : ( i ) denaturation at 95c for 30 s , ( ii ) 40 cycles of 10 s at 95c , 10 s at 50c , 12 s at 72c , ( iii ) melting curve performed for 10 s at 95c , 45c , 95c . transition rates were 5c / s in the 72c segment , 0.1c / s in the 45c segment , and 20c / s for another step . fluorescence was detected at the end of the 72c segment in the pcr step ( single mode ) , and at the 45c segment in the melting step ( continuous mode ) in the f1 channel . during initial optimization of real - time reaction , pcr products were analyzed using agarose gel and by a melting curve analysis to ensure correct sample product size . the amount of bacterial dna in the samples was calculated following sequent formula ( c = q 25 ) , c is the final bacterial concentration ( totals or single periodontal pathogen ) in the specimen ; q is the bacterial number calculated interpolating threshold cycle with a qpcr standard curve . to compare the differences of the periodontal indices such as pi , bop , pd ; and the differences between the microbiological biofilm in the patients treated respectively with invisalign , fixed appliances and control group the mann whitney test was performed to test differences between different time - points in each group . all statistical analyses were run on the statistical package spss ( spss 17.0 ; spss inc . a priori sample size calculation was performed with = 0.05 and a power set at 80% . seventy - seven patients , 52 females and 25 males with a mean age of 24.3 years ( range from 16 to 30 ) were included in this study referring to the department of orthodontics of the university of insubria . sixty - seven patients referred to our clinic for orthodontic treatment and were randomly selected to the test invisalign treatment group and the fixed appliance treatment group . a group of ten patients who did not need any treatment was used as control group . smoking habitpresence of extensive dental restorations in proximity to the gingival marginpresence of fixed bridges / crowns or partial denturesprevious periodontal nonsurgical treatment ( such as full mouth disinfection , quadrant - by - quadrant therapy , full mouth debridement ) within the past yearmedications such as antibiotics , steroids , or nonsteroidal anti - inflammatory drugs within the past 6 months . presence of extensive dental restorations in proximity to the gingival margin presence of fixed bridges / crowns or partial dentures previous periodontal nonsurgical treatment ( such as full mouth disinfection , quadrant - by - quadrant therapy , full mouth debridement ) within the past year medications such as antibiotics , steroids , or nonsteroidal anti - inflammatory drugs within the past 6 months . moreover , the patients used no oral antiseptic solutions or mouthwash during the entire investigation , but who used dietary supplements with antioxidant properties were not excluded . class i skeletal relationshipnormo - divergent frankfort mandibular - plane angleage > 16class i molar relationshipminimal mandibular crowding in a range from 1 to 3 according to little 's index . class i skeletal relationship normo - divergent frankfort mandibular - plane angle class i molar relationship minimal mandibular crowding in a range from 1 to 3 according to little 's index . all patients were informed of the nature of the study to be carried out on an individual basis and read and signed a written consent form . the study protocol was conducted in accordance with the helsinki declaration of 1975 , as revised in 2007 . one - month before orthodontic therapy , professional oral hygiene was performed , and patients were instructed on a standardized oral hygiene protocol . oral hygiene instructions were specified by an experienced dental hygienist before the treatment and recapitulated during all the scheduled check - up . all patients had to use an orthodontic brush ( bass technique for 2 min ) and dental floss three times a day . the fixed orthodontic treatment was performed in all patients by treating the upper and lower arch simultaneously . mini sprint brackets ( forestadent , pforzheim , germany ) and standard elastic ligatures were used on incisors , canines , and premolars ; orthodontic bonded tubes were used for the first molars ( forestadent ) . the bonding procedure was performed with a direct technique using transbond xt ( 3 m , st . the patients in the invisalign group were instructed to wear the aligners 20 h a day . the invisalign aligners were replaced every 2 weeks with a new set , which had been previously developed according to the treatment plan of each single patient . the clinical assessment of the periodontal health status was achieved using the periodontal index , according to the criteria of the modified pi of loe and silness , pocket pd and bop . the pocket pd was measured to the nearest millimeter on the scale of the periodontal probe ( goldman - fox , hu - friedy mfg co. , inc . , chicago , il , usa ) and bop tendency was registered 20 s after probing ( absent = 0 , present = 1 ) . the pi was assessed by observing the plaque accumulation in the gingival area and was classified into one of four grades . scoring criteria were : 0 = no plaque / debris on inspection and probing1 = thin film of plaque only visible after probing2 = ribbon - like layer of plaque covering the gingival sulcus with no involvement of interproximal dental space3 = thick layer of plaque clearly visible at inspection and involving an interproximal dental space . 0 = no plaque / debris on inspection and probing 1 = thin film of plaque only visible after probing 2 = ribbon - like layer of plaque covering the gingival sulcus with no involvement of interproximal dental space 3 = thick layer of plaque clearly visible at inspection and involving an interproximal dental space . these clinical parameters were assessed on the mesio - vestibular surface of the examined teeth : upper right first molar ( site 0 ) and upper left central incisor ( site 1 ) , according to the ramfjord system . this periodontal assessment was performed at the beginning of the orthodontic treatment ( t0 ) , after 1-month ( t1 ) and after 3 months , corresponding to the end of the treatment ( t2 ) . the scoring registrations were executed by a single calibrated examiner while all reviews according to the protocol were carried out by two operators who were unaware of the experimental protocol . the microbiological samples were obtained from the same sites ( site 0 and 1 ) at t0 , t1 , and t3 as previously described in the periodontal assessment . in order to evaluate the biofilm present in the experimental sites , the microbiological investigation was performed to confirm the presence or absence of four periodontopathic anaerobes species : p. intermedia , a. actinomycetemcomitans , p. gingivalis , tannerella forsythia . these samples were collected in dry field conditions by inserting one sterile paper point into the deepest part of the gingival sulcus for 30 s. after insertion , paper points were closed into a test tube , refrigerated at 20c and sent to the dna sequencing service , university of cagliari , italy , where the microbiological analysis was performed . each paper point was suspended in 50 l of pure dimethyl sulfoxide ( dmso ) and centrifuged for 30 s. two microliter was used as dna suspension for real - time pcr reactions . periodontal pathogen and total bacteria enumeration ( biofilm mass ) were detected by real - time pcr procedure and molecular analysis protocols used in this paper has been described in previously published papers . real - time pcr was performed using a lightcycler instrument and a lightcycler dna master sybr green i kit ( roche diagnostics mannheim germany ) , according to the manufacturer 's instructions . among 10 fold serial dilutions of each bacterium in dmso ranging from 10 to 10 cells / ml pcr mixture contained ( 20 l final volume ) : 4 mm mgcl2 , 1 m of each primer , and 2 l of dmso suspension . the pcr program was the following : ( i ) denaturation at 95c for 30 s , ( ii ) 40 cycles of 10 s at 95c , 10 s at 50c , 12 s at 72c , ( iii ) melting curve performed for 10 s at 95c , 45c , 95c . transition rates were 5c / s in the 72c segment , 0.1c / s in the 45c segment , and 20c / s for another step . fluorescence was detected at the end of the 72c segment in the pcr step ( single mode ) , and at the 45c segment in the melting step ( continuous mode ) in the f1 channel . during initial optimization of real - time reaction , pcr products were analyzed using agarose gel and by a melting curve analysis to ensure correct sample product size . the amount of bacterial dna in the samples was calculated following sequent formula ( c = q 25 ) , c is the final bacterial concentration ( totals or single periodontal pathogen ) in the specimen ; q is the bacterial number calculated interpolating threshold cycle with a qpcr standard curve . to compare the differences of the periodontal indices such as pi , bop , pd ; and the differences between the microbiological biofilm in the patients treated respectively with invisalign , fixed appliances and control group the mann whitney test was performed to test differences between different time - points in each group . all statistical analyses were run on the statistical package spss ( spss 17.0 ; spss inc . , a priori sample size calculation was performed with = 0.05 and a power set at 80% . the microbiological analyses detected the presence of a. actinomycetemcomitans only in one patient treated with fixed orthodontic appliances at t1 and t2 . a statistically significant difference ( p < 0.05 ) was found between the invisalign group and the fixed orthodontic appliance group in all periodontal parameters ( bop , pd , and pi ) with the invisalign group scoring lower values compared to the fixed orthodontic appliance group . furthermore , the total biofilm mass showed a statistically significant differences ( p < 0.05 ) between the invisalign group and the fixed orthodontic appliance group . the periodontal parameters showed worst scores in t2 compared to t0 and t1 in the fixed orthodontic appliance group , as well as the total biofilm mass . the invisalign group showed a statistically significant increase in the pi values in the t2 compared to t0 . furthermore , no statistically significant differences in the bop and in the pd were observed . the invisalign group showed a statistically significant difference ( p < 0.05 ) between the t2 and t0 in the total biofilm mass with a lower score in the 90 days follow - up control . in the control group , no statistical differences were found between the follow - up controls . at t2 the mean bacterial concentration c was 104,536,026 ; 2739 and 8187 in the fixed orthodontic , the invisalign and control group , respectively , being significantly lower for the two latter groups . the mean pd in the fixed orthodontic group ; invisalign group and control group at t2 were , respectively , 1.3;1.6;1.7 . the study the mean little 's index score for the invisalign group was 2.3 0.3 , for the fixed appliances group 2.5 0.4 and for the control group 2.3 0.4 . the present study has described microbiological and periodontal changes in two groups of patients treated , respectively , with fixed appliances and invisalign removable aligners . the effect of orthodontic appliances on periodontal health has been evaluated in many studies . a systematic review of the literature about the relationship between orthodontic treatment effect and periodontal health stated that gingivitis and attachment loss were inconsistent across studies , and that there is an absence of evidence supporting positive effects of orthodontic treatment on overall periodontal health , but many data indicate that orthodontic therapy may result in small detrimental effects to the periodontium . plaque accumulation is the main etiological factor , and gingival inflammation enhances the flowing of gingival crevicular fluids that supply plasma proteins , which are essential for the growth of proteolytic anaerobes . our data showed how fixed orthodontic treatment group resulted in higher plaque accumulation compared to invisalign treatment group . this result can be attributable to easier oral hygiene procedures favored by a better accessibility in invisalign patients . furthermore , fixed orthodontic devices present more plaque retention sites that potentially lead to periodontal inflammation . a significant increase of pi and bop was found in patients treated with fixed orthodontic , but pd index had no significant changes . the increase in biofilm mass was a direct consequence of impeded oral hygiene procedures . this clearly show how patients treated with fixed appliances are more likely susceptible to gingival inflammation that eventually could develop to periodontal disease . a recent investigation by ghijselings et al . showed how in the long - term patient treated with fixed orthodontic appliances have a worsening in the periodontal parameters . on the contrary , the microbiological analyses underlined a significantly difference between the aerobe / anaerobe ratio prior and after the treatment . some clinical studies reported poor periodontal health and greater loss of clinical attachment level in the distal area of the dental arches in patients treated with fixed orthodontic treatment . these worse data could be the result of poor oral hygiene in molar regions also due to the presence of molar bands , which favor food entrapment . statistical differences between t0 and t2 of the bop , pd and pi were found in the fixed orthodontic treatment group . these results are similar to the one reported by ristic et al . and by demling et al . that observed an increase in the periodontal indices and a modification of the microbiological composition . the change in the microbiological composition with the shift of microbiological flora is due to the food entrapment that eventually lead to plaque accumulation and inflammation . in our study , we performed a professional cleaning in order to eradicate possibly periodontal pathogens . a recent investigation using atomic force microscope by germano et al . showed how periodontitis bacteria have complex glycocalyx being also able to co - aggregate thus improving their resistance to antibiotics . decreased plaque level were found in the invisalign treatment group and were associated with better periodontal health indices ; these results are in accordance with karkhanechi et al . a possible explanation can be attributable to easier oral hygiene procedures ; the absence of bands , brackets and archwires in the patients treated with invisalign can favor the maintenance of better oral hygiene . a recent systematic review of the literature showed how clear aligner treatments have an improvement in periodontal health indexes compared to fixed orthodontic treatments . the overall higher periodontal indices and microbiological results can be attributable to better compliance in oral hygiene procedures in invisalign group as showed in a previous study . a main difference in our study , is that we did not evaluate the patients ' compliance . as the clear aligners are removable appliances giving to the patients easy access to all teeth surfaces it can be assumed that this treatment option should be a first choice in adult patients and in patients with possible periodontal problems . it is important to stress that a careful hygiene maintenance of the aligners must be performed in order to control the plaque accumulation on the clear aligners . one of the limitations of this study , is the short follow - up period , a longer observational time would be useful for the evaluation of the plaque accumulation and the periodontal indices because the change of the bacterial flora in patients receiving fixed appliances take place in the first 3 months . to our knowledge , this is the first prospective study , to compare the periodontal status and the plaque accumulation via real - time pcr between fixed buccal appliances and removable aligners in the short - term period . although periodontal status showed the worst score in patients receiving the fixed appliance treatment , we do not suggest to avoid this kind of treatment in adult patients . furthermore , there is still uncertainty about the long - term possible negative effect of fixed orthodontic appliance on periodontal health . within the limit of this study , we can state that : patients treated with removable aligners had a better periodontal health status ( pi , pd , bop ) compared to patients treated with fixed appliancesremovable aligners seem to facilitate oral hygiene proceduresabsence of periodontal pathogenic bacteria in invisalign treatment groupreal - time pcr analysis detected a periodontopathic bacteria in one patient treated with fixed orthodontic devicereal - time pcr showed higher level of bacteria concentration in patients treated with fixed orthodontic device . patients treated with removable aligners had a better periodontal health status ( pi , pd , bop ) compared to patients treated with fixed appliances removable aligners seem to facilitate oral hygiene procedures absence of periodontal pathogenic bacteria in invisalign treatment group real - time pcr analysis detected a periodontopathic bacteria in one patient treated with fixed orthodontic device real - time pcr showed higher level of bacteria concentration in patients treated with fixed orthodontic device .

objective : the aim of this prospective study was to compare the periodontal health and the microbiological changes via real - time polymerase chain reaction ( pcr ) in patients treated with fixed orthodontic appliances and invisalign system ( align technology , santa clara , california).materials and methods : seventy - seven patients were enrolled in this study and divided into three groups ( invisalign group , fixed orthodontic appliances group and control group ) . plaque index , probing depth , bleeding on probing were assessed . total biofilm mass and periodontal pathogens were analyzed and detected via real - time pcr . all these data were analyzed at the t0 ( beginning of the treatment ) t1 ( 1-month ) and t2 ( 3 months ) ; and statistically compared using the mann whitney test for independent groups.results:after 1-month and after 3 months of treatment there was only one sample with periodontopathic anaerobes found in patient treated using fixed orthodontic appliances . the invisalign group showed better results in terms of periodontal health and total biofilm mass compared to the fixed orthodontic appliance group . a statistical significant difference ( p < 0.05 ) at the t2 in the total biofilm mass was found between the two groups.conclusion:patients undergoing orthodontic treatment with the invisalign system show a superior periodontal health in the short - term when compared to patients in treatment with fixed orthodontic appliances . invisalign should be considered as a first treatment option in patients with risk of developing periodontal disease .

INTRODUCTION MATERIALS AND METHODS Patient population Exclusion criteria Inclusion criteria Periodontal indices Evaluation of total biofilm mass and periodontopathic bacterial species Molecular analysis Statistical analysis RESULTS DISCUSSION CONCLUSIONS Financial support and sponsorship Conflicts of interest

treatment with fixed orthodontic devices such as brackets and bands creates numerous plaque accumulation sites impeding oral hygiene procedures and thus potentially leading to develop white spot lesions , caries , and periodontitis . it is recognized that microbial dental plaque is the main etiologic factor in the development of dental caries and periodontal disease . plaque accumulation can favor the transition of the microbial biofilm to a more aggressive periodontopathogenic flora in subgingival periodontal pockets and the production of proinflammatory cytokines . during fixed orthodontic treatment inflammation occurs and pathologic phenomena such as gingivitis , gingival bleeding , gingival enlargement , and increased gingival pocket depth are observed . furthermore , microbiological studies revealed significant changes in the bacterial composition of the subgingival dental plaque , so orthodontic treatment may affect the equilibrium of oral microflora and increase bacteria retention . it has been shown that treatment with fixed orthodontic appliances stimulates the growth of a subgingival plaque where some periodontopathogenic bacterial strains are prevalent such as porphyromonas gingivalis , prevotella intermedia , bacteroides forsythus , actinobacillus actinomycetemcomitans , fusobacterium nucleatum , and treponema denticola . in contrast , the use of removable orthodontic appliance is able to allow an adequate oral hygiene and reduce the risk for such negative dental and periodontal complication . orthodontic treatment should be able to expose the patients to none or limited side effects . along with the risk of root resorption periodontal complications are the most reported to occur . periodontal health should be regarded as one of the success criteria in orthodontic treatment . in 1999 , a new orthodontic system based on a polymer composed by a chain of organic units joined with urethane links was introduced ( invisalign , align technology , santa clara , california ) as a removable appliance able to gradually move the teeth to a treatment plan , which was formerly computer designed . few studies evaluated the subgingival pathogenic microflora via real - time polymerase chain reaction ( pcr ) analyses in fixed orthodontic therapy , and there is only one preliminary report evaluating microbiological and periodontal data related to periodontal disease risk development in patient treated with fixed and removable appliances . to date , the literature is scarce about scientific trials that are committed to elucidate the relationship between orthodontic treatment and periodontal health status . to the authors ' knowledge , there are only clinical trials evaluating the periodontal health of clear aligners compared to traditional orthodontic appliances but none compared the microbiological aspect of the invisalign treatment to the fixed orthodontic appliances . the hypothesis that we investigated was that patients treated with invisalign aligners had a better periodontal health compared to patients who were treated with fixed appliances . thus , the aim of this study was to evaluate the total microbiological biofilm mass and the presence of selected bacteria , via real - time pcr , in adults undergoing fixed or removable orthodontic therapy with the invisalign system . the modified plaque index ( pi ) , pocket probing depth ( pd ) , and the bleeding on probing ( bop ) were also evaluated during the entire period of treatment by clinical assessment . seventy - seven patients , 52 females and 25 males with a mean age of 24.3 years ( range from 16 to 30 ) were included in this study referring to the department of orthodontics of the university of insubria . sixty - seven patients referred to our clinic for orthodontic treatment and were randomly selected to the test invisalign treatment group and the fixed appliance treatment group . composition of the groups smoking habitpresence of extensive dental restorations in proximity to the gingival marginpresence of fixed bridges / crowns or partial denturesprevious periodontal nonsurgical treatment ( such as full mouth disinfection , quadrant - by - quadrant therapy , full mouth debridement ) within the past yearmedications such as antibiotics , steroids , or nonsteroidal anti - inflammatory drugs within the past 6 months . all patients were informed of the nature of the study to be carried out on an individual basis and read and signed a written consent form . the fixed orthodontic treatment was performed in all patients by treating the upper and lower arch simultaneously . the bonding procedure was performed with a direct technique using transbond xt ( 3 m , st . the patients in the invisalign group were instructed to wear the aligners 20 h a day . the invisalign aligners were replaced every 2 weeks with a new set , which had been previously developed according to the treatment plan of each single patient . the clinical assessment of the periodontal health status was achieved using the periodontal index , according to the criteria of the modified pi of loe and silness , pocket pd and bop . the pocket pd was measured to the nearest millimeter on the scale of the periodontal probe ( goldman - fox , hu - friedy mfg co. , inc . these clinical parameters were assessed on the mesio - vestibular surface of the examined teeth : upper right first molar ( site 0 ) and upper left central incisor ( site 1 ) , according to the ramfjord system . this periodontal assessment was performed at the beginning of the orthodontic treatment ( t0 ) , after 1-month ( t1 ) and after 3 months , corresponding to the end of the treatment ( t2 ) . the scoring registrations were executed by a single calibrated examiner while all reviews according to the protocol were carried out by two operators who were unaware of the experimental protocol . the microbiological samples were obtained from the same sites ( site 0 and 1 ) at t0 , t1 , and t3 as previously described in the periodontal assessment . in order to evaluate the biofilm present in the experimental sites , the microbiological investigation was performed to confirm the presence or absence of four periodontopathic anaerobes species : p. intermedia , a. actinomycetemcomitans , p. gingivalis , tannerella forsythia . these samples were collected in dry field conditions by inserting one sterile paper point into the deepest part of the gingival sulcus for 30 s. after insertion , paper points were closed into a test tube , refrigerated at 20c and sent to the dna sequencing service , university of cagliari , italy , where the microbiological analysis was performed . each paper point was suspended in 50 l of pure dimethyl sulfoxide ( dmso ) and centrifuged for 30 s. two microliter was used as dna suspension for real - time pcr reactions . periodontal pathogen and total bacteria enumeration ( biofilm mass ) were detected by real - time pcr procedure and molecular analysis protocols used in this paper has been described in previously published papers . real - time pcr was performed using a lightcycler instrument and a lightcycler dna master sybr green i kit ( roche diagnostics mannheim germany ) , according to the manufacturer 's instructions . transition rates were 5c / s in the 72c segment , 0.1c / s in the 45c segment , and 20c / s for another step . fluorescence was detected at the end of the 72c segment in the pcr step ( single mode ) , and at the 45c segment in the melting step ( continuous mode ) in the f1 channel . during initial optimization of real - time reaction , pcr products were analyzed using agarose gel and by a melting curve analysis to ensure correct sample product size . the amount of bacterial dna in the samples was calculated following sequent formula ( c = q 25 ) , c is the final bacterial concentration ( totals or single periodontal pathogen ) in the specimen ; q is the bacterial number calculated interpolating threshold cycle with a qpcr standard curve . to compare the differences of the periodontal indices such as pi , bop , pd ; and the differences between the microbiological biofilm in the patients treated respectively with invisalign , fixed appliances and control group the mann whitney test was performed to test differences between different time - points in each group . seventy - seven patients , 52 females and 25 males with a mean age of 24.3 years ( range from 16 to 30 ) were included in this study referring to the department of orthodontics of the university of insubria . sixty - seven patients referred to our clinic for orthodontic treatment and were randomly selected to the test invisalign treatment group and the fixed appliance treatment group . all patients were informed of the nature of the study to be carried out on an individual basis and read and signed a written consent form . the study protocol was conducted in accordance with the helsinki declaration of 1975 , as revised in 2007 . the fixed orthodontic treatment was performed in all patients by treating the upper and lower arch simultaneously . the patients in the invisalign group were instructed to wear the aligners 20 h a day . the invisalign aligners were replaced every 2 weeks with a new set , which had been previously developed according to the treatment plan of each single patient . the clinical assessment of the periodontal health status was achieved using the periodontal index , according to the criteria of the modified pi of loe and silness , pocket pd and bop . the pocket pd was measured to the nearest millimeter on the scale of the periodontal probe ( goldman - fox , hu - friedy mfg co. , inc . , chicago , il , usa ) and bop tendency was registered 20 s after probing ( absent = 0 , present = 1 ) . these clinical parameters were assessed on the mesio - vestibular surface of the examined teeth : upper right first molar ( site 0 ) and upper left central incisor ( site 1 ) , according to the ramfjord system . this periodontal assessment was performed at the beginning of the orthodontic treatment ( t0 ) , after 1-month ( t1 ) and after 3 months , corresponding to the end of the treatment ( t2 ) . the scoring registrations were executed by a single calibrated examiner while all reviews according to the protocol were carried out by two operators who were unaware of the experimental protocol . the microbiological samples were obtained from the same sites ( site 0 and 1 ) at t0 , t1 , and t3 as previously described in the periodontal assessment . in order to evaluate the biofilm present in the experimental sites , the microbiological investigation was performed to confirm the presence or absence of four periodontopathic anaerobes species : p. intermedia , a. actinomycetemcomitans , p. gingivalis , tannerella forsythia . these samples were collected in dry field conditions by inserting one sterile paper point into the deepest part of the gingival sulcus for 30 s. after insertion , paper points were closed into a test tube , refrigerated at 20c and sent to the dna sequencing service , university of cagliari , italy , where the microbiological analysis was performed . each paper point was suspended in 50 l of pure dimethyl sulfoxide ( dmso ) and centrifuged for 30 s. two microliter was used as dna suspension for real - time pcr reactions . periodontal pathogen and total bacteria enumeration ( biofilm mass ) were detected by real - time pcr procedure and molecular analysis protocols used in this paper has been described in previously published papers . real - time pcr was performed using a lightcycler instrument and a lightcycler dna master sybr green i kit ( roche diagnostics mannheim germany ) , according to the manufacturer 's instructions . fluorescence was detected at the end of the 72c segment in the pcr step ( single mode ) , and at the 45c segment in the melting step ( continuous mode ) in the f1 channel . during initial optimization of real - time reaction , pcr products were analyzed using agarose gel and by a melting curve analysis to ensure correct sample product size . the amount of bacterial dna in the samples was calculated following sequent formula ( c = q 25 ) , c is the final bacterial concentration ( totals or single periodontal pathogen ) in the specimen ; q is the bacterial number calculated interpolating threshold cycle with a qpcr standard curve . to compare the differences of the periodontal indices such as pi , bop , pd ; and the differences between the microbiological biofilm in the patients treated respectively with invisalign , fixed appliances and control group the mann whitney test was performed to test differences between different time - points in each group . , a priori sample size calculation was performed with = 0.05 and a power set at 80% . the microbiological analyses detected the presence of a. actinomycetemcomitans only in one patient treated with fixed orthodontic appliances at t1 and t2 . a statistically significant difference ( p < 0.05 ) was found between the invisalign group and the fixed orthodontic appliance group in all periodontal parameters ( bop , pd , and pi ) with the invisalign group scoring lower values compared to the fixed orthodontic appliance group . furthermore , the total biofilm mass showed a statistically significant differences ( p < 0.05 ) between the invisalign group and the fixed orthodontic appliance group . the periodontal parameters showed worst scores in t2 compared to t0 and t1 in the fixed orthodontic appliance group , as well as the total biofilm mass . the invisalign group showed a statistically significant increase in the pi values in the t2 compared to t0 . the invisalign group showed a statistically significant difference ( p < 0.05 ) between the t2 and t0 in the total biofilm mass with a lower score in the 90 days follow - up control . in the control group , no statistical differences were found between the follow - up controls . at t2 the mean bacterial concentration c was 104,536,026 ; 2739 and 8187 in the fixed orthodontic , the invisalign and control group , respectively , being significantly lower for the two latter groups . the mean pd in the fixed orthodontic group ; invisalign group and control group at t2 were , respectively , 1.3;1.6;1.7 . the study the mean little 's index score for the invisalign group was 2.3 0.3 , for the fixed appliances group 2.5 0.4 and for the control group 2.3 0.4 . the present study has described microbiological and periodontal changes in two groups of patients treated , respectively , with fixed appliances and invisalign removable aligners . the effect of orthodontic appliances on periodontal health has been evaluated in many studies . a systematic review of the literature about the relationship between orthodontic treatment effect and periodontal health stated that gingivitis and attachment loss were inconsistent across studies , and that there is an absence of evidence supporting positive effects of orthodontic treatment on overall periodontal health , but many data indicate that orthodontic therapy may result in small detrimental effects to the periodontium . our data showed how fixed orthodontic treatment group resulted in higher plaque accumulation compared to invisalign treatment group . furthermore , fixed orthodontic devices present more plaque retention sites that potentially lead to periodontal inflammation . a significant increase of pi and bop was found in patients treated with fixed orthodontic , but pd index had no significant changes . the increase in biofilm mass was a direct consequence of impeded oral hygiene procedures . this clearly show how patients treated with fixed appliances are more likely susceptible to gingival inflammation that eventually could develop to periodontal disease . showed how in the long - term patient treated with fixed orthodontic appliances have a worsening in the periodontal parameters . on the contrary , the microbiological analyses underlined a significantly difference between the aerobe / anaerobe ratio prior and after the treatment . some clinical studies reported poor periodontal health and greater loss of clinical attachment level in the distal area of the dental arches in patients treated with fixed orthodontic treatment . statistical differences between t0 and t2 of the bop , pd and pi were found in the fixed orthodontic treatment group . these results are similar to the one reported by ristic et al . that observed an increase in the periodontal indices and a modification of the microbiological composition . the change in the microbiological composition with the shift of microbiological flora is due to the food entrapment that eventually lead to plaque accumulation and inflammation . decreased plaque level were found in the invisalign treatment group and were associated with better periodontal health indices ; these results are in accordance with karkhanechi et al . a possible explanation can be attributable to easier oral hygiene procedures ; the absence of bands , brackets and archwires in the patients treated with invisalign can favor the maintenance of better oral hygiene . a recent systematic review of the literature showed how clear aligner treatments have an improvement in periodontal health indexes compared to fixed orthodontic treatments . as the clear aligners are removable appliances giving to the patients easy access to all teeth surfaces it can be assumed that this treatment option should be a first choice in adult patients and in patients with possible periodontal problems . one of the limitations of this study , is the short follow - up period , a longer observational time would be useful for the evaluation of the plaque accumulation and the periodontal indices because the change of the bacterial flora in patients receiving fixed appliances take place in the first 3 months . to our knowledge , this is the first prospective study , to compare the periodontal status and the plaque accumulation via real - time pcr between fixed buccal appliances and removable aligners in the short - term period . although periodontal status showed the worst score in patients receiving the fixed appliance treatment , we do not suggest to avoid this kind of treatment in adult patients . furthermore , there is still uncertainty about the long - term possible negative effect of fixed orthodontic appliance on periodontal health . within the limit of this study , we can state that : patients treated with removable aligners had a better periodontal health status ( pi , pd , bop ) compared to patients treated with fixed appliancesremovable aligners seem to facilitate oral hygiene proceduresabsence of periodontal pathogenic bacteria in invisalign treatment groupreal - time pcr analysis detected a periodontopathic bacteria in one patient treated with fixed orthodontic devicereal - time pcr showed higher level of bacteria concentration in patients treated with fixed orthodontic device . patients treated with removable aligners had a better periodontal health status ( pi , pd , bop ) compared to patients treated with fixed appliances removable aligners seem to facilitate oral hygiene procedures absence of periodontal pathogenic bacteria in invisalign treatment group real - time pcr analysis detected a periodontopathic bacteria in one patient treated with fixed orthodontic device real - time pcr showed higher level of bacteria concentration in patients treated with fixed orthodontic device .

in october 2011 , an abnormally large morbidity and mortality event was noted in the intensive care unit ( icu ) of a veterinary school hospital in nantes , france . cats , and cats only , transferred from the emergency room presented with fever , ulcers on the tongue and cutaneous lesions around venepuncture or surgical incision sites , leading to suspicion of a feline calicivirus - associated virulent systemic disease confirmed with reverse transcriptase - polymerase chain reaction . fifty - seven percent of the cats were euthanased ( 8/14 ) and 21% died ( 3/14 ) , with a combined mortality of 79% ( 11/14 ) the highest ever reported . eight outbreaks have been reported , in veterinary clinics or in group - housed cats . the main unusual aspects of the present outbreak were : ( 1 ) the extreme flare - up of lesions at sites of skin breach , precluding any puncture / incision ; ( 2 ) the suggested better survival rate at home than in hospital ; and ( 3 ) the immediate control of the outbreak after recognition of the disease . other striking but less unusual features of this outbreak were : ( 4 ) the increasing of the virulence of the calicivirus with the passage of time ; and ( 5 ) the primary role that the caregivers hands played in the spread of the outbreak . at the start of october 2011 , a month after the beginning of classes , an abnormally large morbidity and mortality event was noted in the intensive care unit ( icu ) of a veterinary school hospital . cats , and cats only , transferred from the emergency room ( er ) presented with fever and cutaneous lesions around venepuncture or surgical incision sites . these clinical signs were apparently unrelated to the cause of hospitalisation . at first , no common link was apparent between these cases . ulcers on the tongue were observed , and attributed to a classical calicivirus . by friday 7 october a nosocomial infection was suspected and a census of suspected cases was undertaken . on sunday 9 october lingual ulcerations and very severe generalised signs in a cat that had spent 2 days in the icu led to the hypothesis of a feline calicivirus - associated virulent systemic disease ( fcv - vsd ) , a hypothesis that was subsequently confirmed . that evening ( 9 october 2011 ) hospital personnel were alerted to the situation by email , and invited to attend an informational meeting the next morning , monday 10 october . the decision was made to close the hospital to cats for 48 h ; dogs were still admitted for appointments . the er and icu were closed to all patients , dogs included , for 5 days , to allow for disinfection of the premises and supplies . hospitalised cats were transferred to a building located 500 m away from the hospital , outside of the school s enclosure , separated by a street ; it accepted neither animals nor personnel from the hospital . a staffed telephone line in the er was operated , to inform clients of the closure , and direct sick animals that had recently been in the hospital to the quarantine facility . all owners of cats that been in the er or the icu from 1 september 2011 onwards were contacted by telephone , to question them about their cat s health . biosecurity measures were put in place in the hospital : bleach footbaths , mandatory booties , scrub caps and gloves , disinfection of the hands with an alcohol - based solution ( even if gloves had been worn ) , use of disposable gloves and clothes , and so on . surfaces and supplies were cleaned with an industrial detergent and disinfectant ( surfanios ; laboratoires anios ) , then disinfected with bleach , then with fumigation ( nocolyse ; oxypharm ) . to allow for better cleaning , cages were disassembled . these procedures were carried out throughout the entire small animal hospital , even though only three services received infected cats . the er is located in a separate building located at the entrance to the veterinary school , 200 m away from the rest of the hospital ; it receives practically no animals from the other services . the icu is located at the end of the hospital building ; animals from all the services are hospitalised there , but normally an animal hospitalised in the icu does not return to the original service . on friday 7 october 2011 , the charts of all the cats presented to the er or hospitalised in the icu since the beginning of classes on 1 on monday 10 october , 10 cats were suspected of being infected with a fcv - vsd ; we later added 4 cats diagnosed after that date ( table 1 ) . summary of 14 cats involved in feline calicivirus - associated virulent systemic disease in nantes , france rct - pcr = reverse transcriptase - pcr cat 0 was an apartment cat that presented to the er on sunday 3 september with fever ( hyperthermia , weakness , anorexia ) and lingual ulcers . on monday 4 september an oesophagostomy tube was placed in the cat in the icu . friday 16 september , a check - up established that the cat was progressing well . on september 23 this cat did not exhibit signs of fcv - vsd but rather signs of unremarkable fcv infection . as the hospital was closed for the whole of august after a complete disinfection at the end of july , it is likely that this was the first case of calicivirus - induced disease introduced after the beginning of classes . the hospitalisation of this cat in the icu preceded by 4 days and overlapped by 2 days that of cat 1 , which arrived healthy and developed marked generalised signs that led to its death . cat 1 was in a road accident and presented with a fracture of the radius - ulna to the er on thursday 7 several wound debridements were carried out by the surgical service , and the fracture was reduced with a plate on tuesday 13 september . its stay in the icu overlapped that of cat 0 for 2 days , friday 8 and saturday 9 september . september the cat presented with a fever , and on 19 september lingual ulcers and significant erythema at the surgical site were observed . cat 2 came in to the er on thursday 15 september for a 10 cm inguinal wound , after being impaled on a piece of bamboo . it left on monday 19 september , having spent 4 days in the hospital at the same time as cat 1 . september , the cat presented again to the er , with hyperthermia ( 40c ) , lingual ulcers , diarrhoea and pleural effusion . cat 3 was seen in the er on wednesday 21 september for a fractured tibia . it was never hospitalised in the icu as it was immediately transferred to its own veterinary surgeon for treatment , but on the day it presented cat 2 was present in the er . on monday 26 september , in its own veterinary surgery , the cat presented with fever , lingual ulcers and facial oedema . it underwent debridements for 9 days , and was finally amputated on saturday 1 october , because of significant erythema at the surgical site . it was very ill for the whole of october and received care at home . by the time of a follow - up appointment on tuesday 8 cat 4 , aged 16 years , was seen in the er on monday 26 september for anorexia secondary to dental disease . it was transferred to the icu on wednesday 28 september and underwent dental extractions in the surgery service . the cat spent 24 h in the icu at the same time as cat 2 , wednesday 28 september . on sunday 2 october it presented with fever , lingual ulcers and perineal ulcerations . the cat left the hospital on friday 7 october , 11 days after admission ; it did not relapse . cat 5 presented to the er on tuesday 27 september for forelimb paralysis following a road accident . abandoned by its owner , this cat was looked after by the er staff while waiting to put it up for adoption . beginning friday 30 september , the cat presented with fever , a chin ulcer and tongue ulcers . cat 5 was the origin of the infection for seven or eight cats ( cats 6 , 7 , 9 , 10 , 11 , 12 , 13 , 14 ) . by her own admission , the student who cared for cat 5 was under the impression that she had infected these cats in the course of caring for them . it never stayed in the icu , where the only two infected cats were hospitalised ( cats 2 and 4 ) . no student from the icu was on duty in the er , or vice versa ; the er is located in a building separated from the rest of the hospital . the resistance of calicivirus in the environment in spite of regular cleaning and disinfection procedures might presumably account for infections by fomites . the student who cared for cat 5 also took care of cat 3 , 6 days earlier . cat 3 would reveal the disease it had thus contracted from this student during the 2 h the cat spent in the er . the student was contaminated via parcipitation in the care of cat 2 , which had returned that day to the er after being infected in the icu . cat 6 presented to and was operated on in the er on sunday 2 october for a ruptured bladder . it may have been infected through the intermediary of cat 5 in the er or cat 4 in the icu . on 5 october the cat presented with fever , lingual ulcers , subcutaneous oedema and significant erythema at the surgical site and around its catheters . cat 7 presented to the er on sunday 2 october after a fall from the fourth floor . it was transferred to its veterinary surgeon on tuesday 4 october , after having spent 2 days near cat 5 , where it underwent surgery . the cat returned to the er on sunday 9 october with fever , ptyalism , lingual ulcers , footpad ulcers and oedema of all four limbs . it was this cat that prompted us to consider the hypothesis of fcv - vsd . it was not the source of a secondary outbreak , presumably because its veterinary surgeon was informed , and took appropriate biosecurity measures . this is the only cat in this event that was not seen in the er . it was hospitalised in the icu that day in preparation for surgery , which , in fact , it never received . the cat was euthanased on 10 october , 7 days after admission . with the exception of cat 11 , which broke with signs the day after admission , the following cases all showed clinical signs after monday 10 october , the date of diagnosis ; they were received directly into the quarantine area after a telephone interview established possible infection during a recent stay in the hospital . it stayed in the er for 2 days , 3 and 4 october , at the same time as cat 5 , and then went home . nine days later , on wednesday 12 october , in the course of our telephone call , the owner informed us that the cat was in poor condition . upon its second admission that day , the cat was admitted to the quarantine facility , and presented with fever , lingual ulcers , sneezing , nasal discharge and oedema of the face and limbs . cat 10 presented to the er on wednesday 5 october for dysorexia and exhaustion ( no precise diagnosis was ever established ) . it was never hospitalised , but presented during the time when cat 5 was present . after 9 days , on friday 14 october , it was admitted to the quarantine area for fever , lingual ulceration and icterus . the following day it presented with fever , lingual ulcers and icterus ; there is no doubt as to the diagnosis , as reverse transcriptase pcr ( rt - pcr ) of a blood sample was positive . it was euthanased on 10 october , 5 days after admission . again , cat 5 was the source of the infection . cat 12 presented to the er on wednesday 5 october after a fall of several stories . it left the next day , thursday 6 october , having been infected by cat 5 . it presented with fever , lingual ulcers , ulcers on the face and limbs ( figure 1 ) , oedema of the face and limbs , nasal discharge , sneezing , epistaxis and vomiting . ulcers on the face of cat 12 cat 13 presented to the er saturday 8 october for bacterial cystitis , and was never hospitalised . it was infected during a blood draw in the er , which took place in the radiography suite ; the student who was caring for cat 5 provided restraint . it presented again on monday 10 october for fever , lingual ulcers , haematuria and vomiting . cat 14 belonged to a student who was working in the icu . on saturday 8 october the cat s owner brought the cat in for anorexia of several days duration . after a blood draw and radiograph in the er , where cat 5 was present , cat 14 spent 3 h in the icu in a cage in which cat 8 had been housed . on wednesday 12 october cat 14 presented with fever and ulceration of the tongue , face and limbs . its owner cared for him at home ; the cat was healed by saturday 22 october , 16 days after admission . cats 2 , 3 , 13 and 14 had received one or more vaccines ( protocols unknown ) ; cats 0 , 4 , 5 , 8 , 9 , 10 and 11 had not ; vaccine status was unknown for cats 1 , 6 , 7 and 12 . with a single exception ( cat 14 ) , none of the infected cats was housed in a cage previously occupied by an infected cat . a meticulous investigation established that : ( 1 ) every cat was infected when a previously infected cat was present in the same area ( with the exception of cat 5 ) ; and ( 2 ) every newly infected cat was cared for by a veterinary student who was also caring for an infected cat ( without exception ) . this investigation was not able to resolve arguments in favour of an environmental contamination , even if this is suspected for cat 5 . er = emergency room ; icu = intensive care unit after the day on which biosecurity measures were established , monday 10 october , there were no further infections . rt - pcr confirmation from blood ( not oropharyngeal swabs ) was obtained for 10/14 cats ( cats 3 , 4 , 5 , 7 , 8 , 9 , 10 , 11 , 12 , 13 ) . the blood samples were collected on monday 10 october for hospitalised cats ( cats 5 , 6 , 7 , 8) , and the day of their second admission for cats 9 , 10 and 11 . analysis was performed on stored refrigerated samples for cats that had left the hospital prior to the assay ( cats 3 and 4 ) . the rt - pcr for cat 6 was invalid , but the autopsy and the context were also unequivocal , with subicterus , subcutaneous oedema , pulmonary oedema , hepatitis and a fibrino - necrotic cystitis , while the presenting complaint was traumatic rupture of the bladder . no samples were saved for the first patients ( cats 0 , 1 , 2 ) , while cat 14 , which belonged to the student , was not screened ; however , for these four cases the context and clinical signs were unequivocal . an autopsy was performed on five cats ( cats 6 , 8 , 9 , 11 , 12 ) ; in each case it revealed multi - organ necrotic and inflammatory lesions and oedema . at the time of diagnosis , monday 10 october , the suspected source of the infection was healed ( cat 0 ) , two cats had died ( cats 1 , 2 ) and two cats were recovering at home ( cats 3 , 4 ) . the five cats that were strongly suspected of fcv - vsd were euthanased that day ( cats 5 , 6 , 7 , 8 , 11 ) ; four infections were confirmed by rt - pcr performed on blood , and infection in cat 6 was confirmed by necropsy . six more hospitalised cats were transferred to the quarantine building ; all had negative rt - pcrs and were returned to their owners . cat 14 was treated at home by its owner , a veterinary student , and survived . four cats ( cats 9 , 10 , 12 , 13 ) that had stayed in the hospital presented directly to the quarantine area after 10 october ; all were confirmed by rt - pcr performed on blood . these students did not own cats , were relieved of all other duties and were forbidden access to the rest of the veterinary school . the pain associated with the lesions observed responds poorly to opioids , and as the latter caused nausea and loss of appetite , they were not used . it was quickly noted that any invasive procedure such as a blood draw or catheter placement caused a flare - up of lesions at the site involved . naso - oesophageal tubes were placed , as all the cats were anorexic , and oral intake of water , food or medication was impossible given the oral lesions . all of the cats died despite attentive care ( washing and cleaning ulcerations , feeding by hand and by naso - oesophageal tube , topical analgesia through application of oral anaesthetic dressings , grooming , and so on ) . the last death occurred on thursday 27 october , 43 days ( around 8 weeks ) after the first signs of systemic disease in the first cat ( cat 2).the cost of care and diagnosis for all the animals was born by the hospital . including closing the hospital , labour costs , disinfection products and medical materials used , damage to the hospital s reputation was limited by great transparency with owners and referring veterinarians , by the scope of our response to the outbreak , and by the fact that all of our response was free of charge to our clients and referring veterinarians . fcv is an rna virus common in the cat , which causes a relatively mild range of upper respiratory signs in which ulcers of the end of the tongue figure prominently . the virus is highly mutable and can give rise to a hypervirulent systemic form of the disease so - called fcv - vsd . the first description of a fcv - vsd is attributed to pedersen et al , who in 2000 published an account of an epidemic that occurred in 1998 in california , affecting 11 cats . seven outbreaks have since been reported , in veterinary clinics ( two in the usa , one in france and one in germany ) , or in group - housed cats ( one in the usa , one in england and one in germany ) ( table 2 ) . fcv - vsd has been observed in isolated cats , in a cat infected experimentally with the normal strain and in captive exotic felids . in our outbreak , we propose the hypothesis that cat 0 , infected with a classical calicivirus , infected cat 1 , which developed a lethal hypervirulent form . the calicivirus would have mutated in our icu or in cat 1 . outbreaks of feline calicivirus - associated virulent systemic disease reported in the world as has been observed in previous outbreaks , vaccinated animals may be affected by fcv - vsd . as feline calicivirus has a high mutation rate , vaccine protection depends upon the adequacy between the infecting strain ( regardless of whether or not it is hypervirulent ) and the vaccine strain(s ) administered . the median length of incubation was 4.5 days ( range 19 ) ( table 1 ) , similar to that found in other outbreaks . clinical signs most frequently observed were fever ( 100% ) ( hyperthermia , anorexia , weakness ) , lingual ulcers ( 100% ) , ulcers and/or oedema of the face ( 4/14 ; 29% ) , ulcers and/or oedema of the limbs ( 4/14 ; 29% ) , significant erythema at surgical or venepuncture sites ( 3/14 ; 21% ) , icterus ( 2/14 ; 14% ) and death ( 11/14 ; 79% ) . fever and lingual ulceration were present in all cases . in a cat presenting with lingual ulcers , a hypervirulent strain of calicivirus should be suspected in the presence of cutaneous effects ( oedema or ulcers ) on the face or limbs or the site of incisions or venepuncture , or in the presence of icterus , or in cases of mortality . twenty - five days passed between the first observation of systemic symptoms in cat 1 on wednesday 14 september and the first suspicion of fcv - vsd put forward on sunday 9 october ( cat 7 ) . fcv is such a ubiquitous virus that full demonstration of the epizootic would have required virus gene sequencing establishing that the same strain of fcv infected several of the cats . despite the lack of this data , positive rt - pcrs performed on blood samples ( not on oropharyngeal swabs ) demonstrated the presence of calicivirus in the blood . given the fact that viraemia occurs during fcv infection , even when it causes classical signs , the rt - pcr must be interpreted in the light of epidemiological and clinical data . in the present context , the systemic nature of the infection is doubtless from cat 1 . in this outbreak , the virulence of the calicivirus seems to have increased with the passage of time . the more the outbreak progressed , the more numerous and spectacular the signs became , and the more grave the outcome ( table 1 ) . of the three survivors ( cats 3 , 4 , 14 ) , two were among the first four patients . of the three survivors , two were cats which were cared for in their owners homes ( cats 3 and 14 ) . this is an illness that is care - dependent : lesions flared at surgical incisions and catheter or venepuncture sites , so much so that any break in the skin was precluded and catheters were removed . fifty - seven percent of the cats were euthanased ( 8/14 ) and 21% died ( 3/14 ) , giving a combined mortality rate of 79% ( 11/14 ) , the highest ever reported ( table 2 ) . global mortality rate is , to some extent , dependent on the criteria that led to a decision perform euthanasia . except for cat 8 , whose lesions were only moderate , the serious lesions observed clinically and on autopsy , and the failure of treatment of cats 9 , 10 , 12 and 13 , strengthened the decisions for euthanasia and made us think that the mortality is probably not an overestimate . no euthanasia was implemented as a sanitary measure to control the outbreak , as we have isolated premises and adequate staffing . a detailed review of all the records reassured us that the morbidity was not significantly underestimated . it is probable that among group - housed cats direct transmission from cat to cat by the nasal route predominates , but in a hospital setting , in which there is no direct contact between cats , indirect infection is most plausible . in this outbreak , the investigation led to the conclusion that all infections took place at the hands of the veterinary students who cared for the cats : every newly infected cat was cared for by a student who was caring for a sick cat at the same time . viral contamination of caretakers hands and clothes may occur not only during care with contact with the oral mucosa of an infected cat , but also during simple handling of an affected cat , especially when cutaneous lesions are present . cats may be infected with calicivirus by the nasal , oral or conjunctival route ; the students examine the mucous membranes of the animals at least twice daily , by raising the lips and eyelids of the cats . when a veterinarian examines the oral or conjunctival mucosa , or gives an oral medication to an infected cat , the next cat will be infected if the veterinarian does not disinfect his or her hands . the procedures that are taught and posted require washing of the hands between patients , drying the hands and disinfecting the hands with a hydro - alcohol solution , but these protocols are not always followed . moreover , fcv is quite resistant in the environment and not sensitive to the antiviral effects of all disinfectants , including hand sanitisers . as antimicrobial soaps or alcohol - based hand rubs may not be sufficient a student contaminated her hands while handling cat 2 and probably infected cats 3 , 5 , 6 , 7 , 9 , 10 , 11 , 12 and 13 , and perhaps 14 . she alone may have been the source of 910 ( 6470% ) of the 14 infections . however , cat 14 may have been infected at home by its owner , a student who worked in the icu ; this has been observed previously . for cat 13 , infected during a blood draw in the radiography suite , and for cat 14 , there does not seem to have been any contact with the oral mucosa . the delay of 6 days between the presence of cat 2 in the icu and the infection of cat 5 is surprising . the possibility that the student concerned never used an effective disinfectant on her hands during these 6 days must be considered . it is not excluded that a fomite like a thermometer , a pill gun , a cage or an examination table could have served as the source , despite the fact that they are disinfected after every use and that no other animal has been infected . this outbreak of fcv - vsd had all the characteristics of a nosocomial infection : it was propagated in a hospital setting , in highly active services , in weakened animals , during treatment , by caretaking staff , because of a break in hygiene that could easily have been prevented . the main unusual aspects of the present outbreak were : ( 1 ) the extreme flare - up of lesions at sites of skin breach , precluding any puncture / incision ; ( 2 ) the suggested better survival rate at home than in hospital ; and ( 3 ) the immediate control of the outbreak after recognition of the disease . other striking but less unusual features of this outbreak were : ( 4 ) the increasing of the virulence of the calicivirus with the passage of time ; and ( 5 ) the primary role that the caregivers hands played in the spread of the outbreak . faster recognition of the disease is key to preventing or improving management of such potentially devastating outbreaks .

case series summaryin october 2011 , an abnormally large morbidity and mortality event was noted in the intensive care unit ( icu ) of a veterinary school hospital in nantes , france . cats , and cats only , transferred from the emergency room presented with fever , ulcers on the tongue and cutaneous lesions around venepuncture or surgical incision sites , leading to suspicion of a feline calicivirus - associated virulent systemic disease confirmed with reverse transcriptase - polymerase chain reaction . a total of 14 cats were suspected . the clinical features and the origin of the contamination were described for each cat . the median length of incubation was 4.5 days . fifty - seven percent of the cats were euthanased ( 8/14 ) and 21% died ( 3/14 ) , with a combined mortality of 79% ( 11/14 ) the highest ever reported . median survival was 12 days . the recovery rate was 21% ( 3/14).relevance and novel informationeight outbreaks have been reported , in veterinary clinics or in group - housed cats . the main unusual aspects of the present outbreak were : ( 1 ) the extreme flare - up of lesions at sites of skin breach , precluding any puncture / incision ; ( 2 ) the suggested better survival rate at home than in hospital ; and ( 3 ) the immediate control of the outbreak after recognition of the disease . other striking but less unusual features of this outbreak were : ( 4 ) the increasing of the virulence of the calicivirus with the passage of time ; and ( 5 ) the primary role that the caregivers hands played in the spread of the outbreak .

Case series summary Relevance and novel information Introduction Discussion Conclusions

in october 2011 , an abnormally large morbidity and mortality event was noted in the intensive care unit ( icu ) of a veterinary school hospital in nantes , france . cats , and cats only , transferred from the emergency room presented with fever , ulcers on the tongue and cutaneous lesions around venepuncture or surgical incision sites , leading to suspicion of a feline calicivirus - associated virulent systemic disease confirmed with reverse transcriptase - polymerase chain reaction . fifty - seven percent of the cats were euthanased ( 8/14 ) and 21% died ( 3/14 ) , with a combined mortality of 79% ( 11/14 ) the highest ever reported . eight outbreaks have been reported , in veterinary clinics or in group - housed cats . the main unusual aspects of the present outbreak were : ( 1 ) the extreme flare - up of lesions at sites of skin breach , precluding any puncture / incision ; ( 2 ) the suggested better survival rate at home than in hospital ; and ( 3 ) the immediate control of the outbreak after recognition of the disease . other striking but less unusual features of this outbreak were : ( 4 ) the increasing of the virulence of the calicivirus with the passage of time ; and ( 5 ) the primary role that the caregivers hands played in the spread of the outbreak . at the start of october 2011 , a month after the beginning of classes , an abnormally large morbidity and mortality event was noted in the intensive care unit ( icu ) of a veterinary school hospital . cats , and cats only , transferred from the emergency room ( er ) presented with fever and cutaneous lesions around venepuncture or surgical incision sites . ulcers on the tongue were observed , and attributed to a classical calicivirus . on sunday 9 october lingual ulcerations and very severe generalised signs in a cat that had spent 2 days in the icu led to the hypothesis of a feline calicivirus - associated virulent systemic disease ( fcv - vsd ) , a hypothesis that was subsequently confirmed . that evening ( 9 october 2011 ) hospital personnel were alerted to the situation by email , and invited to attend an informational meeting the next morning , monday 10 october . the er and icu were closed to all patients , dogs included , for 5 days , to allow for disinfection of the premises and supplies . hospitalised cats were transferred to a building located 500 m away from the hospital , outside of the school s enclosure , separated by a street ; it accepted neither animals nor personnel from the hospital . a staffed telephone line in the er was operated , to inform clients of the closure , and direct sick animals that had recently been in the hospital to the quarantine facility . biosecurity measures were put in place in the hospital : bleach footbaths , mandatory booties , scrub caps and gloves , disinfection of the hands with an alcohol - based solution ( even if gloves had been worn ) , use of disposable gloves and clothes , and so on . surfaces and supplies were cleaned with an industrial detergent and disinfectant ( surfanios ; laboratoires anios ) , then disinfected with bleach , then with fumigation ( nocolyse ; oxypharm ) . the er is located in a separate building located at the entrance to the veterinary school , 200 m away from the rest of the hospital ; it receives practically no animals from the other services . the icu is located at the end of the hospital building ; animals from all the services are hospitalised there , but normally an animal hospitalised in the icu does not return to the original service . on friday 7 october 2011 , the charts of all the cats presented to the er or hospitalised in the icu since the beginning of classes on 1 on monday 10 october , 10 cats were suspected of being infected with a fcv - vsd ; we later added 4 cats diagnosed after that date ( table 1 ) . summary of 14 cats involved in feline calicivirus - associated virulent systemic disease in nantes , france rct - pcr = reverse transcriptase - pcr cat 0 was an apartment cat that presented to the er on sunday 3 september with fever ( hyperthermia , weakness , anorexia ) and lingual ulcers . friday 16 september , a check - up established that the cat was progressing well . as the hospital was closed for the whole of august after a complete disinfection at the end of july , it is likely that this was the first case of calicivirus - induced disease introduced after the beginning of classes . the hospitalisation of this cat in the icu preceded by 4 days and overlapped by 2 days that of cat 1 , which arrived healthy and developed marked generalised signs that led to its death . cat 1 was in a road accident and presented with a fracture of the radius - ulna to the er on thursday 7 several wound debridements were carried out by the surgical service , and the fracture was reduced with a plate on tuesday 13 september . september the cat presented with a fever , and on 19 september lingual ulcers and significant erythema at the surgical site were observed . it left on monday 19 september , having spent 4 days in the hospital at the same time as cat 1 . september , the cat presented again to the er , with hyperthermia ( 40c ) , lingual ulcers , diarrhoea and pleural effusion . cat 3 was seen in the er on wednesday 21 september for a fractured tibia . it was never hospitalised in the icu as it was immediately transferred to its own veterinary surgeon for treatment , but on the day it presented cat 2 was present in the er . on monday 26 september , in its own veterinary surgery , the cat presented with fever , lingual ulcers and facial oedema . it underwent debridements for 9 days , and was finally amputated on saturday 1 october , because of significant erythema at the surgical site . by the time of a follow - up appointment on tuesday 8 cat 4 , aged 16 years , was seen in the er on monday 26 september for anorexia secondary to dental disease . it was transferred to the icu on wednesday 28 september and underwent dental extractions in the surgery service . on sunday 2 october it presented with fever , lingual ulcers and perineal ulcerations . beginning friday 30 september , the cat presented with fever , a chin ulcer and tongue ulcers . cat 5 was the origin of the infection for seven or eight cats ( cats 6 , 7 , 9 , 10 , 11 , 12 , 13 , 14 ) . it never stayed in the icu , where the only two infected cats were hospitalised ( cats 2 and 4 ) . no student from the icu was on duty in the er , or vice versa ; the er is located in a building separated from the rest of the hospital . cat 3 would reveal the disease it had thus contracted from this student during the 2 h the cat spent in the er . it may have been infected through the intermediary of cat 5 in the er or cat 4 in the icu . on 5 october the cat presented with fever , lingual ulcers , subcutaneous oedema and significant erythema at the surgical site and around its catheters . the cat returned to the er on sunday 9 october with fever , ptyalism , lingual ulcers , footpad ulcers and oedema of all four limbs . it was not the source of a secondary outbreak , presumably because its veterinary surgeon was informed , and took appropriate biosecurity measures . it was hospitalised in the icu that day in preparation for surgery , which , in fact , it never received . with the exception of cat 11 , which broke with signs the day after admission , the following cases all showed clinical signs after monday 10 october , the date of diagnosis ; they were received directly into the quarantine area after a telephone interview established possible infection during a recent stay in the hospital . it stayed in the er for 2 days , 3 and 4 october , at the same time as cat 5 , and then went home . nine days later , on wednesday 12 october , in the course of our telephone call , the owner informed us that the cat was in poor condition . upon its second admission that day , the cat was admitted to the quarantine facility , and presented with fever , lingual ulcers , sneezing , nasal discharge and oedema of the face and limbs . the following day it presented with fever , lingual ulcers and icterus ; there is no doubt as to the diagnosis , as reverse transcriptase pcr ( rt - pcr ) of a blood sample was positive . again , cat 5 was the source of the infection . it presented with fever , lingual ulcers , ulcers on the face and limbs ( figure 1 ) , oedema of the face and limbs , nasal discharge , sneezing , epistaxis and vomiting . ulcers on the face of cat 12 cat 13 presented to the er saturday 8 october for bacterial cystitis , and was never hospitalised . it presented again on monday 10 october for fever , lingual ulcers , haematuria and vomiting . on wednesday 12 october cat 14 presented with fever and ulceration of the tongue , face and limbs . with a single exception ( cat 14 ) , none of the infected cats was housed in a cage previously occupied by an infected cat . a meticulous investigation established that : ( 1 ) every cat was infected when a previously infected cat was present in the same area ( with the exception of cat 5 ) ; and ( 2 ) every newly infected cat was cared for by a veterinary student who was also caring for an infected cat ( without exception ) . er = emergency room ; icu = intensive care unit after the day on which biosecurity measures were established , monday 10 october , there were no further infections . the blood samples were collected on monday 10 october for hospitalised cats ( cats 5 , 6 , 7 , 8) , and the day of their second admission for cats 9 , 10 and 11 . analysis was performed on stored refrigerated samples for cats that had left the hospital prior to the assay ( cats 3 and 4 ) . the rt - pcr for cat 6 was invalid , but the autopsy and the context were also unequivocal , with subicterus , subcutaneous oedema , pulmonary oedema , hepatitis and a fibrino - necrotic cystitis , while the presenting complaint was traumatic rupture of the bladder . no samples were saved for the first patients ( cats 0 , 1 , 2 ) , while cat 14 , which belonged to the student , was not screened ; however , for these four cases the context and clinical signs were unequivocal . at the time of diagnosis , monday 10 october , the suspected source of the infection was healed ( cat 0 ) , two cats had died ( cats 1 , 2 ) and two cats were recovering at home ( cats 3 , 4 ) . the five cats that were strongly suspected of fcv - vsd were euthanased that day ( cats 5 , 6 , 7 , 8 , 11 ) ; four infections were confirmed by rt - pcr performed on blood , and infection in cat 6 was confirmed by necropsy . cat 14 was treated at home by its owner , a veterinary student , and survived . these students did not own cats , were relieved of all other duties and were forbidden access to the rest of the veterinary school . the pain associated with the lesions observed responds poorly to opioids , and as the latter caused nausea and loss of appetite , they were not used . it was quickly noted that any invasive procedure such as a blood draw or catheter placement caused a flare - up of lesions at the site involved . naso - oesophageal tubes were placed , as all the cats were anorexic , and oral intake of water , food or medication was impossible given the oral lesions . all of the cats died despite attentive care ( washing and cleaning ulcerations , feeding by hand and by naso - oesophageal tube , topical analgesia through application of oral anaesthetic dressings , grooming , and so on ) . the last death occurred on thursday 27 october , 43 days ( around 8 weeks ) after the first signs of systemic disease in the first cat ( cat 2).the cost of care and diagnosis for all the animals was born by the hospital . including closing the hospital , labour costs , disinfection products and medical materials used , damage to the hospital s reputation was limited by great transparency with owners and referring veterinarians , by the scope of our response to the outbreak , and by the fact that all of our response was free of charge to our clients and referring veterinarians . fcv is an rna virus common in the cat , which causes a relatively mild range of upper respiratory signs in which ulcers of the end of the tongue figure prominently . the virus is highly mutable and can give rise to a hypervirulent systemic form of the disease so - called fcv - vsd . seven outbreaks have since been reported , in veterinary clinics ( two in the usa , one in france and one in germany ) , or in group - housed cats ( one in the usa , one in england and one in germany ) ( table 2 ) . fcv - vsd has been observed in isolated cats , in a cat infected experimentally with the normal strain and in captive exotic felids . the calicivirus would have mutated in our icu or in cat 1 . outbreaks of feline calicivirus - associated virulent systemic disease reported in the world as has been observed in previous outbreaks , vaccinated animals may be affected by fcv - vsd . as feline calicivirus has a high mutation rate , vaccine protection depends upon the adequacy between the infecting strain ( regardless of whether or not it is hypervirulent ) and the vaccine strain(s ) administered . the median length of incubation was 4.5 days ( range 19 ) ( table 1 ) , similar to that found in other outbreaks . clinical signs most frequently observed were fever ( 100% ) ( hyperthermia , anorexia , weakness ) , lingual ulcers ( 100% ) , ulcers and/or oedema of the face ( 4/14 ; 29% ) , ulcers and/or oedema of the limbs ( 4/14 ; 29% ) , significant erythema at surgical or venepuncture sites ( 3/14 ; 21% ) , icterus ( 2/14 ; 14% ) and death ( 11/14 ; 79% ) . in a cat presenting with lingual ulcers , a hypervirulent strain of calicivirus should be suspected in the presence of cutaneous effects ( oedema or ulcers ) on the face or limbs or the site of incisions or venepuncture , or in the presence of icterus , or in cases of mortality . twenty - five days passed between the first observation of systemic symptoms in cat 1 on wednesday 14 september and the first suspicion of fcv - vsd put forward on sunday 9 october ( cat 7 ) . fcv is such a ubiquitous virus that full demonstration of the epizootic would have required virus gene sequencing establishing that the same strain of fcv infected several of the cats . despite the lack of this data , positive rt - pcrs performed on blood samples ( not on oropharyngeal swabs ) demonstrated the presence of calicivirus in the blood . in the present context , the systemic nature of the infection is doubtless from cat 1 . in this outbreak , the virulence of the calicivirus seems to have increased with the passage of time . the more the outbreak progressed , the more numerous and spectacular the signs became , and the more grave the outcome ( table 1 ) . of the three survivors ( cats 3 , 4 , 14 ) , two were among the first four patients . of the three survivors , two were cats which were cared for in their owners homes ( cats 3 and 14 ) . this is an illness that is care - dependent : lesions flared at surgical incisions and catheter or venepuncture sites , so much so that any break in the skin was precluded and catheters were removed . fifty - seven percent of the cats were euthanased ( 8/14 ) and 21% died ( 3/14 ) , giving a combined mortality rate of 79% ( 11/14 ) , the highest ever reported ( table 2 ) . except for cat 8 , whose lesions were only moderate , the serious lesions observed clinically and on autopsy , and the failure of treatment of cats 9 , 10 , 12 and 13 , strengthened the decisions for euthanasia and made us think that the mortality is probably not an overestimate . it is probable that among group - housed cats direct transmission from cat to cat by the nasal route predominates , but in a hospital setting , in which there is no direct contact between cats , indirect infection is most plausible . in this outbreak , the investigation led to the conclusion that all infections took place at the hands of the veterinary students who cared for the cats : every newly infected cat was cared for by a student who was caring for a sick cat at the same time . viral contamination of caretakers hands and clothes may occur not only during care with contact with the oral mucosa of an infected cat , but also during simple handling of an affected cat , especially when cutaneous lesions are present . cats may be infected with calicivirus by the nasal , oral or conjunctival route ; the students examine the mucous membranes of the animals at least twice daily , by raising the lips and eyelids of the cats . the procedures that are taught and posted require washing of the hands between patients , drying the hands and disinfecting the hands with a hydro - alcohol solution , but these protocols are not always followed . moreover , fcv is quite resistant in the environment and not sensitive to the antiviral effects of all disinfectants , including hand sanitisers . she alone may have been the source of 910 ( 6470% ) of the 14 infections . however , cat 14 may have been infected at home by its owner , a student who worked in the icu ; this has been observed previously . for cat 13 , infected during a blood draw in the radiography suite , and for cat 14 , there does not seem to have been any contact with the oral mucosa . the delay of 6 days between the presence of cat 2 in the icu and the infection of cat 5 is surprising . this outbreak of fcv - vsd had all the characteristics of a nosocomial infection : it was propagated in a hospital setting , in highly active services , in weakened animals , during treatment , by caretaking staff , because of a break in hygiene that could easily have been prevented . the main unusual aspects of the present outbreak were : ( 1 ) the extreme flare - up of lesions at sites of skin breach , precluding any puncture / incision ; ( 2 ) the suggested better survival rate at home than in hospital ; and ( 3 ) the immediate control of the outbreak after recognition of the disease . other striking but less unusual features of this outbreak were : ( 4 ) the increasing of the virulence of the calicivirus with the passage of time ; and ( 5 ) the primary role that the caregivers hands played in the spread of the outbreak . faster recognition of the disease is key to preventing or improving management of such potentially devastating outbreaks .

a rise in nutrients occurs in the small intestine after meal ingestion , and nutrients are released into the circulation after their absorption . while still in the preabsorptive state , nutrients activate sensing mechanisms in the duodenum to trigger a gut - brain driven negative feedback system to inhibit exogenous nutrient intake and endogenous nutrient production by the liver ( 1012 ) . consequently , metabolic homeostasis is maintained in response to nutrient intake . in diabetes and obesity , intestinal nutrient - sensing mechanisms fail to lower food intake and glucose production ( 13 ) , leading to a disruption in metabolic homeostasis . with an aim to restore glucose and energy homeostasis , laboratories have focused on characterizing duodenal nutrient - sensing mechanisms that alter food intake and glucose production in diabetes and obesity . this article will first highlight the role of duodenal nutrient - sensing mechanisms in the regulation of glucose homeostasis and its relevance in diabetes and obesity . after the absorption of nutrients such as lipids into duodenal enterocytes , long - chain fatty acids ( lcfas ) from lipids are metabolized into lcfa - coa via acyl - coa synthase ( 12 ) . short - term continuous intraduodenal intralipids infusion elevates duodenal lcfa - coa levels and lowers glucose production in healthy rodents while plasma insulin concentrations are maintained at basal levels ( 14 ) ( fig . 1 ) . direct inhibition of duodenal acyl - coa synthase and accumulation of duodenal lcfa - coa negates the glucoregulatory effect of intralipids , and intraduodenal lipid infusion also fails to lower glucose production in high - fat diet these findings illustrate the importance of elucidating the downstream duodenal lipid signaling events that regulate glucose production in normal and high - fat diet high - fat feeding induces duodenal cck resistance and direct activation of pka bypasses the defect . ac , adenylyl cyclase ; cck1-r , cck receptor ; hfd , high - fat diet . intraduodenal lipid administration lowers food intake in rodents and humans through the release of the gut - derived peptide hormone cholecystokinin ( cck ) from the duodenal i cells ( 11 ) . the release of cck induced by lcfa is confirmed in the stc-1 cck - secreting cell line and is protein kinase c ( pkc ) dependent ( 15,16 ) . along with this , activation of duodenal mucosa pkc ( particularly pkc- , expressed in both rodent and human duodenal mucosa ) by pkc activator is sufficient to lower glucose production while molecular and chemical inhibition of duodenal pkc- negate the glucoregulatory effect of duodenal lipids ( 17 ) . further , given that direct inhibition of intestinal cck-1 receptors negates the ability of intraduodenal lipids , pkc activator , or cck-8 infusion to lower glucose production , the subsequent activation of duodenal cck-1 receptors is required for the lcfa pkc-cck signaling axis to lower glucose production in healthy rodents in vivo ( 1719 ) ( fig . it is believed that cck binds to the cck-1 receptors expressed on vagal afferents innervating the small intestine ( 20 ) , and activation of cck-1 receptors triggers a gut - brain - liver axis to lower glucose production in healthy rodents ( 19 ) . although pkc- is colocalized with cck in the duodenal mucosa ( 18 ) , suggesting that the lcfa - coa pkc- cck signaling event takes place in the duodenal i - cells , definitive in vivo experiments are lacking . nonetheless , direct activation of the duodenal pkc- cck signaling axis fails to lower glucose production in high - fat diet fed rodents independent of changes in the duodenal expression of cck-1 receptors ( 18,19,21 ) . addressing the mechanism(s ) underlying duodenal pkc- cck resistance in lowering glucose in high - fat diet is essential for 1 ) evaluating novel fatty acid g - protein receptors coupled to pkc- in duodenal i cells and 2 ) in evaluating cck-1 receptor agonists as antidiabetic therapies . in healthy volunteers , it has also been reported that cck is released in response to intragastric fatty acids with chain lengths matching the ligand profiles of gpr40/ffar1 and gpr120/o3far1 , both of which have been shown to be present in rodent duodenal i cells and to modulate cck release ( 22,23 ) . gpr40 and gpr120 are both gq - coupled receptors activating pkc , but is unknown if these receptors lower glucose production via a duodenual - brain - liver axis in rodents on a high - fat diet or if present in human i cells . recent advances in isolation of human enteroendocrine cells and measuring peptide secretion will directly address the latter ( 24 ) . despite the earlier promising potential of cck-1 receptor agonists in metabolism , selective activation of the receptor has failed to generate a new class of antiobesity or diabetic therapy primarily due to toleration and lack of efficacy . systemic agonists must be minimized to avoid unwanted adverse effects via direct activation on the gall bladder and pancreas , whereas gut - selective approaches have also been dose limited as a result of gastrointestinal side effects such as vomiting ( 25 ) . understanding the endogenous cck tone within the duodenum in addition to a deeper understanding of the cck-1 receptor intracellular coupling mechanisms underlying glucose - lowering efficacy may provide new insights into the doability of cck-1 receptor agonists for diabetes . cck-1 receptors are g - protein coupled and are highly expressed in the gut ( 26 ) . the signaling cascade of cck-1 receptors in the acinar cells involves both protein kinase a ( pka ) and phospholipase c ( plc ) activation , which mediate cck s ability to trigger pancreatic secretion ( 2629 ) . although the cck-1 receptor signaling cascade in the duodenum remains largely undefined , direct activation of duodenal pka signaling has recently been demonstrated to be sufficient and necessary for cck-1 receptor activation to stimulate vagal afferent firing in vivo ( 21 ) . consequently , a gut - brain - liver driven neuronal network is ignited to lower glucose production in both normal and high - fat diet however , duodenal pka is not the sole mediator of this network as inhibition of plc prevents cck from lowering glucose production as well ( 21 ) . given that direct duodenal pka activation bypasses duodenal cck resistance in high - fed feeding to lower glucose production ( 21 ) , high - fat diet induced defects lie within the inability of duodenal cck-1 receptor activation to stimulate pka ( fig . 1 ) . future studies are warranted to clarify the duodenal signaling cascades of the cck-1 receptor that regulate glucose production such that novel and effective antidiabetic therapeutic targets could be revealed . recently , it has been shown that glucose sensing in l - cells , enterochromaffin cells and activation of neural pathways is impaired in diabetes as determined using phosphorylated calcium calmodulin - dependent kinase ii as a marker of cellular activation in vivo ( 30 ) . could activation of duodenal pka signaling mediate other gut - derived peptide hormones such as glucagon - like peptide-1 ( glp-1 ) and gip to lower glucose levels in diabetes and obesity ? glp-1 and gip receptors signal via pka ( 32,33 ) , and selective inhibition of intestinal dipeptidyl peptidase - iv ( dpp - iv ) activity , which enhances glp-1 and gip , can trigger vagal firing and improves glucose homeostasis in diet - induced obese rodents in a glp-1 receptor dependent manner ( 34 ) . although the specific site(s ) of glp-1 action in the gut remain to be clarified , a possibility remains that the local release of intestinal glp-1 activates glp-1 receptors expressed on the vagus ( 32 ) and triggers a gut - brain driven system to lower blood glucose levels in diabetes and obesity . of note , it is unknown how the glp-1 receptor ( 34 ) but not cck-1 receptor ( 19,21 ) signaling in the gut remains intact in diet - induced obesity to lower blood glucose levels . nonetheless , we propose that pka signaling ( likely located on the vagal terminals that innervate the intestine ) mediates multiple gut - derived hormones to trigger a gut - brain driven system to lower blood glucose levels in diabetes ( fig . targeted research may prove useful to unveil novel antidiabetic targets , but on the other hand , examining whether different types of fatty acids or nutrients other than lipids may trigger the gut - brain axis to lower glucose production may be equally important . cck signaling in the gut is not only triggered by lipids ( 19 ) , but also by refeeding ( 17,19 ) to regulate glucose production and homeostasis , respectively . although the ability of the duodenal lipid - sensing pathway to regulate glucose production is assessed under the pancreatic euglycemic clamp conditions to ensure that any changes in glucose metabolism are due primarily to the treatment rather than changes in circulating glucoregulatory hormones , direct inhibition of the duodenal lipid - sensing pathway is also found to disrupt glucose homeostasis following a fasting refeeding nonclamp protocol ( 17,19 ) . these findings highlight the physiological relevance of a gut - brain driven feedback system that is activated by duodenal nutrient sensing after a meal to maintain circulating nutrient homeostasis . it is currently unknown whether other classes of nutrients , in addition to lipids , regulate glucose production in the proximal intestine . however , duodenal glucose sensing inhibits feeding ( 35,36 ) while enteric glucose sensing ( 37 ) ( which includes intestinal , mesenteric , and hepatoportal sensing systems ) or , specifically , jejunal glucose sensing ( 6 ) regulates peripheral glucose homeostasis via a gut - brain driven system . glucose is absorbed through the proximal intestine into the portal vein where selective portal glucose sensing triggers a gut - brain - liver axis to inhibit glucose production and mediate the glucose - lowering effect of bariatric surgery in high - fat diet hence , the essential glucoregulatory role of nutrient - sensing mechanisms in the gut is highlighted and a possibility emerges that nutrient - sensing mechanisms may be conserved throughout the gut for glucose regulation . nutrient absorption occurs predominantly in the duodenum , and it is believed that only under malabsorptive conditions undigested nutrients reach the distal gut ( 39 ) . however , studies in animals ( 4043 ) and humans ( 44,45 ) illustrate that ingested nutrients reach the distal gut during early phases of food ingestion , opening up the possibility that nutrient - sensing mechanisms in the jejunum or ileum are activated shortly after a meal to regulate peripheral metabolic homeostasis . in fact , direct jejunal lipid ( 4650 ) or glucose ( 47 ) but not protein ( 47 ) administration lowers food intake . the underlying mechanisms of jejunal nutrient sensing remain unclear , but the roles of peptide yy ( 46,47 ) , glp-1 ( 4648 ) , cck ( 46 ) , and/or a gut - brain neuronal axis ( 46,51,52 ) have been implicated . nonetheless , given that jejunal nutrient sensing regulates feeding ( 4650 ) and that , as discussed above , duodenal lipid sensing regulates glucose production ( 12 ) , a potential glucoregulatory role of jejunal nutrient sensing has been examined as well ( 6 ) ( fig . an influx of nutrients into the duodenum or jejunum activates a complex biochemical , molecular , neuronal , and physiological network to lower hepatic glucose production . although there are some parallels regarding nutrient sensing in the duodenum and jejunum , it appears that unlike in the duodenum , jejunal nutrient sensing is pka independent . acs , acyl - coa synthetase ; nts nmda , nucleus of the solitary tract n - methyl - d - aspartate ; pyy , peptide yy . direct administration of glucose or lipids ( the main sources of food energy ) into the jejunum lowers glucose production in the absence of a lowering of food intake ( 6 ) . the effect of nutrient sensing is ignited within the jejunum since direct inhibition of glucose sensing mechanisms in the duodenum or ileum fails to negate the ability of a jejunal glucose infusion to lower glucose production ( 6 ) . in addition , an equimolar infusion of glucose into the portal vein , as in the jejunum , fails to lower glucose production ( 6 ) . these experiments do not exclude the glucoregulatory role of nutrient sensing in the duodenum , ileum , or portal vein but rather illustrate that direct infusion of glucose into the jejunum triggers signaling mechanisms within the jejunum to lower glucose production . nutrient sensing in the jejunum appears to share similar mechanisms with the duodenum , since both segments lower glucose production in response to lipids , require the formation of lcfa - coa , and trigger a gut - brain - liver neuronal relay to lower glucose production ( 6,53 ) . however , evidence suggests that important differences may exist between the two because direct administration of a pka agonist ( which activates downstream cck - pka signaling ) into the duodenum , but not the jejunum , lowers glucose production ( 21 ) , although cck is also synthesized in the jejunum of animals and humans ( 5456 ) but to a much lower extent ( 57 ) . given that cck mediates duodenal lipid sensing mechanisms via pka and/or plc signaling pathways to lower glucose production ( 53 ) ( fig . 1 ) , these findings suggest that either jejunal nutrient sensing is not cck dependent or may potentially signal via the cck - plc signaling pathway . out of all the gut - derived satiety factors studied , apolipoprotein a - iv is principally synthesized and secreted from the jejunum , although its secretion is thought to be specific to lipids and not glucose ( 11 ) . the potential role of apolipoprotein a - iv in jejunal regulation of glucose homeostasis remains unknown . similarly , other major distal gut peptides that have documented anorectic effects mediated by the vagus are glp-1 and peptide yy ( 58 ) , either of which remain possible mediator(s ) in maintaining glucose homeostasis triggered by jejunal nutrient sensing . future investigation is required to dissect the downstream jejunal signaling pathway(s ) of nutrient sensing that regulate peripheral metabolic homeostasis . inhibition of the sodium - glucose transporter proteins ( sglts ) in the jejunum negates the ability of intrajejunal glucose administration to lower glucose production ( 6 ) , indicating that jejunal glucose uptake from the lumen is a necessary step for jejunal glucose sensing to regulate peripheral glucose homeostasis . since galactose , like glucose , is also absorbed into the intestinal enterocyte from the lumen via the same glucose transporter ( 59 ) , it would be important to assess whether galactose , at an equimolar concentration of glucose in the jejunum , also lowers glucose production . another monosaccharide , fructose , is absorbed into the intestine via glucose transporter-5 ( 59 ) , and the potential glucoregulatory role of fructose sensing in the jejunum deserves future attention . we propose that comparing the glucoregulatory impact of sensing mechanisms potentially triggered by any of these monosaccharides will be crucial in revealing novel nutrient - sensing mechanisms in the jejunum . in parallel , accumulation of jejunal lcfa - coa is required for intrajejunal lipid administration to lower glucose production ( 6 ) . because the accumulation of lcfa - coa in the brain and the liver are sufficient and necessary to mediate both lipid and glucose sensing mechanisms to regulate hepatic glucose metabolism ( 60 ) , this raises the possibility that lipid and glucose sensing mechanisms within the jejunum may converge . however , this postulation remains to be confirmed . in a physiological nonclamp ( i.e. , glucoregulatory hormones change at will ) condition when nutrient - sensing mechanisms are activated by a fasting - refeeding protocol , direct disruption of duodenal nutrient - sensing mechanisms increases plasma glucose levels during refeeding and disrupts glucose homeostasis as compared with rodents with intact duodenal nutrient sensing ( 17,19 ) . in contrast , in the same experimental conditions , plasma glucose levels are not altered when jejunal nutrient - sensing mechanisms are disrupted ( 6 ) . these data suggest that the key benefits of nutrient - sensing mechanisms in the jejunum to regulate glucose homeostasis are revealed under conditions in which normal nutrient flow is disrupted , for example , when sections of the upper intestine are removed in cases of intestinal cancer or inflammation ( where short bowel resection is recommended ) or after bariatric surgery in obese patients . several key aspects of gut nutrient - sensing mechanisms and the regulation of glucose production have been uncovered , yet several questions remain . first , given the fact that direct nutrient administration into the duodenum or jejunum exerts similar effects on glucose production , would an additive or possibly synergistic effect on glucose control be observed if both duodenal and jejunal nutrient sensing were stimulated in parallel with a matched caloric load ? is there a humoral or neuronal crosstalk between the duodenum and jejunum ? given that the duodenum is exposed to the highest influx of nutrients from the stomach , are duodenal nutrient - sensing mechanisms dominant over jejunal nutrient sensing ? if so , what is the functional relevance of jejunal nutrient sensing regarding glucose control ? the concept of a defect in duodenal nutrient sensing resulting in an increase in glucose production raises many fundamental questions when translating to a new antidiabetic therapy . do the duodenal nutrient - sensing defects trigger the first step in the journey of diabetes or is this defect responsible for a select subset of patients with diabetes who need to be defined ? how can defects in duodenal nutrient sensing in patients be identified early as part of an intervention strategy ? pharmacologically , any gut - targeted therapy to lower blood glucose will access both the duodenum and jejunum , so is there a predominant selective gut mechanism of action to sufficiently lower blood glucose in diabetes ? alternatively what combination approaches should be pursued to maximize efficacy while maintaining a well - tolerated safety profile ? although the primary goal of bariatric procedures is weight loss , for which it is currently the most effective intervention , it also remains the only known treatment to induce remission of type 2 diabetes resulting in euglycemia . bariatric surgery normalizes blood glucose concentrations in the majority of type 2 diabetic humans independent of weight loss ( 6264 ) . furthermore , the effectiveness on glucose control induced by bariatric surgery is superior to conventional pharmacological therapy and lifestyle modifications ( 6264 ) . one additional study even indicates that bariatric surgery induces diabetes remission up to 6 years ( 65 ) . although the effectiveness of bariatric surgery has set the standard for goals in glucose control for nonsurgical treatments , replicating its effect with nonsurgical tools has been relatively unsuccessful . perhaps this is partly due to the complex interactions of numerous signaling pathways affected by the procedure in combination with the equally complex nature of the targeted diseases . clearly , diabetes remission induced by bariatric surgery is associated with an enhancement of nutrient delivery to the distal small intestine . could the enhancement of nutrient flux into the distal small intestine after bariatric surgery lead to an antidiabetic effect ( fig . 3 ) ? given that both the duodenum and jejunum are independently capable of sensing nutrients and generating a gut - derived signal mediated by vagal nerves to trigger a gut - brain - liver axis to lower glucose production independent of weight loss ( as discussed above ) , a clear possibility remains . this hypothesis is also supported by medical devices that are being investigated as noninvasive alternatives to bariatric surgery . a trial using a novel device that electrically alters the vagal nerve firing as a treatment for morbid obesity reports a fast decrease in fasting plasma glucose within the first week in patients with type 2 diabetes ( enable trial ; http://ir.enteromedics.com/releasedetail.cfm?releaseid=610191 ) . likewise a tube device that is endoscopically inserted in the gut extending from the duodenum to the proximal jejunum to prevent nutrients being absorbed within this region also reports rapid changes in glucose ( http://www.gidynamics.com/endobarrier-overview.php ) . bariatric surgery bypasses the duodenum , and nutrient flow is enhanced into the jejunum , resulting in activation of jejunal nutrient - sensing mechanisms involving a series of biochemical reactions , gut - derived hormones , and neuronal circuitry to lower glucose production and plasma glucose levels . djb , a nonrestrictive experimental form of bariatric surgery ( 66 ) , lowers glucose levels in nonobese rodents ( 66 ) and nonobese / mild - obese humans with type 2 diabetes ( 67,68 ) independent of weight loss . this surgical procedure redirects nutrient flow from the stomach to the distal jejunum and thus bypasses the duodenum . can the enhancement of nutrient flux into the jejunum trigger a neurocircuit to lower glucose levels in diabetes with djb ? first , djb is performed in two independent rat models in which diabetes is induced by destruction of the pancreatic -cells by either a toxic injection of streptozotocin or a genetic autoimmune response ( biobreeding rats ) ( 6 ) . the use of these uncontrolled diabetic models enables the assessment of the effects of djb in a nonobese setting with impaired insulin secretion . within 2 days , djb lowers blood glucose concentrations independent of lowering food intake / body weight or increasing insulin secretion in both uncontrolled diabetic models ( 6 ) . importantly , direct inhibition of jejunal nutrient - sensing mechanisms disrupts peripheral glucose control triggered by djb during refeeding ( 6 ) . of note , the glucose response during refeeding in the djb diabetic rodents with or without intact jejunal nutrient - sensing mechanisms closely resembles those observed in relatively mild - obese type 2 diabetic humans with or without djb surgery , respectively ( 67 ) . together with the ability of jejunal nutrient sensing to trigger a gut - brain - liver neuronal relay to lower glucose production in normal rodents ( see above ) , these findings collectively suggest that increased delivery of nutrients to the jejunum after djb triggers a neuronal - mediated signaling mechanism to lower glucose production and hence blood glucose concentrations in diabetes . the ability of djb to rapidly lower blood glucose concentrations is potentially via a cns - mediated reduction in hepatic glucose production ( 6 ) . in fact , other reports have published similar results that djb ( 7 ) or a variant of djb ( 38 ) triggers the cns to lower hepatic glucose production in obese type 2 diabetic rodents . of note , djb lowers blood glucose concentrations in insulin - deficient rodents ( 6 ) and lowers glucose production in obese type 2 diabetic rodents independent of an improvement on insulin action ( 7 ) . thus , we propose that insulin action is not required for the rapid glucose - lowering effect induced by djb in diabetes . however , these findings do not exclude the possibility that djb could also lower blood glucose concentrations via increased insulin - dependent or independent glucose uptake , although studies have indicated that djb does not improve insulin action in rodents with diabetes and/or obesity ( 7,69,70 ) . in addition , improvement in -cell function occurs after bariatric surgery in obese type 2 diabetic subjects ( 61 ) . thus , a potential increase in glucose uptake , improvement in insulin action and/or increase in insulin secretion could play important roles in the sustained glucose - lowering effect triggered by djb . of note , changes in insulin secretion after bariatric procedures in obese type 2 diabetes however , changes in circulating glp-1 are not consistent between models of diabetes that received djb ( 6 ) . moreover , bariatric surgery still improves glucose tolerance in glp-1 receptor deficient high - fat diet fed mice ( 71 ) . in addition , clinical use of glp-1 agonists and dpp - iv inhibitors does not mirror the full glycemic benefits observed after bariatric surgery , and although controversial , it is possible that other incretin hormones such as gip may be involved in enhancing insulin secretion . it is well known that bariatric surgery results in significant health benefits in obesity with type 2 diabetes , whereas only a few studies have demonstrated that bariatric surgery lowers glucose concentrations in mild / nonobese type 2 diabetic subjects ( 67,68 ) and in obese type 1 diabetic subjects ( 72 ) . in clinical practice , there is much debate regarding the appropriate bmi level to determine eligibility for bariatric procedures . therefore , until this debate is resolved , determining whether bariatric surgery could exert significant benefit in nonobese humans with type 1 diabetes will remain speculative . despite this , the existence of a gut - brain - liver axis regulating glucose homeostasis and its relevance in the antidiabetic effect of bariatric surgery suggests that targeting the gut with drug therapy to influence the liver could be a valid therapeutic approach and would potentially mimic the antidiabetic effect of bariatric surgery . given the accessibility of the gut to pharmacotherapy , we propose targeting nutrient sensing molecules in the gut will have therapeutic potential in lowering blood glucose concentrations in diabetes . the intestine has developed mechanisms to sense nutrients after a meal and trigger a negative feedback system to maintain glucose homeostasis . this triggering leads to the rapid glucose lowering effect of djb in diabetes ( figs . 2 and 3 ) . however , we must keep in mind that the intestinal environment is complex , given that an abundant collection of microorganisms ( predominantly bacteria ) termed microbiota resides within the gut . in the human intestine approximately 400 bacterial species are present and together resemble a multicellular organ that has evolved to provide metabolic functions ( 73 ) . these bacteria belong to three main groups ( phyla ) : firmicutes , bacteriodetes , and actinobacteria , which represent > 95% of the total bacteria in the intestine . disruption in this symbiotic relationship can disturb host functions and in extreme cases can cause serious disease such as irritable bowel disease ( 74 ) , stressing the importance of these microorganisms in maintaining the intestinal environment and the roles they play in helping the host . the gut microbiota is a dynamic organ that is constantly adapting to changes in its environment , and it is believed that the progression of obesity / diabetes can be attributed to the intestinal microbiota host relationship . in mice switched from a regular chow to a high - fat diet , the gut microbiota composition is altered in as little as 24 h ( 75 ) , resulting in increased levels of firmicutes ( 76 ) . this change is secondary to increased fat content rather than the obese state , rendering the microbiota to be more obesogenic and resulting in increased energy harvest from the diet in the distal gut . in addition , germ - free mice are protected from developing diet - induced obesity ( 77 ) , further highlighting the relationship between the gut microbiome and obesity . of note , it has recently been demonstrated that genetically identical mice fed the same high - fat diet differed in their metabolic phenotype due to differences in their microbiota composition ( 78 ) . thus , the relative contribution of diet versus the gut microbiota profile in causing metabolic diseases remains to be addressed . although a larger bacterial population resides in the more distal portion of the small intestine and in the colon , bacteria are also present in the upper intestine , including the duodenum and jejunum . in fact , direct administration of microbiota from lean donors into the duodenum of humans with metabolic syndrome significantly improves insulin sensitivity independent of weight loss ( 79 ) , consistent with the view that the duodenum regulates peripheral glucose homeostasis ( fig . 1 ) . furthermore , gut microbiota alters intestinal permeability ( 80,81 ) and regulates both the synthesis and production of bile acids ( 82,83 ) , which are important for fat emulsification and absorption in the intestine . gut microbiota also regulates hormone release from enteroendocrine cells ( 84,85 ) and interestingly , ingestion of bacteria activates vagal afferent signaling ( 86 ) . in addition , germ - free mice have altered cd36 expression ( a proximal fatty acid transporter ) ( 85 ) and sglt-1 expression ( 84 ) , indicating that gut bacteria affects nutrient absorption / uptake . further , high - fat feeding promotes an obesogenic bacterial profile , which is linked to increased inflammation in the intestine and whole - body insulin resistance ( 80 ) . obesogenic mirobiota induces inflammation potentially via bacterial fragments ( 87 ) . in light of these findings , we put forward a working hypothesis that the obesogenic microbiome disrupts nutrient - sensing cck signaling mechanisms in the duodenum , leading to a dysregulation of glucose homeostasis ( fig . changes in microbiota also occur after bariatric surgery in which the obese bacterial profile is altered to reflect a more lean profile ( 88 ) . whether these changes are associated with a decrease in food intake or are due to it has also been suggested that this change in microbiota after surgery may lead to changes in enteroendocrine cell function ( 85,89 ) . thus , the link between bariatric surgery and changes in gut microbiota as well as its eventual impact on metabolic regulation warrants further investigation . nevertheless , a possibility remains that the improvement of glucose homeostasis observed following bariatric surgery is mediated by a change in gut microbiota , leading to an enhancement of nutrient - sensing mechanisms in the jejunum and an improvement in glucose tolerance ( fig . the gut possesses a unique role in regulating glucose and energy homeostasis whereby the presence of ingested nutrients into the small intestine activates sensing mechanisms that affect both glucose production and food intake . the nutrient - sensing mechanisms are conserved from the duodenum to the jejunum , and although it is evident that nutrient uptake and a neuronal network are required in both regions to lower glucose production , whether additional parallel mechanisms exist is not yet known . duodenal nutrient sensing acts as a protective mechanism under normal physiological conditions to maintain glucose homeostasis during nutrient ingestion by ensuring that glucose production is decreased ; however , this mechanism appears to be impaired after excess caloric intake . on the other hand , jejunal nutrient sensing is important in mediating the glucose lowering effect ( via decreased glucose production ) of bariatric surgery where nutrient influx to the jejunum is enhanced . emerging evidence also suggests a relationship between gut microbiota and intestinal nutrient - sensing mechanisms ; however , this concept has not been directly tested . although many questions remain unanswered , recent advances in our understanding of the pathways regulating gut nutrient sensing provide compelling support for potential new therapeutic targets to restore glucose homeostasis in diabetes .

the small intestine is traditionally viewed as an organ that mediates nutrient digestion and absorption . this view has recently been revised owing to the ability of the duodenum to sense nutrient influx and trigger negative feedback loops to inhibit glucose production and food intake to maintain metabolic homeostasis . further , duodenal nutrient - sensing defects are acquired in diabetes and obesity , leading to increased glucose production . in contrast , jejunal nutrient sensing inhibits glucose production and mediates the early antidiabetic effect of bariatric surgery , and gut microbiota composition may alter intestinal nutrient - sensing mechanisms to regain better control of glucose homeostasis in diabetes and obesity in the long term . this perspective highlights nutrient - sensing mechanisms in the gut that regulate glucose homeostasis and the potential of targeting gut nutrient - sensing mechanisms as a therapeutic strategy to lower blood glucose concentrations in diabetes .

Nutrient-sensing mechanisms in the duodenum Nutrient-sensing mechanisms in the jejunum Gut nutrient-sensing mechanisms in bariatric surgery Gut microbiota and nutrient sensing SUMMARY

a rise in nutrients occurs in the small intestine after meal ingestion , and nutrients are released into the circulation after their absorption . while still in the preabsorptive state , nutrients activate sensing mechanisms in the duodenum to trigger a gut - brain driven negative feedback system to inhibit exogenous nutrient intake and endogenous nutrient production by the liver ( 1012 ) . in diabetes and obesity , intestinal nutrient - sensing mechanisms fail to lower food intake and glucose production ( 13 ) , leading to a disruption in metabolic homeostasis . with an aim to restore glucose and energy homeostasis , laboratories have focused on characterizing duodenal nutrient - sensing mechanisms that alter food intake and glucose production in diabetes and obesity . this article will first highlight the role of duodenal nutrient - sensing mechanisms in the regulation of glucose homeostasis and its relevance in diabetes and obesity . direct inhibition of duodenal acyl - coa synthase and accumulation of duodenal lcfa - coa negates the glucoregulatory effect of intralipids , and intraduodenal lipid infusion also fails to lower glucose production in high - fat diet these findings illustrate the importance of elucidating the downstream duodenal lipid signaling events that regulate glucose production in normal and high - fat diet high - fat feeding induces duodenal cck resistance and direct activation of pka bypasses the defect . intraduodenal lipid administration lowers food intake in rodents and humans through the release of the gut - derived peptide hormone cholecystokinin ( cck ) from the duodenal i cells ( 11 ) . along with this , activation of duodenal mucosa pkc ( particularly pkc- , expressed in both rodent and human duodenal mucosa ) by pkc activator is sufficient to lower glucose production while molecular and chemical inhibition of duodenal pkc- negate the glucoregulatory effect of duodenal lipids ( 17 ) . further , given that direct inhibition of intestinal cck-1 receptors negates the ability of intraduodenal lipids , pkc activator , or cck-8 infusion to lower glucose production , the subsequent activation of duodenal cck-1 receptors is required for the lcfa pkc-cck signaling axis to lower glucose production in healthy rodents in vivo ( 1719 ) ( fig . it is believed that cck binds to the cck-1 receptors expressed on vagal afferents innervating the small intestine ( 20 ) , and activation of cck-1 receptors triggers a gut - brain - liver axis to lower glucose production in healthy rodents ( 19 ) . nonetheless , direct activation of the duodenal pkc- cck signaling axis fails to lower glucose production in high - fat diet fed rodents independent of changes in the duodenal expression of cck-1 receptors ( 18,19,21 ) . although the cck-1 receptor signaling cascade in the duodenum remains largely undefined , direct activation of duodenal pka signaling has recently been demonstrated to be sufficient and necessary for cck-1 receptor activation to stimulate vagal afferent firing in vivo ( 21 ) . consequently , a gut - brain - liver driven neuronal network is ignited to lower glucose production in both normal and high - fat diet however , duodenal pka is not the sole mediator of this network as inhibition of plc prevents cck from lowering glucose production as well ( 21 ) . future studies are warranted to clarify the duodenal signaling cascades of the cck-1 receptor that regulate glucose production such that novel and effective antidiabetic therapeutic targets could be revealed . could activation of duodenal pka signaling mediate other gut - derived peptide hormones such as glucagon - like peptide-1 ( glp-1 ) and gip to lower glucose levels in diabetes and obesity ? although the specific site(s ) of glp-1 action in the gut remain to be clarified , a possibility remains that the local release of intestinal glp-1 activates glp-1 receptors expressed on the vagus ( 32 ) and triggers a gut - brain driven system to lower blood glucose levels in diabetes and obesity . of note , it is unknown how the glp-1 receptor ( 34 ) but not cck-1 receptor ( 19,21 ) signaling in the gut remains intact in diet - induced obesity to lower blood glucose levels . nonetheless , we propose that pka signaling ( likely located on the vagal terminals that innervate the intestine ) mediates multiple gut - derived hormones to trigger a gut - brain driven system to lower blood glucose levels in diabetes ( fig . targeted research may prove useful to unveil novel antidiabetic targets , but on the other hand , examining whether different types of fatty acids or nutrients other than lipids may trigger the gut - brain axis to lower glucose production may be equally important . cck signaling in the gut is not only triggered by lipids ( 19 ) , but also by refeeding ( 17,19 ) to regulate glucose production and homeostasis , respectively . although the ability of the duodenal lipid - sensing pathway to regulate glucose production is assessed under the pancreatic euglycemic clamp conditions to ensure that any changes in glucose metabolism are due primarily to the treatment rather than changes in circulating glucoregulatory hormones , direct inhibition of the duodenal lipid - sensing pathway is also found to disrupt glucose homeostasis following a fasting refeeding nonclamp protocol ( 17,19 ) . it is currently unknown whether other classes of nutrients , in addition to lipids , regulate glucose production in the proximal intestine . however , duodenal glucose sensing inhibits feeding ( 35,36 ) while enteric glucose sensing ( 37 ) ( which includes intestinal , mesenteric , and hepatoportal sensing systems ) or , specifically , jejunal glucose sensing ( 6 ) regulates peripheral glucose homeostasis via a gut - brain driven system . glucose is absorbed through the proximal intestine into the portal vein where selective portal glucose sensing triggers a gut - brain - liver axis to inhibit glucose production and mediate the glucose - lowering effect of bariatric surgery in high - fat diet hence , the essential glucoregulatory role of nutrient - sensing mechanisms in the gut is highlighted and a possibility emerges that nutrient - sensing mechanisms may be conserved throughout the gut for glucose regulation . nutrient absorption occurs predominantly in the duodenum , and it is believed that only under malabsorptive conditions undigested nutrients reach the distal gut ( 39 ) . however , studies in animals ( 4043 ) and humans ( 44,45 ) illustrate that ingested nutrients reach the distal gut during early phases of food ingestion , opening up the possibility that nutrient - sensing mechanisms in the jejunum or ileum are activated shortly after a meal to regulate peripheral metabolic homeostasis . nonetheless , given that jejunal nutrient sensing regulates feeding ( 4650 ) and that , as discussed above , duodenal lipid sensing regulates glucose production ( 12 ) , a potential glucoregulatory role of jejunal nutrient sensing has been examined as well ( 6 ) ( fig . an influx of nutrients into the duodenum or jejunum activates a complex biochemical , molecular , neuronal , and physiological network to lower hepatic glucose production . although there are some parallels regarding nutrient sensing in the duodenum and jejunum , it appears that unlike in the duodenum , jejunal nutrient sensing is pka independent . direct administration of glucose or lipids ( the main sources of food energy ) into the jejunum lowers glucose production in the absence of a lowering of food intake ( 6 ) . the effect of nutrient sensing is ignited within the jejunum since direct inhibition of glucose sensing mechanisms in the duodenum or ileum fails to negate the ability of a jejunal glucose infusion to lower glucose production ( 6 ) . in addition , an equimolar infusion of glucose into the portal vein , as in the jejunum , fails to lower glucose production ( 6 ) . these experiments do not exclude the glucoregulatory role of nutrient sensing in the duodenum , ileum , or portal vein but rather illustrate that direct infusion of glucose into the jejunum triggers signaling mechanisms within the jejunum to lower glucose production . nutrient sensing in the jejunum appears to share similar mechanisms with the duodenum , since both segments lower glucose production in response to lipids , require the formation of lcfa - coa , and trigger a gut - brain - liver neuronal relay to lower glucose production ( 6,53 ) . however , evidence suggests that important differences may exist between the two because direct administration of a pka agonist ( which activates downstream cck - pka signaling ) into the duodenum , but not the jejunum , lowers glucose production ( 21 ) , although cck is also synthesized in the jejunum of animals and humans ( 5456 ) but to a much lower extent ( 57 ) . given that cck mediates duodenal lipid sensing mechanisms via pka and/or plc signaling pathways to lower glucose production ( 53 ) ( fig . similarly , other major distal gut peptides that have documented anorectic effects mediated by the vagus are glp-1 and peptide yy ( 58 ) , either of which remain possible mediator(s ) in maintaining glucose homeostasis triggered by jejunal nutrient sensing . future investigation is required to dissect the downstream jejunal signaling pathway(s ) of nutrient sensing that regulate peripheral metabolic homeostasis . inhibition of the sodium - glucose transporter proteins ( sglts ) in the jejunum negates the ability of intrajejunal glucose administration to lower glucose production ( 6 ) , indicating that jejunal glucose uptake from the lumen is a necessary step for jejunal glucose sensing to regulate peripheral glucose homeostasis . since galactose , like glucose , is also absorbed into the intestinal enterocyte from the lumen via the same glucose transporter ( 59 ) , it would be important to assess whether galactose , at an equimolar concentration of glucose in the jejunum , also lowers glucose production . another monosaccharide , fructose , is absorbed into the intestine via glucose transporter-5 ( 59 ) , and the potential glucoregulatory role of fructose sensing in the jejunum deserves future attention . we propose that comparing the glucoregulatory impact of sensing mechanisms potentially triggered by any of these monosaccharides will be crucial in revealing novel nutrient - sensing mechanisms in the jejunum . because the accumulation of lcfa - coa in the brain and the liver are sufficient and necessary to mediate both lipid and glucose sensing mechanisms to regulate hepatic glucose metabolism ( 60 ) , this raises the possibility that lipid and glucose sensing mechanisms within the jejunum may converge . , glucoregulatory hormones change at will ) condition when nutrient - sensing mechanisms are activated by a fasting - refeeding protocol , direct disruption of duodenal nutrient - sensing mechanisms increases plasma glucose levels during refeeding and disrupts glucose homeostasis as compared with rodents with intact duodenal nutrient sensing ( 17,19 ) . in contrast , in the same experimental conditions , plasma glucose levels are not altered when jejunal nutrient - sensing mechanisms are disrupted ( 6 ) . these data suggest that the key benefits of nutrient - sensing mechanisms in the jejunum to regulate glucose homeostasis are revealed under conditions in which normal nutrient flow is disrupted , for example , when sections of the upper intestine are removed in cases of intestinal cancer or inflammation ( where short bowel resection is recommended ) or after bariatric surgery in obese patients . several key aspects of gut nutrient - sensing mechanisms and the regulation of glucose production have been uncovered , yet several questions remain . first , given the fact that direct nutrient administration into the duodenum or jejunum exerts similar effects on glucose production , would an additive or possibly synergistic effect on glucose control be observed if both duodenal and jejunal nutrient sensing were stimulated in parallel with a matched caloric load ? given that the duodenum is exposed to the highest influx of nutrients from the stomach , are duodenal nutrient - sensing mechanisms dominant over jejunal nutrient sensing ? do the duodenal nutrient - sensing defects trigger the first step in the journey of diabetes or is this defect responsible for a select subset of patients with diabetes who need to be defined ? pharmacologically , any gut - targeted therapy to lower blood glucose will access both the duodenum and jejunum , so is there a predominant selective gut mechanism of action to sufficiently lower blood glucose in diabetes ? bariatric surgery normalizes blood glucose concentrations in the majority of type 2 diabetic humans independent of weight loss ( 6264 ) . clearly , diabetes remission induced by bariatric surgery is associated with an enhancement of nutrient delivery to the distal small intestine . could the enhancement of nutrient flux into the distal small intestine after bariatric surgery lead to an antidiabetic effect ( fig . given that both the duodenum and jejunum are independently capable of sensing nutrients and generating a gut - derived signal mediated by vagal nerves to trigger a gut - brain - liver axis to lower glucose production independent of weight loss ( as discussed above ) , a clear possibility remains . likewise a tube device that is endoscopically inserted in the gut extending from the duodenum to the proximal jejunum to prevent nutrients being absorbed within this region also reports rapid changes in glucose ( http://www.gidynamics.com/endobarrier-overview.php ) . bariatric surgery bypasses the duodenum , and nutrient flow is enhanced into the jejunum , resulting in activation of jejunal nutrient - sensing mechanisms involving a series of biochemical reactions , gut - derived hormones , and neuronal circuitry to lower glucose production and plasma glucose levels . within 2 days , djb lowers blood glucose concentrations independent of lowering food intake / body weight or increasing insulin secretion in both uncontrolled diabetic models ( 6 ) . importantly , direct inhibition of jejunal nutrient - sensing mechanisms disrupts peripheral glucose control triggered by djb during refeeding ( 6 ) . of note , the glucose response during refeeding in the djb diabetic rodents with or without intact jejunal nutrient - sensing mechanisms closely resembles those observed in relatively mild - obese type 2 diabetic humans with or without djb surgery , respectively ( 67 ) . together with the ability of jejunal nutrient sensing to trigger a gut - brain - liver neuronal relay to lower glucose production in normal rodents ( see above ) , these findings collectively suggest that increased delivery of nutrients to the jejunum after djb triggers a neuronal - mediated signaling mechanism to lower glucose production and hence blood glucose concentrations in diabetes . the ability of djb to rapidly lower blood glucose concentrations is potentially via a cns - mediated reduction in hepatic glucose production ( 6 ) . in fact , other reports have published similar results that djb ( 7 ) or a variant of djb ( 38 ) triggers the cns to lower hepatic glucose production in obese type 2 diabetic rodents . of note , djb lowers blood glucose concentrations in insulin - deficient rodents ( 6 ) and lowers glucose production in obese type 2 diabetic rodents independent of an improvement on insulin action ( 7 ) . however , these findings do not exclude the possibility that djb could also lower blood glucose concentrations via increased insulin - dependent or independent glucose uptake , although studies have indicated that djb does not improve insulin action in rodents with diabetes and/or obesity ( 7,69,70 ) . in addition , clinical use of glp-1 agonists and dpp - iv inhibitors does not mirror the full glycemic benefits observed after bariatric surgery , and although controversial , it is possible that other incretin hormones such as gip may be involved in enhancing insulin secretion . it is well known that bariatric surgery results in significant health benefits in obesity with type 2 diabetes , whereas only a few studies have demonstrated that bariatric surgery lowers glucose concentrations in mild / nonobese type 2 diabetic subjects ( 67,68 ) and in obese type 1 diabetic subjects ( 72 ) . despite this , the existence of a gut - brain - liver axis regulating glucose homeostasis and its relevance in the antidiabetic effect of bariatric surgery suggests that targeting the gut with drug therapy to influence the liver could be a valid therapeutic approach and would potentially mimic the antidiabetic effect of bariatric surgery . given the accessibility of the gut to pharmacotherapy , we propose targeting nutrient sensing molecules in the gut will have therapeutic potential in lowering blood glucose concentrations in diabetes . the intestine has developed mechanisms to sense nutrients after a meal and trigger a negative feedback system to maintain glucose homeostasis . this triggering leads to the rapid glucose lowering effect of djb in diabetes ( figs . the gut microbiota is a dynamic organ that is constantly adapting to changes in its environment , and it is believed that the progression of obesity / diabetes can be attributed to the intestinal microbiota host relationship . in mice switched from a regular chow to a high - fat diet , the gut microbiota composition is altered in as little as 24 h ( 75 ) , resulting in increased levels of firmicutes ( 76 ) . of note , it has recently been demonstrated that genetically identical mice fed the same high - fat diet differed in their metabolic phenotype due to differences in their microbiota composition ( 78 ) . although a larger bacterial population resides in the more distal portion of the small intestine and in the colon , bacteria are also present in the upper intestine , including the duodenum and jejunum . furthermore , gut microbiota alters intestinal permeability ( 80,81 ) and regulates both the synthesis and production of bile acids ( 82,83 ) , which are important for fat emulsification and absorption in the intestine . further , high - fat feeding promotes an obesogenic bacterial profile , which is linked to increased inflammation in the intestine and whole - body insulin resistance ( 80 ) . in light of these findings , we put forward a working hypothesis that the obesogenic microbiome disrupts nutrient - sensing cck signaling mechanisms in the duodenum , leading to a dysregulation of glucose homeostasis ( fig . nevertheless , a possibility remains that the improvement of glucose homeostasis observed following bariatric surgery is mediated by a change in gut microbiota , leading to an enhancement of nutrient - sensing mechanisms in the jejunum and an improvement in glucose tolerance ( fig . the gut possesses a unique role in regulating glucose and energy homeostasis whereby the presence of ingested nutrients into the small intestine activates sensing mechanisms that affect both glucose production and food intake . the nutrient - sensing mechanisms are conserved from the duodenum to the jejunum , and although it is evident that nutrient uptake and a neuronal network are required in both regions to lower glucose production , whether additional parallel mechanisms exist is not yet known . duodenal nutrient sensing acts as a protective mechanism under normal physiological conditions to maintain glucose homeostasis during nutrient ingestion by ensuring that glucose production is decreased ; however , this mechanism appears to be impaired after excess caloric intake . on the other hand , jejunal nutrient sensing is important in mediating the glucose lowering effect ( via decreased glucose production ) of bariatric surgery where nutrient influx to the jejunum is enhanced . emerging evidence also suggests a relationship between gut microbiota and intestinal nutrient - sensing mechanisms ; however , this concept has not been directly tested . although many questions remain unanswered , recent advances in our understanding of the pathways regulating gut nutrient sensing provide compelling support for potential new therapeutic targets to restore glucose homeostasis in diabetes .

selective logging is the dominant commercial activity in forested areas of the tropics with strong conservation potential ( frumhoff , 1995 ; struhsaker , 1997 ) . this potential is arguably strongest in central africa , where a combination of weak communications and infrastructure , lack of logistics systems , and high transportation costs result in highly selective timber extraction , focusing on a few economically valuable species occurring at low densities ( boardman et al . , 2007 ; clark et al . , 2009 ) . during the last decade , the timber industry has thrived in central africa and many of the remaining forests have been allocated to timber concessions . with extraction rates of 14 trees harvested per hectare , damage to remaining forest this is compared to extraction rates of 510 trees per hectare and associated disruption of 2550% of the canopy typical of other forested regions of the tropics ( skorupa , 1987 ; chapman et al . , 2000 ) . whereas some species of wildlife thrive in such low - intensity operations , even low - level extraction appears to have a negative effect on some primate species , including chimpanzees ( white , 1992 ; barnes and lahm , 1997 ; white and tutin , 2001 ; matthews , 2004 ) . although logging is known to reduce the abundance of some primate species due to associated hunting and the loss of food sources for frugivores ( wilkie and carpenter , 1998 ; chapman et al . , 2000 ) , the potential role of parasite infections in such primate population declines remains largely unexplored . logging results in a suite of alterations in host ecology forest structure and human - wildlife overlap that may alter infection prevalence and infection risk in resident populations . patterns of parasitism in wildlife populations are influenced by host ranging patterns , density , intraspecific and interspecific contact rates , diet , and immune function ( hudson et al . , 2001 ; nunn et al . , 2003 ; gillespie et al . , 2005 , 2008 ; beldomenico et al . , 2008 ) . studies on a variety of species have demonstrated that these characteristics can be affected by changes in forest structure ( olupot et al . , 1994 ; heydon and bulloh , 1997 ; patriquin and barclay , 2003 ) . giardia and cryptosporidium are protozoal pathogens that infect humans , domestic animals , and wildlife worldwide ( appelbee et al . , 2005 ) . in humans and livestock , these parasites cause diarrhea and other enteric disorders that can contribute to nutritional deficiencies and impaired weight gain ( savioli et al . , 2006 ; thompson , 2004 ) . little information exists about the clinical affects of these parasites on their wildlife hosts ; however , environmental pollution with human fecal material is recognized as a potential pathogen pathway for wildlife infections with zoonotic protozoal parasites , such as giardia and cryptosporidium ( appelbee et al . , 2005 ) . considering the capacity of such infections to put wildlife populations at risk , it is important to monitor the health status of wildlife in general and endangered species in particular . it is equally important to identify potential pathogen transmission pathways from humans and domestic animals to wildlife . to better understand the affect of selective logging on the transmission and persistence of these protozoal pathogens , we examined noninvasively collected fecal samples from 134 sympatric western lowland gorillas ( gorilla gorilla gorilla ) and chimpanzees ( pan troglodytes troglodytes ) in the northern republic of congo . we compared patterns of infection between ape samples from the kabo logging concession with those sampled from the adjoining undisturbed forest of the nouabal - ndoki national park . the sangha river tri - national conservation area ( 36,236 km ) is notable for its extensive tracks of forest and transboundary protected area network : nouabal - ndoki national park in republic of congo , lobk national park in cameroon , and dzanga - ndoki national park and dzanga - sangha special reserve in central african republic ( fig . 1 ) . this region is largely unpopulated with human densities estimated at 0.7 to 0.8 people per km ( auzel and wilkie , 2000 ) . semi - nomadic baaka pygmies and bantu agriculturalist - fishermen live in complex , interdependent economic and social relationships ( eves and ruggiero , 2000 ) . most villages are along major waterways leaving the remote interior forest with little human development and associated pressures . however , commercial logging is rapidly expanding along the periphery of the tri - national conservation area . timber exploitation in these remote areas is relatively labor - intensive and can result in the transient relocation of skilled workers and their families , introducing the risk of novel opportunities for pathogen transmission from people to resident wildlife.figure 1land use in the sangha tri - national conservation area at the boundary of republic of congo , cameroon , and the central african republic . the goualougo triangle is located in the southern portion of the nouabal - ndoki national park . land use in the sangha tri - national conservation area at the boundary of republic of congo , cameroon , and the central african republic . the goualougo triangle is located in the southern portion of the nouabal - ndoki national park . the kabo logging concession ( 2800 km ) , operated by the congolaise industrielle des bois , has been logged to varying degrees for the past 40 years . the pristine forests of the adjoining goualougo triangle ( 310 km ) were initially part of the kabo concession until biological surveys by the wildlife conservation society revealed the conservation significance of this region s great ape populations and their habitat . in june 2003 , the congolese government announced that it would annex the majority of the goualougo triangle to the nouabal - ndoki national park ( 4400 km , 2.053.03n , 16.5116.56e ) . however , the adjacent forest remains in an active logging concession . as part of the goualougo triangle ape project s mandate to evaluate the impacts of logging on wild apes , this area was stratified into study zones ( a , b1 , b2 , c , and d ) with relation to the national park boundaries and logging activities ( fig . 2 ) . zone d was logged in the early 1970s , and zone c is slated for logging in the near future . all study zones are characterized by similar terra firma forest dominated by mixed species interspersed with monodominant gilbertiodendron dewevrei , seasonally flooded forest , and open swamp forests ( morgan et al . , 2006).figure 2location of study zones within the goualougo triangle ( a , b1 , and b2 ) and adjacent logging concession ( c and d ) in republic of congo . location of study zones within the goualougo triangle ( a , b1 , and b2 ) and adjacent logging concession ( c and d ) in republic of congo . between 2004 and 2009 , we conducted repeated reconnaissance and line transect surveys for ape signs in zone d of the kabo logging concession and zones a and b1 within the goualougo triangle ( morgan et al . our first surveys of zone d were conducted after forestry activities had been dormant for more than 30 years . ape fecal samples were collected opportunistically in the field and preserved in 10% neutral buffered formalin ( gillespie , 2006 ) . fecal samples were characterized as chimpanzee or gorilla based on morphological features . at similar sites using similar methods , samples characterized in this fashion were verified to the correct species more than 94% of the time by genetic methods , with field researchers able to correctly identify gorilla dung ( 318/324 = 98% ) more often than chimpanzee fecal remains ( 200/211 = 94% ) ( arandjelovic , 2009 , personal communication ) . when collected , fecal material was characterized as soft or firm depending on the consistency , assuming that sick apes will evacuate soft stools ( gillespie et al . , 2008 ) . a merifluor cryptosporidium / giardia direct immunofluorescent detection kit ( meridian bioscience , inc . , cincinnati , oh ) was used to detect both parasites ( johnston et al . , 2003 ) . fecal samples were scored both for presence or absence of the pathogens as well as quantification of cryptosporidium sp . oocysts and giardia sp . cysts in feces . counts were calculated by analyzing 10 l of the 1 g / ml solution of concentrated feces from each sample . oocysts or cysts were then quantified by counting total numbers in 150 microscope fields ( 400 magnification ) and extrapolating results to the entire sample . this method was validated by spiking negative fecal samples with known numbers of cryptosporidium sp oocysts and giardia sp . results were analyzed using epiinfo , version 3.3.2 ( centers for disease control , atlanta , ga ) . a power analysis was conducted using the finite population correction for proportions ( israel 1992 ) . the corrected sample sizes are calculated using the original sample size results calculated using the equation for a large population . these numbers are based on a confidence level of 95% and level of precision of 0.05 . the sangha river tri - national conservation area ( 36,236 km ) is notable for its extensive tracks of forest and transboundary protected area network : nouabal - ndoki national park in republic of congo , lobk national park in cameroon , and dzanga - ndoki national park and dzanga - sangha special reserve in central african republic ( fig . 1 ) . this region is largely unpopulated with human densities estimated at 0.7 to 0.8 people per km ( auzel and wilkie , 2000 ) . semi - nomadic baaka pygmies and bantu agriculturalist - fishermen live in complex , interdependent economic and social relationships ( eves and ruggiero , 2000 ) . most villages are along major waterways leaving the remote interior forest with little human development and associated pressures . however , commercial logging is rapidly expanding along the periphery of the tri - national conservation area . timber exploitation in these remote areas is relatively labor - intensive and can result in the transient relocation of skilled workers and their families , introducing the risk of novel opportunities for pathogen transmission from people to resident wildlife.figure 1land use in the sangha tri - national conservation area at the boundary of republic of congo , cameroon , and the central african republic . the goualougo triangle is located in the southern portion of the nouabal - ndoki national park . land use in the sangha tri - national conservation area at the boundary of republic of congo , cameroon , and the central african republic . the goualougo triangle is located in the southern portion of the nouabal - ndoki national park . the kabo logging concession ( 2800 km ) , operated by the congolaise industrielle des bois , has been logged to varying degrees for the past 40 years . the pristine forests of the adjoining goualougo triangle ( 310 km ) were initially part of the kabo concession until biological surveys by the wildlife conservation society revealed the conservation significance of this region s great ape populations and their habitat . in june 2003 , the congolese government announced that it would annex the majority of the goualougo triangle to the nouabal - ndoki national park ( 4400 km , 2.053.03n , 16.5116.56e ) . however , the adjacent forest remains in an active logging concession . as part of the goualougo triangle ape project s mandate to evaluate the impacts of logging on wild apes , this area was stratified into study zones ( a , b1 , b2 , c , and d ) with relation to the national park boundaries and logging activities ( fig . 2 ) . zone d was logged in the early 1970s , and zone c is slated for logging in the near future . all study zones are characterized by similar terra firma forest dominated by mixed species interspersed with monodominant gilbertiodendron dewevrei , seasonally flooded forest , and open swamp forests ( morgan et al . , 2006).figure 2location of study zones within the goualougo triangle ( a , b1 , and b2 ) and adjacent logging concession ( c and d ) in republic of congo . location of study zones within the goualougo triangle ( a , b1 , and b2 ) and adjacent logging concession ( c and d ) in republic of congo . between 2004 and 2009 , we conducted repeated reconnaissance and line transect surveys for ape signs in zone d of the kabo logging concession and zones a and b1 within the goualougo triangle ( morgan et al . , 2006 , 2009 ) . our first surveys of zone d were conducted after forestry activities had been dormant for more than 30 years . ape fecal samples were collected opportunistically in the field and preserved in 10% neutral buffered formalin ( gillespie , 2006 ) . fecal samples were characterized as chimpanzee or gorilla based on morphological features . at similar sites using similar methods , samples characterized in this fashion were verified to the correct species more than 94% of the time by genetic methods , with field researchers able to correctly identify gorilla dung ( 318/324 = 98% ) more often than chimpanzee fecal remains ( 200/211 = 94% ) ( arandjelovic , 2009 , personal communication ) . when collected , fecal material was characterized as soft or firm depending on the consistency , assuming that sick apes will evacuate soft stools ( gillespie et al . , 2008 ) . a merifluor cryptosporidium / giardia direct immunofluorescent detection kit ( meridian bioscience , inc . , cincinnati , oh ) was used to detect both parasites ( johnston et al . , fecal samples were scored both for presence or absence of the pathogens as well as quantification of cryptosporidium sp . oocysts and giardia sp . cysts in feces . counts were calculated by analyzing 10 l of the 1 g / ml solution of concentrated feces from each sample . oocysts or cysts were then quantified by counting total numbers in 150 microscope fields ( 400 magnification ) and extrapolating results to the entire sample . this method was validated by spiking negative fecal samples with known numbers of cryptosporidium sp oocysts and giardia sp . cysts . results were analyzed using epiinfo , version 3.3.2 ( centers for disease control , atlanta , ga ) . a power analysis was conducted using the finite population correction for proportions ( israel 1992 ) . the corrected sample sizes are calculated using the original sample size results calculated using the equation for a large population . these numbers are based on a confidence level of 95% and level of precision of 0.05 . three of 34 chimpanzee samples ( 8.82% ) from the logged site , 1 of 33 chimpanzee samples from the undisturbed site ( 3.03% ) , 0 of 31 gorilla samples from the logged site ( 0% ) , and 1 of 36 gorilla samples from the undisturbed sites ( 2.78% ) were infected with giardia sp . ( table 1 ) . prevalence of infection with giardia sp . was not significantly different between logged and undisturbed forest for chimpanzees ( = 1.00 , p = 0.32 , degrees of freedom ( df ) = 1 ) or gorillas ( = 0.87 , p = 0.35 , df = 1).table 1prevalence and intensity of cryptosporidium and giardia spp . in chimpanzees and gorillas in the goualougo triangle of nouabal - ndoki national park and kabo logging concession , republic of congospeciespopulationsamplescryptosporidiumgiardiaprevalence ( % ) mean intensityprevalence ( % ) mean intensitychimpanzeegoualougo33003.031000kabo34008.821833 573.49gorillagoualougo36009.78900kabo310000mean intensities are expressed as oocysts ( cryptosporidium ) or cysts ( giardia ) per gram , standard error of the mean ; negative samples are not included in this calculation . prevalence and intensity of cryptosporidium and giardia spp . in chimpanzees and gorillas in the goualougo triangle of nouabal - ndoki national park and kabo logging concession , republic of congo mean intensities are expressed as oocysts ( cryptosporidium ) or cysts ( giardia ) per gram , standard error of the mean ; negative samples are not included in this calculation . of all 67 chimpanzee samples , one was characterized as soft ( 1.5% ) and the remainder as firm ( 98.5% ) . of all 67 gorilla samples , 3 were characterized as soft ( 4.5% ) and the remainder as firm ( 95.5% ) . there was no relationship between fecal consistency and giardia infection status for chimpanzees ( = 0.00 , p = 0.78 , df = 1 ) or gorillas ( = 0.07 , p = 0.78 , df = 1 ) . our power analysis demonstrated that additional zone d chimpanzee sampling would be required to ensure that the lack of significance observed in intersite comparisons of chimpanzees giardia sp . prevalence is indeed valid ( 47 zone d compared with the 34 collected thus far ) . samples were collected during repeated line transect surveys during a period of 5 years . three to five samples can be collected in a day by a team of five trackers . samples are collected by backtracking sighted apes to find feces voided within the past hour . aged feces , such as those from night nests are not collected . due to these limitations , we regard the lack of significant differences in giardia sp . prevalence between sites for chimpanzees to be preliminary . we also compared the intensity of infection for each ape species between logged and undisturbed sites . the mean intensity of infection was highest in chimpanzees from the disturbed site ( 1833 573.49 cysts / g ) , followed by chimpanzees in the undisturbed site ( 1000 cysts / g ) . mean intensity of infection was slightly lower for gorillas from the undisturbed site ( 900 cysts / g ) , and no infections were found in gorillas from the disturbed site , resulting in an intensity of zero ( table 1 ) ; mean intensities of infection could not be compared statistically across sites due to rarity of infection . our results demonstrate that giardia sp . is present at low prevalence in chimpanzees and lowland gorillas in undisturbed forests and forests with a history of selective logging . in these same populations a lack of cryptosporidium sp . in all samples examined suggests that cryptosporidium sp . is not a natural component of ape parasite communities at this site and that historical land - use patterns are not exposing apes to zoonotic sources of this pathogen . regardless of historical land - use patterns suggest that a low level of circulating giardia is a natural component of congolese ape parasite communities and that historical land - use patterns are not exposing apes to zoonotic sources of this pathogen . our study area is located only 32 km from the nearest village and the large river that serves as a major transportation source for the region . however , the forest is continuous and the encounter rate of human signs in the study zones was low , which confirms that these forests have remained relatively undisturbed despite past timber exploitation . this post - logging scenario is much different than the high human densities and forest fragmentation typical in east and west africa . giardia and cryptosporidium are notable for cross - species transmission ( thompson , 2004 ) . the propensity of these protozoans to cross species barriers results from the host nonspecificity of trophozoites of either genus , and the high infectivity and environmental stability of giardia cyst and cryptosporidium oocysts ( smith and nichols , 2006 ) . previous field - based studies confirm this pattern , demonstrating that high levels of primate ( nizeyi et al . , 2002 ; graczyk et al . , 2001a , b ; graczyk et al . the results of the current study are the first to examine the long - term effects of historical primate human overlap on contemporary patterns of infection with these pathogens . considered among the most common human intestinal protozoa , giardia ranges in clinical presentation from asymptomatic to highly pathogenic , causing chronic malabsorptive diarrhea in some patients ( thompson , 2004 ) . both host factors ( e.g. , nutrition , immunity , coinfection with other agents ) and pathogen factors ( e.g. , strain , infectious dose ) are thought to contribute to the severity of clinical disease ( thompson , 2000 ) . giardiasis is now considered by the world health organization to be a neglected disease , due to its ubiquity in equatorial latitudes , its chronic detrimental health effects ( especially in children ) , its propensity to infect economically disadvantaged populations , and the paradoxical availability of relatively inexpensive and effective pharmaceuticals for its treatment ( savioli et al . , 2006 ) . in our study , we found no relationship between infection status and fecal consistency ( soft vs. firm ) . the lack of a strong measurable association between infection and acute gastrointestinal symptoms in our study population was surprising , but it is concordant with recent studies of humans . although giardia is associated with clinical diseases in developed economies , evidence is nevertheless mounting that it may be commensal in rural settings where people may normally be exposed to diverse parasite communities from an early age . for example , perz cordn et al . ( 2008 ) found giardia at 28.1% prevalence in diarrheic peruvian children , as well as in 19.5% of nondiarrheic children , emphasizing the importance of asymptomatic patients in giardia transmission where hygiene and sanitation are poor ( perz cordn et al . , 2008 ) . ethiopia ( ayalew et al . , 2008 ) , bangladesh ( dib et al . , 2008 ) , and peru ( hollm - delgado et al . , 2008 ) have found similarly weak or nonexistent associations between g. duodenalis infection status and gastrointestinal symptoms , suggesting that g. duodenalis may coexist benignly with human hosts under conditions where acquired immunity to the pathogen can develop . lack of early exposure and acquired immunity to g. duodenalis may , in fact , account for the role of the pathogen as a cause of sporadic diarrheal outbreaks in developed countries ( istre et al . , 1984 ) , as well as a major cause of traveler s diarrhea for people from developed countries who visit endemic areas ( reinthaler et al . we hope to explore in detail the association between symptom and infection in apes in more detail in future work . our results from the goualougo triangle provide a rare opportunity to observe baseline patterns of infection in undisturbed ape populations . baseline patterns of parasitism are necessary to understand both individual animal health status and to assess what would constitute an outbreak : a level of disease or parasitism in excess of what is normally observed . thus , these results provide a first step toward an index of population health and disease risk assessment for conservation and management plans of these endangered african ape populations . our results from the kabo concession suggest that a legacy of low - intensity selective logging did not result in the persistence of elevated prevalence of cryptosporidium sp . and giardia sp . in these ape populations . it is encouraging that the resident ape populations are not suffering from infections of these protozoa , because this suggests such potential health threats may be mitigated . however , if these protozoa are exceptionally pathogenic in wild ape populations , infected individuals may have been removed from the population long ago due to high associated mortality . we have yet to examine how patterns and risk of parasitism may differ during the various stages of the active logging process ( i.e. , exploration , road construction , and timber harvesting ) from those that are the product of a legacy of selective logging . the expansion of timber activities in the neighboring undisturbed forests of the kabo concession will enable us to address these questions through an ongoing progressive monitoring program that involves documenting ape populations , human disturbance , and the health implications during their interactions .

many studies have examined the long - term effects of selective logging on the abundance and diversity of free - ranging primates . logging is known to reduce the abundance of some primate species through associated hunting and the loss of food trees for frugivores ; however , the potential role of pathogens in such primate population declines is largely unexplored . selective logging results in a suite of alterations in host ecology and forest structure that may alter pathogen dynamics in resident wildlife populations . in addition , environmental pollution with human fecal material may present a risk for wildlife infections with zoonotic protozoa , such as cryptosporidium and giardia . to better understand this interplay , we compared patterns of infection with these potentially pathogenic protozoa in sympatric western lowland gorillas ( gorilla gorilla gorilla ) and chimpanzees ( pan troglodytes troglodytes ) in the undisturbed goualougo triangle of nouabal - ndoki national park and the adjacent previously logged kabo concession in northern republic of congo . no cryptosporidium infections were detected in any of the apes examined and prevalence of infection with giardia was low ( 3.73% overall ) and did not differ between logged and undisturbed forest for chimpanzees or gorillas . these results provide a baseline for prevalence of these protozoa in forest - dwelling african apes and suggest that low - intensity logging may not result in long - term elevated prevalence of potentially pathogenic protozoa .

Introduction Methods Study Site Sample Collection and Analysis Results Discussion

selective logging is the dominant commercial activity in forested areas of the tropics with strong conservation potential ( frumhoff , 1995 ; struhsaker , 1997 ) . this potential is arguably strongest in central africa , where a combination of weak communications and infrastructure , lack of logistics systems , and high transportation costs result in highly selective timber extraction , focusing on a few economically valuable species occurring at low densities ( boardman et al . during the last decade , the timber industry has thrived in central africa and many of the remaining forests have been allocated to timber concessions . with extraction rates of 14 trees harvested per hectare , damage to remaining forest this is compared to extraction rates of 510 trees per hectare and associated disruption of 2550% of the canopy typical of other forested regions of the tropics ( skorupa , 1987 ; chapman et al . whereas some species of wildlife thrive in such low - intensity operations , even low - level extraction appears to have a negative effect on some primate species , including chimpanzees ( white , 1992 ; barnes and lahm , 1997 ; white and tutin , 2001 ; matthews , 2004 ) . although logging is known to reduce the abundance of some primate species due to associated hunting and the loss of food sources for frugivores ( wilkie and carpenter , 1998 ; chapman et al . , 2000 ) , the potential role of parasite infections in such primate population declines remains largely unexplored . logging results in a suite of alterations in host ecology forest structure and human - wildlife overlap that may alter infection prevalence and infection risk in resident populations . patterns of parasitism in wildlife populations are influenced by host ranging patterns , density , intraspecific and interspecific contact rates , diet , and immune function ( hudson et al . studies on a variety of species have demonstrated that these characteristics can be affected by changes in forest structure ( olupot et al . , 2006 ; thompson , 2004 ) . little information exists about the clinical affects of these parasites on their wildlife hosts ; however , environmental pollution with human fecal material is recognized as a potential pathogen pathway for wildlife infections with zoonotic protozoal parasites , such as giardia and cryptosporidium ( appelbee et al . considering the capacity of such infections to put wildlife populations at risk , it is important to monitor the health status of wildlife in general and endangered species in particular . it is equally important to identify potential pathogen transmission pathways from humans and domestic animals to wildlife . to better understand the affect of selective logging on the transmission and persistence of these protozoal pathogens , we examined noninvasively collected fecal samples from 134 sympatric western lowland gorillas ( gorilla gorilla gorilla ) and chimpanzees ( pan troglodytes troglodytes ) in the northern republic of congo . we compared patterns of infection between ape samples from the kabo logging concession with those sampled from the adjoining undisturbed forest of the nouabal - ndoki national park . the sangha river tri - national conservation area ( 36,236 km ) is notable for its extensive tracks of forest and transboundary protected area network : nouabal - ndoki national park in republic of congo , lobk national park in cameroon , and dzanga - ndoki national park and dzanga - sangha special reserve in central african republic ( fig . 1 ) . this region is largely unpopulated with human densities estimated at 0.7 to 0.8 people per km ( auzel and wilkie , 2000 ) . semi - nomadic baaka pygmies and bantu agriculturalist - fishermen live in complex , interdependent economic and social relationships ( eves and ruggiero , 2000 ) . however , commercial logging is rapidly expanding along the periphery of the tri - national conservation area . timber exploitation in these remote areas is relatively labor - intensive and can result in the transient relocation of skilled workers and their families , introducing the risk of novel opportunities for pathogen transmission from people to resident wildlife.figure 1land use in the sangha tri - national conservation area at the boundary of republic of congo , cameroon , and the central african republic . the goualougo triangle is located in the southern portion of the nouabal - ndoki national park . land use in the sangha tri - national conservation area at the boundary of republic of congo , cameroon , and the central african republic . the goualougo triangle is located in the southern portion of the nouabal - ndoki national park . the pristine forests of the adjoining goualougo triangle ( 310 km ) were initially part of the kabo concession until biological surveys by the wildlife conservation society revealed the conservation significance of this region s great ape populations and their habitat . in june 2003 , the congolese government announced that it would annex the majority of the goualougo triangle to the nouabal - ndoki national park ( 4400 km , 2.053.03n , 16.5116.56e ) . however , the adjacent forest remains in an active logging concession . as part of the goualougo triangle ape project s mandate to evaluate the impacts of logging on wild apes , this area was stratified into study zones ( a , b1 , b2 , c , and d ) with relation to the national park boundaries and logging activities ( fig . zone d was logged in the early 1970s , and zone c is slated for logging in the near future . , 2006).figure 2location of study zones within the goualougo triangle ( a , b1 , and b2 ) and adjacent logging concession ( c and d ) in republic of congo . location of study zones within the goualougo triangle ( a , b1 , and b2 ) and adjacent logging concession ( c and d ) in republic of congo . between 2004 and 2009 , we conducted repeated reconnaissance and line transect surveys for ape signs in zone d of the kabo logging concession and zones a and b1 within the goualougo triangle ( morgan et al . ape fecal samples were collected opportunistically in the field and preserved in 10% neutral buffered formalin ( gillespie , 2006 ) . at similar sites using similar methods , samples characterized in this fashion were verified to the correct species more than 94% of the time by genetic methods , with field researchers able to correctly identify gorilla dung ( 318/324 = 98% ) more often than chimpanzee fecal remains ( 200/211 = 94% ) ( arandjelovic , 2009 , personal communication ) . when collected , fecal material was characterized as soft or firm depending on the consistency , assuming that sick apes will evacuate soft stools ( gillespie et al . a merifluor cryptosporidium / giardia direct immunofluorescent detection kit ( meridian bioscience , inc . fecal samples were scored both for presence or absence of the pathogens as well as quantification of cryptosporidium sp . oocysts and giardia sp . counts were calculated by analyzing 10 l of the 1 g / ml solution of concentrated feces from each sample . oocysts or cysts were then quantified by counting total numbers in 150 microscope fields ( 400 magnification ) and extrapolating results to the entire sample . this method was validated by spiking negative fecal samples with known numbers of cryptosporidium sp oocysts and giardia sp . the sangha river tri - national conservation area ( 36,236 km ) is notable for its extensive tracks of forest and transboundary protected area network : nouabal - ndoki national park in republic of congo , lobk national park in cameroon , and dzanga - ndoki national park and dzanga - sangha special reserve in central african republic ( fig . this region is largely unpopulated with human densities estimated at 0.7 to 0.8 people per km ( auzel and wilkie , 2000 ) . however , commercial logging is rapidly expanding along the periphery of the tri - national conservation area . timber exploitation in these remote areas is relatively labor - intensive and can result in the transient relocation of skilled workers and their families , introducing the risk of novel opportunities for pathogen transmission from people to resident wildlife.figure 1land use in the sangha tri - national conservation area at the boundary of republic of congo , cameroon , and the central african republic . the goualougo triangle is located in the southern portion of the nouabal - ndoki national park . land use in the sangha tri - national conservation area at the boundary of republic of congo , cameroon , and the central african republic . the goualougo triangle is located in the southern portion of the nouabal - ndoki national park . the pristine forests of the adjoining goualougo triangle ( 310 km ) were initially part of the kabo concession until biological surveys by the wildlife conservation society revealed the conservation significance of this region s great ape populations and their habitat . in june 2003 , the congolese government announced that it would annex the majority of the goualougo triangle to the nouabal - ndoki national park ( 4400 km , 2.053.03n , 16.5116.56e ) . however , the adjacent forest remains in an active logging concession . as part of the goualougo triangle ape project s mandate to evaluate the impacts of logging on wild apes , this area was stratified into study zones ( a , b1 , b2 , c , and d ) with relation to the national park boundaries and logging activities ( fig . zone d was logged in the early 1970s , and zone c is slated for logging in the near future . all study zones are characterized by similar terra firma forest dominated by mixed species interspersed with monodominant gilbertiodendron dewevrei , seasonally flooded forest , and open swamp forests ( morgan et al . , 2006).figure 2location of study zones within the goualougo triangle ( a , b1 , and b2 ) and adjacent logging concession ( c and d ) in republic of congo . location of study zones within the goualougo triangle ( a , b1 , and b2 ) and adjacent logging concession ( c and d ) in republic of congo . between 2004 and 2009 , we conducted repeated reconnaissance and line transect surveys for ape signs in zone d of the kabo logging concession and zones a and b1 within the goualougo triangle ( morgan et al . ape fecal samples were collected opportunistically in the field and preserved in 10% neutral buffered formalin ( gillespie , 2006 ) . at similar sites using similar methods , samples characterized in this fashion were verified to the correct species more than 94% of the time by genetic methods , with field researchers able to correctly identify gorilla dung ( 318/324 = 98% ) more often than chimpanzee fecal remains ( 200/211 = 94% ) ( arandjelovic , 2009 , personal communication ) . when collected , fecal material was characterized as soft or firm depending on the consistency , assuming that sick apes will evacuate soft stools ( gillespie et al . , 2008 ) . , cincinnati , oh ) was used to detect both parasites ( johnston et al . , fecal samples were scored both for presence or absence of the pathogens as well as quantification of cryptosporidium sp . oocysts and giardia sp . cysts in feces . counts were calculated by analyzing 10 l of the 1 g / ml solution of concentrated feces from each sample . oocysts or cysts were then quantified by counting total numbers in 150 microscope fields ( 400 magnification ) and extrapolating results to the entire sample . this method was validated by spiking negative fecal samples with known numbers of cryptosporidium sp oocysts and giardia sp . a power analysis was conducted using the finite population correction for proportions ( israel 1992 ) . three of 34 chimpanzee samples ( 8.82% ) from the logged site , 1 of 33 chimpanzee samples from the undisturbed site ( 3.03% ) , 0 of 31 gorilla samples from the logged site ( 0% ) , and 1 of 36 gorilla samples from the undisturbed sites ( 2.78% ) were infected with giardia sp . prevalence of infection with giardia sp . was not significantly different between logged and undisturbed forest for chimpanzees ( = 1.00 , p = 0.32 , degrees of freedom ( df ) = 1 ) or gorillas ( = 0.87 , p = 0.35 , df = 1).table 1prevalence and intensity of cryptosporidium and giardia spp . in chimpanzees and gorillas in the goualougo triangle of nouabal - ndoki national park and kabo logging concession , republic of congospeciespopulationsamplescryptosporidiumgiardiaprevalence ( % ) mean intensityprevalence ( % ) mean intensitychimpanzeegoualougo33003.031000kabo34008.821833 573.49gorillagoualougo36009.78900kabo310000mean intensities are expressed as oocysts ( cryptosporidium ) or cysts ( giardia ) per gram , standard error of the mean ; negative samples are not included in this calculation . prevalence and intensity of cryptosporidium and giardia spp . in chimpanzees and gorillas in the goualougo triangle of nouabal - ndoki national park and kabo logging concession , republic of congo mean intensities are expressed as oocysts ( cryptosporidium ) or cysts ( giardia ) per gram , standard error of the mean ; negative samples are not included in this calculation . of all 67 chimpanzee samples , one was characterized as soft ( 1.5% ) and the remainder as firm ( 98.5% ) . of all 67 gorilla samples , 3 were characterized as soft ( 4.5% ) and the remainder as firm ( 95.5% ) . there was no relationship between fecal consistency and giardia infection status for chimpanzees ( = 0.00 , p = 0.78 , df = 1 ) or gorillas ( = 0.07 , p = 0.78 , df = 1 ) . samples were collected during repeated line transect surveys during a period of 5 years . three to five samples can be collected in a day by a team of five trackers . samples are collected by backtracking sighted apes to find feces voided within the past hour . aged feces , such as those from night nests are not collected . due to these limitations , we regard the lack of significant differences in giardia sp . prevalence between sites for chimpanzees to be preliminary . we also compared the intensity of infection for each ape species between logged and undisturbed sites . the mean intensity of infection was highest in chimpanzees from the disturbed site ( 1833 573.49 cysts / g ) , followed by chimpanzees in the undisturbed site ( 1000 cysts / g ) . mean intensity of infection was slightly lower for gorillas from the undisturbed site ( 900 cysts / g ) , and no infections were found in gorillas from the disturbed site , resulting in an intensity of zero ( table 1 ) ; mean intensities of infection could not be compared statistically across sites due to rarity of infection . is present at low prevalence in chimpanzees and lowland gorillas in undisturbed forests and forests with a history of selective logging . regardless of historical land - use patterns suggest that a low level of circulating giardia is a natural component of congolese ape parasite communities and that historical land - use patterns are not exposing apes to zoonotic sources of this pathogen . our study area is located only 32 km from the nearest village and the large river that serves as a major transportation source for the region . however , the forest is continuous and the encounter rate of human signs in the study zones was low , which confirms that these forests have remained relatively undisturbed despite past timber exploitation . this post - logging scenario is much different than the high human densities and forest fragmentation typical in east and west africa . the propensity of these protozoans to cross species barriers results from the host nonspecificity of trophozoites of either genus , and the high infectivity and environmental stability of giardia cyst and cryptosporidium oocysts ( smith and nichols , 2006 ) . the results of the current study are the first to examine the long - term effects of historical primate human overlap on contemporary patterns of infection with these pathogens . considered among the most common human intestinal protozoa , giardia ranges in clinical presentation from asymptomatic to highly pathogenic , causing chronic malabsorptive diarrhea in some patients ( thompson , 2004 ) . , nutrition , immunity , coinfection with other agents ) and pathogen factors ( e.g. giardiasis is now considered by the world health organization to be a neglected disease , due to its ubiquity in equatorial latitudes , its chronic detrimental health effects ( especially in children ) , its propensity to infect economically disadvantaged populations , and the paradoxical availability of relatively inexpensive and effective pharmaceuticals for its treatment ( savioli et al . , 2006 ) . in our study , we found no relationship between infection status and fecal consistency ( soft vs. firm ) . , 2008 ) . , 2008 ) have found similarly weak or nonexistent associations between g. duodenalis infection status and gastrointestinal symptoms , suggesting that g. duodenalis may coexist benignly with human hosts under conditions where acquired immunity to the pathogen can develop . lack of early exposure and acquired immunity to g. duodenalis may , in fact , account for the role of the pathogen as a cause of sporadic diarrheal outbreaks in developed countries ( istre et al . our results from the goualougo triangle provide a rare opportunity to observe baseline patterns of infection in undisturbed ape populations . baseline patterns of parasitism are necessary to understand both individual animal health status and to assess what would constitute an outbreak : a level of disease or parasitism in excess of what is normally observed . thus , these results provide a first step toward an index of population health and disease risk assessment for conservation and management plans of these endangered african ape populations . our results from the kabo concession suggest that a legacy of low - intensity selective logging did not result in the persistence of elevated prevalence of cryptosporidium sp . and giardia sp . it is encouraging that the resident ape populations are not suffering from infections of these protozoa , because this suggests such potential health threats may be mitigated . however , if these protozoa are exceptionally pathogenic in wild ape populations , infected individuals may have been removed from the population long ago due to high associated mortality . we have yet to examine how patterns and risk of parasitism may differ during the various stages of the active logging process ( i.e. , exploration , road construction , and timber harvesting ) from those that are the product of a legacy of selective logging . the expansion of timber activities in the neighboring undisturbed forests of the kabo concession will enable us to address these questions through an ongoing progressive monitoring program that involves documenting ape populations , human disturbance , and the health implications during their interactions .

there has been a notable shift in eating culture in the past 40 years . since the late 1970s , consumption of foods prepared outside the home has steadily grown , from one - sixth to almost one - third of an individual 's daily dietary intake [ 14 ] . a 2012 analysis of 2007 - 2008 national health and nutrition examination survey ( nhanes ) data found that 41% of adults consumed foods and/or beverages from fast food - type restaurants and 27% from full - service restaurants during the previous 24 hours . with fewer meals being prepared at home , not surprisingly , individuals who consume more fafh are reported to have poorer diet quality . higher fruit and vegetable intake is associated with better dietary quality [ 5 , 6 ] , and some studies have found an inverse relationship between frequency of fast food use and daily servings or meal density of fruit and vegetables [ 712 ] . the healthy eating index ( hei ) is a measure of diet quality , with total fruit and total vegetable intake as two of the main components of this composite measure . economic research service reports using the 199496 continuing survey of food intakes by individuals ( csfii ) and 2003 - 04 nhanes data found that one meal away from home lowered the daily hei score enough to shift the average adult 's diet quality from a classification of fair to poor [ 12 , 14 ] . another study using the csfii 199496 data reported that more days of fast food consumption resulted in increased energy and macronutrient intakes and decreased micronutrient density . more specifically , eating fast food was associated with lower intakes of vitamin a , carotene , vitamin c , calcium , and magnesium , high amounts of nondiet carbonated soft drinks , and insignificant amounts of fruit . other studies have found that those who consume fafh more frequently have higher intakes of total energy , total fat , saturated fat , cholesterol , sodium , sugar - sweetened beverages , and sugar and lower intakes of fiber and some micronutrients [ 10 , 1419 ] . cross - sectional and prospective studies have also found that frequent fafh consumption is associated with higher body mass index ( bmi ) and percent body fat as well as increased risk for overweight / obesity , cardiometabolic risk factors , and type 2 diabetes [ 7 , 10 , 15 , 16 , 1940 ] . the coronary artery risk development in young adults ( cardia ) study found that change in fast - food frequency over 15 years was directly associated with changes in body weight . similarly , a national study of australian young adults reported that , after adjusting for confounding variables including age , leisure time physical activity , tv viewing , and employment status , consuming takeaway food twice a week or more was associated with a 31% higher prevalence of moderate abdominal obesity in men and a 25% higher prevalence in women , compared to those who ate takeaway food less than twice a week . many of the studies linking fafh with overweight and obesity were conducted with children , adolescents , or young adults [ 9 , 10 , 28 , 29 , 34 , 3639 , 4244 ] . the shift that has occurred in consumption frequency of fafh has had important implications in terms of diet quality , including fruit and vegetable intake , and obesity . this cross - sectional study aimed to examine the relationship between fafh frequency and fruit and vegetable intake as well as the relationship between fafh frequency and bmi among a representative sample of adult men and women king county , washington . as most previous studies have focused on younger age groups , the present study fills an important gap in the literature . the seattle obesity study was a cross - sectional study of 2,001 adult male and female residents of king county , washington . a stratified random sampling scheme was used to ensure a representative sample by income and race / ethnicity . detailed study methodology has previously been published [ 45 , 46 ] and discussed here briefly . to develop the sampling frame , a prenotification letter was mailed out to alert potential respondents that their household had been randomly selected for this study . telephone calls were made to every household to randomly select an adult member as a survey respondent . exclusion criteria were age less than 18 years , discordance between addresses and telephone numbers obtained from the commercial database , and those self - reported by the respondent . after screening for eligibility , study procedures were explained to the potential respondent and a verbal consent was obtained over the phone and formally recorded . the sos sample was demographically comparable to 2007 behavioral risk factor surveillance system ( brfss ) and king county census data . all procedures involving human subjects / patients were approved by the university of washington institutional review board . self - reported data on frequency of fafh was available from 1,976 of 2,001 phone survey respondents . respondents were asked to report number of meals eaten out each week , including breakfast and lunch . frequency of fafh was categorized as 0 - 1 , 24 , or 5 or more times per week based on the distribution of the data obtained . the telephone survey asked a series of questions to collect self - reported data on socioeconomic status ( ses ) and demographic and lifestyle variables . income data collected using 9 categories was recombined into three categories for analytical purpose : < $ 50,000 , $ 50,000$100,000 , and > $ 100,000 , herein $ 50k , $ 50$100k , and > $ 100k . the 6-category education variable was recombined into three groups : high school or less , some college , and college graduates or higher . demographic variables of interest were age , gender , race / ethnicity , and household size . physical activity was captured using the standard question from brfss ( behavioral risk factor surveillance system)during the past month , other than your regular job , did you participate in any physical activity or exercise in your leisure time apart from your work ? for analytical purpose , it was treated as a dichotomous variable : yes / no . one thousand six hundred ninety - seven respondents had complete data for the demographic and lifestyle variables . self - reported data on weight and height was available from 1,877 survey respondents and was used to compute bmi . bmi was calculated as weight ( in kilograms ) divided by square of height ( in meters ) . standard dietary questions from brfss were used to capture the intake of fruits and vegetables , and other foods for the present study . each respondent was asked to report the frequency of consumption of fruit juices , fruits , green salad , carrots , and other vegetables by per day / week / month or year basis . the reported frequency for each of these items was added together to compute the total fruit and vegetable intake per person per day . all statistical analyses were conducted using spss 21.0 for windows ( version 2012 , ibm - spss , inc . ) . first , univariate descriptive statistics were conducted for each variable ( means , standard deviations , and frequencies ) . bivariate associations were then examined between frequency of fafh and each of the sociodemographic variables , bmi , and fruit and vegetable consumption , separately for males and females as well as for both genders combined . chi - square tests of independence or analysis of variance tests , followed by post hoc comparisons , were run to test the association of these demographic variables with fafh . finally , general linear models ( glm ) were used to assess the relationship between bmi and fafh , and fruit and vegetable consumption and fafh . the analyses were conducted separately for each gender as well as for both genders combined . the independent variables tested in the model were fafh , gender , age , education , income , marital status , physical activity , and smoking . for the model for both genders combined , interaction terms between gender and other relevant independent variables were tested . sample characteristics of the 1570 adults with complete data for all of the above described variables are presented in table 1 . a higher percentage of females participated than males ( 60% versus 40% , resp . ) . the sample was more likely to be white ( 84% ) , educated ( 57% college graduate ) , and with annual household income of > $ 50k ( 60% ) . about half the sample was married / member of an unmarried couple ( 57% ) , and half of the sample never smoked ( 53% ) . half of the sample consumed fafh 0 - 1 time per week , with 34% of the sample consuming 24 times per week , and 16% eating out 5 or more meals per week . average fv consumption was 4.19 servings per day , and the average bmi was 26.6 . females and males were comparable by income , education , race , marital status , and physical activity . average bmi was much higher among males ( 27.3 kg / m ) as compared to females ( 26.1 kg / m ) . overall , fafh frequency was higher among males than females ( 43% versus 54% eating out 0 - 1 meal per week , resp . ) . a significantly higher proportion of males ate out 5 or more meals per week ( 22% versus 12% among females ) ( p value < 0.001 ) . on average , females reported eating approximately half a serving more of fruits and vegetables per day than males ( 4.45 versus 3.81 servings per day , resp . ; p < 0.001 ) . bivariate associations between fafh frequency and key sociodemographic variables are presented in table 2 . significant bivariate trends were observed in frequency of fafh with selected sociodemographic variables , health indicators , and fv consumption . higher frequency of fafh was associated with being younger in age and higher bmi both among females and males ( p < 0.05 ) . for example , females with fafh frequency 5 + times per week were on average 50 y old as compared to 57 y old for those never or rarely eating out ( defined as 0 - 1 time per week ) . higher frequency of fafh was associated with relatively higher bmi both among females and males . for example , mean bmi was 27.4 kg / m among females with frequency of fafh 5 + times / week as compared to 25.8 kg / m among those who never or rarely ate out ( 0 - 1 time per week ) ( p < 0.05 ) . for females , frequency of fafh was higher among college graduates ( 15% with fafh frequency 5 + times per week ) as compared to those with high school or some college ( only 7% ) . similar trends were seen among males ( 25% versus 15% , resp . ) , although the relationship was not significant . for example , among females , 12% of those with household income of $ 50$100k and 20% of those with income > $ 100k ate out five or more times per week as compared to only 7% of those with income < $ 50k . for example , for males , while there were significant differences between fruit and vegetable intake for those who ate out 0 - 1 or 24 times per week versus 5 or more times per week , the overall association was not significant ( 3.98 , 3.78 , and 3.53 , p value = 0.093 ) . no significant differences were observed in frequency of fafh by race , marital status , or smoking status . table 3 shows associations between bmi and frequency of fafh , separately among females and males as well as for both genders combined , after adjusting for covariates . there were no significant interactions between gender and other relevant independent variables . among females , frequency of fafh 5 or more times per week was associated with significantly higher bmi as compared to those who eat out only once per week or never ( = 2.19 ; se = 0.60 ; p value < 0.001 ) . however , no significant difference in bmi was observed among females with frequency of fafh 24 times per week as compared to the reference category . among males , eating out 24 times per week and 5 times per week or more were associated with significantly higher bmi ( = 1.27 ; se = 0.44 ; p value = 0.004 and = 1.34 ; se = 0.52 ; p value = 0.010 , resp . ) , as compared to those who only eat out only once a week or never . these associations were observed even after adjusting for covariates : income , education , race / ethnicity , marital status , and lifestyle variables ( figure 1 ) . significant associations were also observed between key sociodemographic and lifestyle variables and bmi , both among females and males . for example , higher income was associated with significantly lower bmi among females ( as compared to the reference category of < $ 50k , = 1.87 , p value 0.001 among those with income $ 50$100k ; and = 1.35 , p value 0.008 among those with income > $ 100k ) . for males , a significant relation was observed with education . as compared to college graduates , having lower education was associated with higher bmi ( = 1.23 , p value : 0.028 ) . physical activity had a significant negative association with bmi for both females and males ( among females , = 2.61 , p value 0.001 for those who were not physically active outside work ; among males , = 1.33 , p value = 0.006 ) . for the models with gender combined , eating out 24 times per week and 5 or more times per week were associated with significantly higher bmi ( = 0.97 , p value = 0.001 and = 1.88 , p value < 0.001 , resp . ) , as compared to those who eat out only once a week or never . table 4 displays the multivariate associations between fv consumption and frequency of fafh , separately for females and males . among females , those who ate fafh 24 times per week consumed significantly fewer servings of fruit and vegetables per day than those who ate out 0 - 1 time per week ( = 0.36 ; se = 0.15 ; p value = 0.018 ) , adjusted for income , education , race / ethnicity , marital status , and lifestyle variables . however , no significant difference in fv consumption was observed among females who ate fafh 5 or more times per week compared to those who ate out 0 - 1 time per week . among males for the models with gender combined , eating fafh 24 times per week and 5 or more times per week was associated with significantly less fv consumption ( = 0.32 , p value = 0.007 and = 0.46 , p value = 0.003 , resp . ) , compared to eating fafh 0 - 1 time per week . in the current study , higher frequency of fafh was associated with higher bmi among both females and males . a 2012 review reported that 7 out of 8 cohort studies found a positive association between the frequency of eating away from home and body weight . among cross - sectional studies , the same review reported that , of 27 cross - sectional studies , 9 showed no association or a negative association among women and 11 showed no association or a negative association among men . in those studies that found no relationship between fafh frequency and body weight , the authors of the review suggested that lack of a common definition of the out - of - home eating concept , food intake assessment method , small sample size , and , in a spanish study , frequency of consumption of food from healthy restaurants versus fast food establishments may have contributed to the lack of association . in general , a number of studies have found fafh to be energy dense , and with higher amounts of total fat and sugar than home - prepared meals [ 12 , 15 , 47 , 48 ] . portion sizes at fast food chains and restaurants might be another factor that contributes to unintended consumption of excess energy . for example , young and nestle found that current sizes for french fries , hamburgers , and sodas are 2 to 5 times larger than the original sizes offered . a number of experiments have determined that people eat more when they are served larger portions [ 5056 ] . energy content and portion size in foods away from home likely contribute to excess energy intake in those who often eat out . this excess energy intake may lead to weight gain , since individuals often do not compensate for consuming larger portions by decreasing caloric intake or increasing physical activity [ 50 , 54 , 55 ] . influences on food choice , including the choices to eat out , eat less , or eat more healthful foods , are complex and include food upbringing , family role , health , ethnic identity and traditions , food environment , and resources , including knowledge and skills ; time , space , and finances ; social networks and supports ; and cultural and social skills . other models of food choice include explanations of value negotiations , weighing sensory perceptions , monetary considerations , health and nutrition beliefs and concerns , convenience , and social relationships . recent evidence indicates that a greater amount of time spent on home food preparation is associated with more frequent intake of fruits and vegetables [ 60 , 61 ] . although there were mixed results for the associations between fafh frequency and fv consumption in this study , the findings suggest a negative correlation . most studies have found eating out to be associated with poor diet quality , including lower consumption of fruits and vegetables [ 10 , 1419 ] . a few previous studies found that eating out was associated with higher vegetable consumption [ 8 , 10 , 62 ] . in one of these , the authors suggested the categorization of french fries as a vegetable may have contributed to this finding . two studies found that eating at full service restaurants was associated with higher vegetable consumption , but eating at burger - and - fries or non - fast food restaurants was not associated or negatively associated with vegetable consumption [ 10 , 62 ] . a few studies have found frequency of fruit consumption to be higher among those who ate out more frequently , at least in some subpopulations [ 6264 ] . befort and colleagues found that , for black adolescents , fruit intake was positively associated with sit - down , menu - based restaurant use , but this was not the case for white adolescents or for fast food restaurant use . a study of korean housewives found that , in the middle income class , frequency of fruit consumption was significantly higher with more frequent eating out , but there were no associations in the lower or upper classes . another korean study reported that people who ate out more frequently consumed more fruit ; the authors noted that this could be explained by korean restaurants serving fresh fruit as dessert . in the current analysis , eating away from home included eating at full service and fast food restaurants , but , as mentioned , the relationship between frequency of eating away from home and poor diet quality may vary according to restaurant type [ 10 , 62 ] . that is , food eaten at full service restaurants may differ in key nutrients from food obtained from fast food restaurants . for example , in a study of young adults , larson and colleagues found more frequent use of full service restaurants was associated with higher vegetable and fiber consumption . one of the reasons for the negative relation between fafh frequency and fv consumption may be the lack of demand for , and thus lack of options offered of , healthy food at fast food restaurants . an australian study found that , out of nearly 1500 meal purchases at mcdonald 's stores in a variety of socioeconomic areas , only 1.5% were healthy , which was defined by a symbol denoting approval from the national heart foundation of australia . another study of fast food patrons found only 58% rated nutrition as important when buying fast food . likewise , interviews with senior menu development and marketing executives at leading casual dining and fast - food restaurant chains revealed they believe demand for healthier foods is not widespread . additional obstacles to including healthier menu items mentioned by the executives were the short shelf life of produce , increased preparation time , low sales , and high labor costs . the authors noted that profit margins are the primary determinants of restaurants ' choice to continue to serve healthier food options and that without an increase in consumer demand it is unlikely the restaurant industry will increase their offering of healthy food choices . overall , in this study 's sample , frequency of fafh was higher among males than females . this finding is consistent with previous research [ 41 , 68 , 69 ] . on average , females in the current study reported eating approximately half a serving more of fruits and vegetables per day than males . this finding is also consistent with previous research [ 7072 ] . in the current study , younger individuals ate out more often than older individuals , a finding supported by previous research [ 24 , 73 , 74 ] . the inclusion of both cheaper and more expensive restaurants in the definition of fafh in this study may have contributed to the finding that those with higher incomes ate out more frequently . although eating out is generally associated with eating more unhealthful food and more calories in general , some restaurants offer healthier options and not all consumers eat more at a meal away from home compared to an at - home meal . among the recommendations in a 2006 report requested by the fda were that food establishments should increase the availability of low - calorie menu items , provide consumers with caloric and nutrient information in a standard , easily accessible format , and develop and promote food and beverage portion size options that help consumers in their efforts to balance their energy intake and output . environmental changes may be an option . a review of worksite health promotion programs found that fruit , vegetable , and fat intake can be positively influenced by environmental strategies including labeling at the point of purchase , promotional materials , expanded availability of healthy foods , and targeted food placement . in school settings , successful environmental strategies for increasing healthy food purchasing include food pricing , availability , and promotion . restaurant managers interested in promoting healthy food choices , while continuing to make a profit , could implement some of these strategies . other strategies concentrate more on consumers making healthy decisions when they choose to eat out . usda recommendations include selecting water over sugar - sweetened beverages and opting for small or medium portions and dishes that have been steamed , grilled , or broiled versus fried or sauted . at fast food restaurants , recommendations include using available nutrition information to inform decisions and passing on super - sizing . as mentioned , one reason people buy fast food is the notion that it is cheaper than home - prepared food . articles in the popular press have attempted to refute this idea . if this were true , one reason for eating away from home could be eliminated . unfortunately , previous analyses of seattle obesity study data suggest that , based on the monetary value of individual diets assessed by food frequency questionnaire , higher - cost diets were significantly higher in nutrient density [ 46 , 81 , 82 ] . other studies support the finding that higher cost diets are more nutritionally dense [ 8386 ] . first , the self - reported nature of the data for the key outcomes of interest is prone to error and bias . next , due to the cross - sectional nature of the study , causality can not be determined . it is possible that eating foods away from home more frequently results in higher overall calorie consumption and therefore higher bmi as well as lower fruit and vegetable intake ; however , it is also possible that individuals of higher body weight and those who consumer fewer fruits and vegetables are more likely to consume away from home meals . finally , while this was a representative sample in seattle , washington , these findings may not be generalizable to all u.s . in this study of 1570 adults , a positive relationship was found between fafh and bmi in both men and women . those who ate out more frequently tended to consume fewer fruits and vegetables , although the results were not consistent among all analyses . overall , frequency of fafh was higher among males than females . on average , females reported eating approximately half a serving more of fruits and vegetables per day than males . eating out less frequently and choosing healthy , low calorie meals when away from home may help reduce overconsumption of energy - dense , nutrient - poor foods . recommendations include both food establishments and consumers taking responsibility for decreasing these trends ; food establishments could provide and promote healthier foods , while consumers could choose more nutrient - dense options .

introduction . consumption of foods prepared away from home ( fafh ) has grown steadily since the 1970s . we examined the relationship between fafh and body mass index ( bmi ) and fruit and vegetable ( fv ) consumption . methods . frequency of fafh , daily fv intake , height and weight , and sociodemographic data were collected using a telephone survey in 2008 - 2009 . participants included a representative sample of 2,001 adult men and women ( mean age 54 15 years ) residing in king county , wa , with an analytical sample of 1,570 . frequency of fafh was categorized as 0 - 1 , 24 , or 5 + times per week . bmi was calculated from self - reported height and weight . we examined the relationship between fafh with fv consumption and bmi using multivariate models . results . higher frequency of fafh was associated with higher bmi , after adjusting for age , income , education , race , smoking , marital status , and physical activity ( women : p = 0.001 ; men : p = 0.003 ) . there was a negative association between frequency of fafh and fv consumption . fafh frequency was significantly ( p < 0.001 ) higher among males than females ( 43.1% versus 54.0% eating out 0 - 1 meal per week , resp . ) . females reported eating significantly ( p < 0.001 ) more fv than males . conclusion . among adults , higher frequency of fafh was related to higher bmi and less fv consumption .

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

since the late 1970s , consumption of foods prepared outside the home has steadily grown , from one - sixth to almost one - third of an individual 's daily dietary intake [ 14 ] . higher fruit and vegetable intake is associated with better dietary quality [ 5 , 6 ] , and some studies have found an inverse relationship between frequency of fast food use and daily servings or meal density of fruit and vegetables [ 712 ] . the healthy eating index ( hei ) is a measure of diet quality , with total fruit and total vegetable intake as two of the main components of this composite measure . economic research service reports using the 199496 continuing survey of food intakes by individuals ( csfii ) and 2003 - 04 nhanes data found that one meal away from home lowered the daily hei score enough to shift the average adult 's diet quality from a classification of fair to poor [ 12 , 14 ] . more specifically , eating fast food was associated with lower intakes of vitamin a , carotene , vitamin c , calcium , and magnesium , high amounts of nondiet carbonated soft drinks , and insignificant amounts of fruit . other studies have found that those who consume fafh more frequently have higher intakes of total energy , total fat , saturated fat , cholesterol , sodium , sugar - sweetened beverages , and sugar and lower intakes of fiber and some micronutrients [ 10 , 1419 ] . cross - sectional and prospective studies have also found that frequent fafh consumption is associated with higher body mass index ( bmi ) and percent body fat as well as increased risk for overweight / obesity , cardiometabolic risk factors , and type 2 diabetes [ 7 , 10 , 15 , 16 , 1940 ] . the coronary artery risk development in young adults ( cardia ) study found that change in fast - food frequency over 15 years was directly associated with changes in body weight . similarly , a national study of australian young adults reported that , after adjusting for confounding variables including age , leisure time physical activity , tv viewing , and employment status , consuming takeaway food twice a week or more was associated with a 31% higher prevalence of moderate abdominal obesity in men and a 25% higher prevalence in women , compared to those who ate takeaway food less than twice a week . many of the studies linking fafh with overweight and obesity were conducted with children , adolescents , or young adults [ 9 , 10 , 28 , 29 , 34 , 3639 , 4244 ] . the shift that has occurred in consumption frequency of fafh has had important implications in terms of diet quality , including fruit and vegetable intake , and obesity . this cross - sectional study aimed to examine the relationship between fafh frequency and fruit and vegetable intake as well as the relationship between fafh frequency and bmi among a representative sample of adult men and women king county , washington . the seattle obesity study was a cross - sectional study of 2,001 adult male and female residents of king county , washington . a stratified random sampling scheme was used to ensure a representative sample by income and race / ethnicity . exclusion criteria were age less than 18 years , discordance between addresses and telephone numbers obtained from the commercial database , and those self - reported by the respondent . after screening for eligibility , study procedures were explained to the potential respondent and a verbal consent was obtained over the phone and formally recorded . the sos sample was demographically comparable to 2007 behavioral risk factor surveillance system ( brfss ) and king county census data . all procedures involving human subjects / patients were approved by the university of washington institutional review board . self - reported data on frequency of fafh was available from 1,976 of 2,001 phone survey respondents . respondents were asked to report number of meals eaten out each week , including breakfast and lunch . frequency of fafh was categorized as 0 - 1 , 24 , or 5 or more times per week based on the distribution of the data obtained . the telephone survey asked a series of questions to collect self - reported data on socioeconomic status ( ses ) and demographic and lifestyle variables . income data collected using 9 categories was recombined into three categories for analytical purpose : < $ 50,000 , $ 50,000$100,000 , and > $ 100,000 , herein $ 50k , $ 50$100k , and > $ 100k . the 6-category education variable was recombined into three groups : high school or less , some college , and college graduates or higher . demographic variables of interest were age , gender , race / ethnicity , and household size . physical activity was captured using the standard question from brfss ( behavioral risk factor surveillance system)during the past month , other than your regular job , did you participate in any physical activity or exercise in your leisure time apart from your work ? self - reported data on weight and height was available from 1,877 survey respondents and was used to compute bmi . bmi was calculated as weight ( in kilograms ) divided by square of height ( in meters ) . standard dietary questions from brfss were used to capture the intake of fruits and vegetables , and other foods for the present study . each respondent was asked to report the frequency of consumption of fruit juices , fruits , green salad , carrots , and other vegetables by per day / week / month or year basis . the reported frequency for each of these items was added together to compute the total fruit and vegetable intake per person per day . first , univariate descriptive statistics were conducted for each variable ( means , standard deviations , and frequencies ) . bivariate associations were then examined between frequency of fafh and each of the sociodemographic variables , bmi , and fruit and vegetable consumption , separately for males and females as well as for both genders combined . finally , general linear models ( glm ) were used to assess the relationship between bmi and fafh , and fruit and vegetable consumption and fafh . the independent variables tested in the model were fafh , gender , age , education , income , marital status , physical activity , and smoking . a higher percentage of females participated than males ( 60% versus 40% , resp . ) the sample was more likely to be white ( 84% ) , educated ( 57% college graduate ) , and with annual household income of > $ 50k ( 60% ) . about half the sample was married / member of an unmarried couple ( 57% ) , and half of the sample never smoked ( 53% ) . half of the sample consumed fafh 0 - 1 time per week , with 34% of the sample consuming 24 times per week , and 16% eating out 5 or more meals per week . average fv consumption was 4.19 servings per day , and the average bmi was 26.6 . females and males were comparable by income , education , race , marital status , and physical activity . average bmi was much higher among males ( 27.3 kg / m ) as compared to females ( 26.1 kg / m ) . overall , fafh frequency was higher among males than females ( 43% versus 54% eating out 0 - 1 meal per week , resp . ) a significantly higher proportion of males ate out 5 or more meals per week ( 22% versus 12% among females ) ( p value < 0.001 ) . on average , females reported eating approximately half a serving more of fruits and vegetables per day than males ( 4.45 versus 3.81 servings per day , resp . ; p < 0.001 ) . bivariate associations between fafh frequency and key sociodemographic variables are presented in table 2 . significant bivariate trends were observed in frequency of fafh with selected sociodemographic variables , health indicators , and fv consumption . higher frequency of fafh was associated with being younger in age and higher bmi both among females and males ( p < 0.05 ) . for example , females with fafh frequency 5 + times per week were on average 50 y old as compared to 57 y old for those never or rarely eating out ( defined as 0 - 1 time per week ) . higher frequency of fafh was associated with relatively higher bmi both among females and males . for example , mean bmi was 27.4 kg / m among females with frequency of fafh 5 + times / week as compared to 25.8 kg / m among those who never or rarely ate out ( 0 - 1 time per week ) ( p < 0.05 ) . for females , frequency of fafh was higher among college graduates ( 15% with fafh frequency 5 + times per week ) as compared to those with high school or some college ( only 7% ) . similar trends were seen among males ( 25% versus 15% , resp . ) , although the relationship was not significant . for example , among females , 12% of those with household income of $ 50$100k and 20% of those with income > $ 100k ate out five or more times per week as compared to only 7% of those with income < $ 50k . for example , for males , while there were significant differences between fruit and vegetable intake for those who ate out 0 - 1 or 24 times per week versus 5 or more times per week , the overall association was not significant ( 3.98 , 3.78 , and 3.53 , p value = 0.093 ) . no significant differences were observed in frequency of fafh by race , marital status , or smoking status . table 3 shows associations between bmi and frequency of fafh , separately among females and males as well as for both genders combined , after adjusting for covariates . among females , frequency of fafh 5 or more times per week was associated with significantly higher bmi as compared to those who eat out only once per week or never ( = 2.19 ; se = 0.60 ; p value < 0.001 ) . however , no significant difference in bmi was observed among females with frequency of fafh 24 times per week as compared to the reference category . among males , eating out 24 times per week and 5 times per week or more were associated with significantly higher bmi ( = 1.27 ; se = 0.44 ; p value = 0.004 and = 1.34 ; se = 0.52 ; p value = 0.010 , resp . ) these associations were observed even after adjusting for covariates : income , education , race / ethnicity , marital status , and lifestyle variables ( figure 1 ) . significant associations were also observed between key sociodemographic and lifestyle variables and bmi , both among females and males . for example , higher income was associated with significantly lower bmi among females ( as compared to the reference category of < $ 50k , = 1.87 , p value 0.001 among those with income $ 50$100k ; and = 1.35 , p value 0.008 among those with income > $ 100k ) . as compared to college graduates , having lower education was associated with higher bmi ( = 1.23 , p value : 0.028 ) . physical activity had a significant negative association with bmi for both females and males ( among females , = 2.61 , p value 0.001 for those who were not physically active outside work ; among males , = 1.33 , p value = 0.006 ) . for the models with gender combined , eating out 24 times per week and 5 or more times per week were associated with significantly higher bmi ( = 0.97 , p value = 0.001 and = 1.88 , p value < 0.001 , resp . ) , as compared to those who eat out only once a week or never . table 4 displays the multivariate associations between fv consumption and frequency of fafh , separately for females and males . among females , those who ate fafh 24 times per week consumed significantly fewer servings of fruit and vegetables per day than those who ate out 0 - 1 time per week ( = 0.36 ; se = 0.15 ; p value = 0.018 ) , adjusted for income , education , race / ethnicity , marital status , and lifestyle variables . however , no significant difference in fv consumption was observed among females who ate fafh 5 or more times per week compared to those who ate out 0 - 1 time per week . among males for the models with gender combined , eating fafh 24 times per week and 5 or more times per week was associated with significantly less fv consumption ( = 0.32 , p value = 0.007 and = 0.46 , p value = 0.003 , resp . ) , compared to eating fafh 0 - 1 time per week . in the current study , higher frequency of fafh was associated with higher bmi among both females and males . a 2012 review reported that 7 out of 8 cohort studies found a positive association between the frequency of eating away from home and body weight . among cross - sectional studies , the same review reported that , of 27 cross - sectional studies , 9 showed no association or a negative association among women and 11 showed no association or a negative association among men . in those studies that found no relationship between fafh frequency and body weight , the authors of the review suggested that lack of a common definition of the out - of - home eating concept , food intake assessment method , small sample size , and , in a spanish study , frequency of consumption of food from healthy restaurants versus fast food establishments may have contributed to the lack of association . in general , a number of studies have found fafh to be energy dense , and with higher amounts of total fat and sugar than home - prepared meals [ 12 , 15 , 47 , 48 ] . portion sizes at fast food chains and restaurants might be another factor that contributes to unintended consumption of excess energy . for example , young and nestle found that current sizes for french fries , hamburgers , and sodas are 2 to 5 times larger than the original sizes offered . energy content and portion size in foods away from home likely contribute to excess energy intake in those who often eat out . this excess energy intake may lead to weight gain , since individuals often do not compensate for consuming larger portions by decreasing caloric intake or increasing physical activity [ 50 , 54 , 55 ] . influences on food choice , including the choices to eat out , eat less , or eat more healthful foods , are complex and include food upbringing , family role , health , ethnic identity and traditions , food environment , and resources , including knowledge and skills ; time , space , and finances ; social networks and supports ; and cultural and social skills . other models of food choice include explanations of value negotiations , weighing sensory perceptions , monetary considerations , health and nutrition beliefs and concerns , convenience , and social relationships . recent evidence indicates that a greater amount of time spent on home food preparation is associated with more frequent intake of fruits and vegetables [ 60 , 61 ] . although there were mixed results for the associations between fafh frequency and fv consumption in this study , the findings suggest a negative correlation . most studies have found eating out to be associated with poor diet quality , including lower consumption of fruits and vegetables [ 10 , 1419 ] . a few previous studies found that eating out was associated with higher vegetable consumption [ 8 , 10 , 62 ] . two studies found that eating at full service restaurants was associated with higher vegetable consumption , but eating at burger - and - fries or non - fast food restaurants was not associated or negatively associated with vegetable consumption [ 10 , 62 ] . a few studies have found frequency of fruit consumption to be higher among those who ate out more frequently , at least in some subpopulations [ 6264 ] . befort and colleagues found that , for black adolescents , fruit intake was positively associated with sit - down , menu - based restaurant use , but this was not the case for white adolescents or for fast food restaurant use . a study of korean housewives found that , in the middle income class , frequency of fruit consumption was significantly higher with more frequent eating out , but there were no associations in the lower or upper classes . in the current analysis , eating away from home included eating at full service and fast food restaurants , but , as mentioned , the relationship between frequency of eating away from home and poor diet quality may vary according to restaurant type [ 10 , 62 ] . for example , in a study of young adults , larson and colleagues found more frequent use of full service restaurants was associated with higher vegetable and fiber consumption . one of the reasons for the negative relation between fafh frequency and fv consumption may be the lack of demand for , and thus lack of options offered of , healthy food at fast food restaurants . an australian study found that , out of nearly 1500 meal purchases at mcdonald 's stores in a variety of socioeconomic areas , only 1.5% were healthy , which was defined by a symbol denoting approval from the national heart foundation of australia . additional obstacles to including healthier menu items mentioned by the executives were the short shelf life of produce , increased preparation time , low sales , and high labor costs . overall , in this study 's sample , frequency of fafh was higher among males than females . on average , females in the current study reported eating approximately half a serving more of fruits and vegetables per day than males . in the current study , younger individuals ate out more often than older individuals , a finding supported by previous research [ 24 , 73 , 74 ] . the inclusion of both cheaper and more expensive restaurants in the definition of fafh in this study may have contributed to the finding that those with higher incomes ate out more frequently . although eating out is generally associated with eating more unhealthful food and more calories in general , some restaurants offer healthier options and not all consumers eat more at a meal away from home compared to an at - home meal . among the recommendations in a 2006 report requested by the fda were that food establishments should increase the availability of low - calorie menu items , provide consumers with caloric and nutrient information in a standard , easily accessible format , and develop and promote food and beverage portion size options that help consumers in their efforts to balance their energy intake and output . a review of worksite health promotion programs found that fruit , vegetable , and fat intake can be positively influenced by environmental strategies including labeling at the point of purchase , promotional materials , expanded availability of healthy foods , and targeted food placement . in school settings , successful environmental strategies for increasing healthy food purchasing include food pricing , availability , and promotion . usda recommendations include selecting water over sugar - sweetened beverages and opting for small or medium portions and dishes that have been steamed , grilled , or broiled versus fried or sauted . if this were true , one reason for eating away from home could be eliminated . unfortunately , previous analyses of seattle obesity study data suggest that , based on the monetary value of individual diets assessed by food frequency questionnaire , higher - cost diets were significantly higher in nutrient density [ 46 , 81 , 82 ] . first , the self - reported nature of the data for the key outcomes of interest is prone to error and bias . it is possible that eating foods away from home more frequently results in higher overall calorie consumption and therefore higher bmi as well as lower fruit and vegetable intake ; however , it is also possible that individuals of higher body weight and those who consumer fewer fruits and vegetables are more likely to consume away from home meals . finally , while this was a representative sample in seattle , washington , these findings may not be generalizable to all u.s . in this study of 1570 adults , a positive relationship was found between fafh and bmi in both men and women . overall , frequency of fafh was higher among males than females . on average , females reported eating approximately half a serving more of fruits and vegetables per day than males . eating out less frequently and choosing healthy , low calorie meals when away from home may help reduce overconsumption of energy - dense , nutrient - poor foods .

kayaking originated from the indigenous inuit tribe . the male members of the tribe utilised the skills of kayaking for hunting on rough seas ( heath & arima , 2004 ; mattos , 2009 ; petersen , 1985 ; winning , 2002 ) . in britain , john macgregor , also known as rob roy , was the man who can be credited for making the sport more popular , writing a bestselling book about his first voyage ( winning , 2002 ) . this initiation of kayaking has resulted in males dominating the sport throughout its history ( winning , 2002 ) . in a kayak , the participant is in a seated position with their legs inside the cockpit and extended in an anterior position ( michael , smith , & rooney , 2009 ) . it is recreational rather than a performance paddlesport under the guidance of the british canoe union ( bcu ) definitions ( taylor , 2009 ) . the aim of white water kayaking is to navigate a river whilst descending rapids ( bcu , 2012 ) , with the forward stroke being the primary stroke used ( wassinger et al . , 2011 ) . the active people survey indicates that the participation in kayaking remains overwhelmingly towards the male demographic , with 35,400 males participating at least once per week in the sport over the october 2012 to october 2013 period , compared to 7600 females ( sport england , 2013 ) . participation in canoeing and kayaking has risen in both the male and female demographics over the past two years , however female participation has risen significantly and more so than males ( sport england , 2013 ) . this largely male dominated history of the sport has resulted in kayaks being designed around the male specification ( levesque , 2008a , 2008b ; manchester , 2008 ) . therefore females have struggled to find suitably fitting boats ( levesque , 2008a ) ; white water boats tend to be too big to be comfortable for women and smaller people ( manchester , 2008 ) . although females have utilised the equipment available to them , it is clear from previous research that there is a large difference between male and female anthropometry in kayaking . ridge , broad , kerr , and ackland ( 2007 ) investigated the anthropometry of male and female slalom paddlers . slalom paddling is similar in its aims to white water kayaking but with a competitive element , thus allowing a clear comparison . ( 2007 ) discovered that for all but the skinfold tests and thigh girth measurement , males recorded larger measurements than their female colleagues across all anthropometric tests carried out . it was also identified that although females tend to have a longer trunk than their male counterparts when comparing their sitting height to their stature ( 53.5% to 52.4% respectively ) , the average sitting height for females remains shorter than their male colleagues ( 89.7 to 92.5 cm respectively ) . ( 2007 ) identified , within the paper , that the anthropometrics measured for the elite slalom paddlers did not differ largely from a non - athlete reference population . this contradicts the findings of ackland , ong , kerr and ridge s ( 2003 ) investigation into olympic sprint canoe and kayak paddlers in which it was identified that this group of kayakers displayed characteristics not often observed in the general population . this finding suggests that larger sample sizes ( ridge et al . , 2007 ) or other kayaking populations might also display measurements further from the non - athlete reference population . this is important to appreciate when discussing white water kayakers because there is no data available on the anthropometrics of the population in this recreational sport . therefore , it is unclear from where the measurements utilised to design kayaks were obtained and how these relate to the population as a whole , particularly how the male measurements relate to their female counterparts . this lack of knowledge has increased importance when it comes to utilising male anthropometrics to design kayaks that females must use ( levesque , 2008b ) . despite this lack of information on what measurements are used by the manufacturers of kayaks , the findings of ridge et al . ( 2007 ) suggest that , if general population anthropometrics are used , there is still a large difference between male and female sitting height , as seen in the results of their slalom paddlers who did not differ from a general population reference sample . although the manufacturers of kayaks will not provide their sources of measurements due to rival companies potentially utilising their data , it is evident from a number of sources that boats are designed around the male specification ( levesque , 2008a , 2008b ; manchester , 2008 ) . the measurements the kayak design is based upon and how this relates to the paddlers themselves is important because the internal structure of the kayak must fit the paddler s body dimensions ( ong et al . , 2005 ) . it is identified in other sports that equipment setup is imperative for both comfort and efficient performance ( burke & pruitt , 2003 ) and also that due to anatomical differences seen between males and females , that equipment is becoming more comfortable with more specific function ( jinhua & yun , 2006 ) . this separation of male and female equipment is becoming more common place in sports ( swedan , 2001 ) , but has not yet reached the white water kayaking domain . for a kayaker to fit their boat effectively a number of contact points within the boat are necessary in order to aid control . these are the lumbar back , gluteal region , hips , thighs , knees , and toes ( whiting & varette , 2004 ) . the design of the boat with respect to the paddler will affect these contact points and therefore the ability to apply a propulsive force to the boat ( ong et al . , 2005 ) . ong et al . ( 2005 ) also noted that many slalom paddlers set their boats up based on comfort rather than the mechanical advantage that the boat setup may afford them . with this in mind , boat manufacturers must make their boats as mechanically efficient as possible allowing paddlers to focus on comfort . paddler efficiency can be identified through consistent boat velocity ( michael et al . , 2009 ) and four key boat movements : boat - centre bouncing centre of the boat moving up and down ( kemecsey & lauder , 1998).boat - end bouncing either end of the boat moving up and down ( kemecsey & lauder , 1998).boat rocking simultaneous submerging and rising of the boat sides about a longitudinal axis ( lauder & kemecsey , 1999 ; loschner , smith , & galloway , 2000).boat snaking sideways boat movement about a vertical axis through the centre of the boat ( lauder & kemecsey , 1999 ) . centre of the boat moving up and down ( kemecsey & lauder , 1998 ) . boat - end bouncing either end of the boat moving up and down ( kemecsey & lauder , 1998 ) . boat rocking simultaneous submerging and rising of the boat sides about a longitudinal axis ( lauder & kemecsey , 1999 ; loschner , smith , & galloway , 2000 ) . boat snaking sideways boat movement about a vertical axis through the centre of the boat ( lauder & kemecsey , 1999 ) . reduction of these movements would indicate an improvement in efficiency ( kemecsey & lauder , 1998 ) due to the work done increasing in relation to the energy cost ( stainsby , gladden , barclay , & wilson , 1980 ; whipp & wasserman , 1969 ) as a result of the decrease in drag forces . in order to improve efficiency , the paddler requires boat control via the contact points previously mentioned ( whiting & varette , 2004 ) . within boats that are too big for females ( manchester , 2008 ) , it can be hypothesised that by raising the sitting height to better reflect male measurements ( ridge et al . , 2007 ) and consequently altering the subsequent contact points , the paddler contact with the boat and thus the paddler s boat control should be improved . if a seat height increase is too much , then the paddler s centre of gravity will be too high , putting the paddler off balance and therefore they will have less control over the boat ( levesque , 2008b ) and therefore , the efficiency measures previously stated will be impacted , thus if the seat raise is not an appropriate height for the individual , either not enough or too much then the paddler will be measured as being inefficient . therefore the aims of this study were to identify the sitting height of the female white water paddlers and to use three - dimensional kinematics and performance measures to identify differences in paddle stroke efficiency when the seat was raised for female white water paddlers . it was hypothesised that the seat raise would result in a more efficient forward paddle stroke . with institutional ethical approval , six female white water kayakers from a uk , south coast kayak club consented to participate in the investigation . the participants were required to have at least two years paddling experience on a variety of rivers and to be over 18 years of age ( 35.6 9.7 years ) . the data capture space was calibrated prior to data collection using a 31 point floating calibration frame . the calibration frame was 5 m by 1.8 m by 2.5 m giving a capture space of 22.5 m. direct linear transform reconstruction of the calibration frame showed less than 1% error of the calibrated volume for all of the resultant errors ( brown , 2009 ) . two peak high speed cameras ( peak performance technologies inc . , colorado , usa ) were placed 14.5 m apart filming at an angle greater than 100 to each other and recording at 200 hz . after capturing the calibration frame , on arrival , the participant s anthropometric measures were taken ( table i ) and specifically sitting height was measured to enable their seat raise to be made out of high density foam , and designed to fit the participant s own boat . the seat raise height was 3.5% of the participant s sitting height to the nearest 0.5 cm . the required seat raise height was determined using a combination of research and empirical evidence . research identified that the average sitting height of female slalom kayakers was 89.7 cm ( ridge et al . , 2007 ) , and the empirical evidence from the canadian freestyle champion suggests that females should raise their seat height by one to one and a half inches ( manchester , 2008 ) . therefore 3.5% of 89.7 cm is 3.14 cm which equates to 1.24 inches , a value within manchester s ( 2008 ) recommendations . the participants were randomly assigned to start with one of the two conditions ; either with seat or no seat , and then prepared for analysis.table i. anthropometric data for participants and a comparison to slalom paddlers . participantfemale white water kayakers ( n = 6)female slalom kayakers ( n = 12 ) * 123456meansrangemeansrangeage54233734372835.510.623.054.026.34.820.035.0weight ( kg)6063.565.463.5546361.64.154.065.459.04.553.368.6height ( cm)166.1162.2164.9165.4152.6154.6161.05.9152.6166.1168.00.05158.0176.0sitting height ( cm)8587.784.985.279.88284.12.879.887.789.73.384.795.1arm span ( cm)177163.5168.3165161157165.36.9157.0177.0167.64.8161.6177.1upper arm length ( cm)34.53031.529.227.43130.62.427.434.531.51.030.333.6forearm length ( cm)27.323.324.222.324.122.524.01.822.327.324.00.722.624.6thigh length ( cm)4438.543.535.437.63639.23.735.444.044.12.440.348.5lower leg length ( cm)43.736.637.437.936.836.638.22.836.643.743.81.342.146.1shoulder breadth ( cm)43.145.345.339.13745.842.63.737.045.837.41.235.939.4flexed upper arm girth ( cm)30.629.630.629282929.51.028.030.630.11.028.131.9chest girth ( cm)94.883.989.193.690.68990.23.983.994.891.03.684.196.1waist girth ( cm)71.872.274.177.36973.673.02.869.077.369.92.665.873.4hip girth ( cm)95.994.194.793.8809291.85.980.095.989.72.785.393.5thigh girth ( cm)48.551.352.550.843.55149.63.343.552.552.92.149.956.6calf girth ( cm)36.637.636.636.731.935.535.82.031.937.634.11.232.336.4 note : * data taken from adapted table in ridge et al . participant s major visible joints , left and right trunk , head and hands were marked using black fabric markers with a white centre circle . the boats were marked on the bow , stern , and behind the cockpit on the left and right side using black markers with yellow tips . the paddle was marked at the point of shaft meeting blade with contrasting coloured tape . after warming up , the participants paddled at a comfortable pace over 50 m through the data capture area , the capture area was between 35 and 40 m of the 50 m paddle stretch . a butterworth filter with a cut - off of 6 hz was applied to the data . one full stroke cycle ( left paddle entry to left paddle entry ) was analysed for each participant under each condition . the reach was measured by taking the paddle at its furthest point forward and measured to the marker on the trunk on the same side of the body . there were two left reach measures ( l reach and l reach 2 ) and one right reach measure due to the nature of the stroke cycle analysed . stroke length of the participants was measured from paddle entering the water to paddle exit from the water . consistency of boat velocity ( michael et al . , 2009 ) was measured from the marker on the bow of the boat . the four boat movements were analysed using the following methods : centre bouncing : for each time point , the vertical movement at the stern was added to the vertical movement at the bow and a scatter graph was plotted . maximum deviation from regression line was calculated.end bouncing : scatter graph for bow movement in the vertical plane against time was translated onto the graph for vertical movement of the stern . maximum difference between bow and stern was recorded.rocking : the vertical movements of the left and right cockpit markers against time were plotted on a scatter graph , and the maximum difference between the two markers was calculated.snaking : lateral movement of the bow and stern were plotted on a scatter graph against time . centre bouncing : for each time point , the vertical movement at the stern was added to the vertical movement at the bow and a scatter graph was plotted . end bouncing : scatter graph for bow movement in the vertical plane against time was translated onto the graph for vertical movement of the stern . rocking : the vertical movements of the left and right cockpit markers against time were plotted on a scatter graph , and the maximum difference between the two markers was calculated . snaking : lateral movement of the bow and stern the measures of efficiency : four boat movements , reach , stroke length , average velocity , and velocity standard deviation , were statistically analysed utilising statistics package for the social sciences ( v18.0 ) . a paired samples t - test was carried out for each measure of efficiency , comparing the mean of the seat condition to the mean of the no seat condition . with institutional ethical approval , six female white water kayakers from a uk , south coast kayak club consented to participate in the investigation . the participants were required to have at least two years paddling experience on a variety of rivers and to be over 18 years of age ( 35.6 9.7 years ) . the data capture space was calibrated prior to data collection using a 31 point floating calibration frame . the calibration frame was 5 m by 1.8 m by 2.5 m giving a capture space of 22.5 m. direct linear transform reconstruction of the calibration frame showed less than 1% error of the calibrated volume for all of the resultant errors ( brown , 2009 ) . , colorado , usa ) were placed 14.5 m apart filming at an angle greater than 100 to each other and recording at 200 hz . after capturing the calibration frame , on arrival , the participant s anthropometric measures were taken ( table i ) and specifically sitting height was measured to enable their seat raise to be made out of high density foam , and designed to fit the participant s own boat . the seat raise height was 3.5% of the participant s sitting height to the nearest 0.5 cm . the required seat raise height was determined using a combination of research and empirical evidence . research identified that the average sitting height of female slalom kayakers was 89.7 cm ( ridge et al . , 2007 ) , and the empirical evidence from the canadian freestyle champion suggests that females should raise their seat height by one to one and a half inches ( manchester , 2008 ) . therefore 3.5% of 89.7 cm is 3.14 cm which equates to 1.24 inches , a value within manchester s ( 2008 ) recommendations . the participants were randomly assigned to start with one of the two conditions ; either with seat or no seat , and then prepared for analysis.table i. anthropometric data for participants and a comparison to slalom paddlers . participantfemale white water kayakers ( n = 6)female slalom kayakers ( n = 12 ) * 123456meansrangemeansrangeage54233734372835.510.623.054.026.34.820.035.0weight ( kg)6063.565.463.5546361.64.154.065.459.04.553.368.6height ( cm)166.1162.2164.9165.4152.6154.6161.05.9152.6166.1168.00.05158.0176.0sitting height ( cm)8587.784.985.279.88284.12.879.887.789.73.384.795.1arm span ( cm)177163.5168.3165161157165.36.9157.0177.0167.64.8161.6177.1upper arm length ( cm)34.53031.529.227.43130.62.427.434.531.51.030.333.6forearm length ( cm)27.323.324.222.324.122.524.01.822.327.324.00.722.624.6thigh length ( cm)4438.543.535.437.63639.23.735.444.044.12.440.348.5lower leg length ( cm)43.736.637.437.936.836.638.22.836.643.743.81.342.146.1shoulder breadth ( cm)43.145.345.339.13745.842.63.737.045.837.41.235.939.4flexed upper arm girth ( cm)30.629.630.629282929.51.028.030.630.11.028.131.9chest girth ( cm)94.883.989.193.690.68990.23.983.994.891.03.684.196.1waist girth ( cm)71.872.274.177.36973.673.02.869.077.369.92.665.873.4hip girth ( cm)95.994.194.793.8809291.85.980.095.989.72.785.393.5thigh girth ( cm)48.551.352.550.843.55149.63.343.552.552.92.149.956.6calf girth ( cm)36.637.636.636.731.935.535.82.031.937.634.11.232.336.4 note : * data taken from adapted table in ridge et al . ( 2007 , p. 110 ) . participant s major visible joints , left and right trunk , head and hands were marked using black fabric markers with a white centre circle . the boats were marked on the bow , stern , and behind the cockpit on the left and right side using black markers with yellow tips . the paddle was marked at the point of shaft meeting blade with contrasting coloured tape . after warming up , the participants paddled at a comfortable pace over 50 m through the data capture area , the capture area was between 35 and 40 m of the 50 m paddle stretch . a butterworth filter with a cut - off of 6 hz was applied to the data . one full stroke cycle ( left paddle entry to left paddle entry ) was analysed for each participant under each condition . the reach was measured by taking the paddle at its furthest point forward and measured to the marker on the trunk on the same side of the body . there were two left reach measures ( l reach and l reach 2 ) and one right reach measure due to the nature of the stroke cycle analysed . stroke length of the participants was measured from paddle entering the water to paddle exit from the water . consistency of boat velocity ( michael et al . , 2009 ) was measured from the marker on the bow of the boat . the four boat movements were analysed using the following methods : centre bouncing : for each time point , the vertical movement at the stern was added to the vertical movement at the bow and a scatter graph was plotted . maximum deviation from regression line was calculated.end bouncing : scatter graph for bow movement in the vertical plane against time was translated onto the graph for vertical movement of the stern . maximum difference between bow and stern was recorded.rocking : the vertical movements of the left and right cockpit markers against time were plotted on a scatter graph , and the maximum difference between the two markers was calculated.snaking : lateral movement of the bow and stern were plotted on a scatter graph against time . maximum deviation from the regression line for each marker centre bouncing : for each time point , the vertical movement at the stern was added to the vertical movement at the bow and a scatter graph was plotted . end bouncing : scatter graph for bow movement in the vertical plane against time was translated onto the graph for vertical movement of the stern . rocking : the vertical movements of the left and right cockpit markers against time were plotted on a scatter graph , and the maximum difference between the two markers was calculated . snaking : lateral movement of the bow and stern the measures of efficiency : four boat movements , reach , stroke length , average velocity , and velocity standard deviation , were statistically analysed utilising statistics package for the social sciences ( v18.0 ) . a paired samples t - test was carried out for each measure of efficiency , comparing the mean of the seat condition to the mean of the no seat condition . the results from the sitting height of the female white water paddlers ( table i ) show that on average they sit at 84.1 cm , shorter than the female slalom paddlers ( 89.7 cm ) measured by ridge et al . ( 2007 ) . a positive change due to introduction of seat raise was observed for all participants in at least four of the 12 efficiency measures ( table ii ) , for three participants there was an improvement for 6 of the measures . the seat raise also displayed an improvement for each efficiency measure for at least two participants . however , there was limited consistent improvement for either the individual participants or for each measure , with some individuals showing a decrease in efficiency with the seat raise . there was no statistically significant ( p < .05 ) difference between the conditions for any of the measures ( table ii ) . however , the result with the highest value of significance ( p = 0.146 one - tailed ) was for the snaking stern pair where there was a decrease in stern snaking from the no seat condition ( m = 0.1788 , s = 0.053 ) to the seat condition ( m = 0.1574 , s = 0.044 ) . the means for each of the no seat and seat conditions ( 0.1788 and 0.1574 , respectively ) can be used to calculate an average of 11.98% reduction in movement at the stern for the seat condition . the largest difference between the two conditions these results indicate that the seat raise reduced the amount of snaking at the stern on average for the participants . however , two participants ( participants 1 and 5 ) showed a negative result , which explained the lack of a significant result in this measure . these two participants ( 1 and 5 ) that showed a decrease in efficiency in the seat raise condition were also the lightest paddlers in the sample ( table i ) . efficiency data for each participant.participant123456meanp-value ( effect size)confidence intervals ( lower / upper ) ns centre bounce ( m ) 0.0190.0180.0210.024 0.023 0.0190.021 0.347 0.003 s centre bounce ( m)0.025 0.014 * 0.017 * 0.0220.0230.020 * 0.020 ( 0.18 ) 0.005ns end bounce ( m)0.0280.0200.0650.0270.0170.0260.0300.2740.008s end bounce ( m)0.036 * 0.015 * 0.045 * 0.0240.0200.027 * 0.028(0.28)0.013 ns rock ( m ) 0.017 xxx xxx 0.0580.0300.036 0.035 0.335 0.023 s rock ( m)0.023 xxx xxx0.077 * 0.028 * 0.0250.038 ( 0.21 ) 0.017ns snake stern ( m)0.1640.1710.2140.2630.1070.1540.1790.1460.025s snake stern ( m)0.220 * 0.135 * 0.165 * 0.1960.110 * 0.118 * 0.157(0.47)0.068 ns snake bow ( m ) 0.0900.1270.113 0.160 0.0530.120 0.111 0.475 0.031 s snake bow ( m)0.111 * 0.081 * 0.1040.1750.089 * 0.109 0.111 ( 0.03 ) 0.030ns l stroke length ( m)0.7200.7920.6541.0260.7820.7130.7810.4790.074s l stroke length ( m)*0.781 * 0.872 * 0.6930.9250.7260.6800.780(0.25)0.077 ns r stroke length ( m)0.722 0.6720.8830.962 0.693 0.7270.777 0.395 0.052 s r stroke length ( m ) 0.716 * 0.708 0.771 0.927 * 0.745 * 0.733 0.767 ( 0.18 ) 0.072ns mean velocity ( ms)1.3101.3601.5501.6401.3601.4001.4370.4830.341s mean velocity ( ms)1.310 * 1.4041.510 * 1.680 * 1.4001.3101.436(0.42)0.804 ns sd velocity ( ms ) 0.0600.0700.1600.120 0.060 0.0700.090 0.283 0.267 s sd velocity ( ms)0.080 * 0.069 * 0.11 * 0.090.0700.080 * 0.083 ( 0.37 ) 0.130ns l reach ( m)1.4871.7121.6821.6021.6821.6661.6390.3690.061s l reach ( m)*1.506 * 1.7321.606 * 1.6821.6811.5661.629(0.16)0.080 ns r reach ( m ) 1.5331.7801.7851.728 1.7141.669 1.702 0.382 0.047 s r reach ( m)*1.577 1.770 1.739 1.724 * 1.775 1.655 * 1.707 ( 0.14 ) 0.037ns l reach 2 ( m)1.4281.6631.6021.5871.5721.5961.5750.3220.053s l reach 2 ( m)*1.465 * 1.6941.5781.572 * 1.5971.4671.562(0.21)0.078 note s : bold text indicates larger number for each participant in each measure comparing no seat ( ns ) and seat ( s ) conditions . left ( l ) right ( r ) . a negative result denotes a decrease in stern snaking for the no seat condition . note s : bold text indicates larger number for each participant in each measure comparing no seat ( ns ) and seat ( s ) conditions . left ( l ) right ( r ) . when looking at the impact of the seat raise on paddling efficiency , it is clear there have been improvements although these are not consistent across the measures or the participants . however for the stern snaking efficiency measure , the paired t - test returned a result with the highest value of significance ( p = 0.1455 ) for the difference between the two conditions . to offer an improvement in efficiency , the forward propulsion of the kayak and paddler must increase in relation to the energy cost ( stainsby et al . , 1980 ; whipp & wasserman , 1969 ) this can be achieved partly through a reduction in drag . the results show that stern snaking values were higher than the bow snaking values , for all participants , across both conditions . however , with the seat raised , there was an average of 11.98% reduction in the stern movement for all participants . this reduction in stern movement should lead to a reduction in drag at the stern , therefore improving efficiency ( lauder & kemecsey , 1999 ) . this result for the stern snaking suggests a positive result for introducing the seat raise , however the subsequent findings have proven to be more individualistic . the height of each participant s seat raise was based on experiential evidence , recommending that females should raise their seat height by one to one and a half inches this was further supported by the average sitting height data gathered for slalom paddlers ( ridge et al . , 2007 ) . having since determined the average sitting height of the white water paddlers as shorter than that of the female slalom paddlers , it may be that the seat raise was not sufficiently high to display the changes expected for some participants or to affect the measures used . further to this , the female slalom paddlers ( 89.7 cm sitting height ) are still shorter than their male slalom colleagues at 92.8 cm sitting height ( ridge et al . , 2007 ) and also the male general population ( 91.5 cm sitting height ) ( pheasant , 1996 ) . although the data used to design current white water kayaks was unavailable due to being proprietary information , it can be assumed that this would closer reflect either the male general population or the male slalom population rather than either the female slalom paddlers or female white water kayakers ( levesque , 2008a , 2008b ; manchester , 2008 ) . if this was the case then it can be suggested that the seat raise should have been increased further , whilst not sacrificing balance ( burkett , 2010 ) due to a raised centre of gravity , to be more representative of the male sitting height . it is also the case that the empirical evidence provided by manchester ( 2008 ) could be incorrect as no scientific research has been carried out in this area . despite the possibility that the seat raise height was insufficient , three of the participants in the study did show improvements for 6 of the 12 efficiency measures when paddling with the seat raise , showing that this seat raise height was more effective for some participants than others this is key when looking at some of the boat movements ; it is clear that for some participants there was an efficiency reduction with the introduction of a seat raise , whereas for others there is an efficiency improvement . this could be , as discussed above , due to an insufficient seat raise , or alternatively it could be due to the seat raise being too high . this would cause the centre of gravity to be too high and then the paddler would become off balance ( burkett , 2010 ) this lack of balance and control of the boat ( whiting & varette , 2004 ) would result in a lower measure of efficiency from the boat movements . ( 2007 ) also identified that female slalom paddlers had shorter upper limb lengths than their male counterparts , therefore in order to interact with water they would have to lean towards the stroke side causing a moment around the longitudinal axis of the boat if the seat raise was too high , therefore reducing the efficiency measures seen in the boat movements . if the boat movements alone are examined ( table ii ) then it can be seen that for participant 1 ( the tallest participant ) there is no improvement in efficiency seen for any of the boat movements . it can also be seen that for participant 5 ( the shortest participant ) there is only one improvement in efficiency for boat movements . this would suggest that participant 1 was possibly sitting too high , impacting the centre of gravity and therefore balance ( burkett , 2010 ) , whereas participant 5 was sitting too low , suggesting that the seat raise was not enough to elicit an improvement in the efficiency and control over the boat movements ( whiting & varette , 2004 ) . this leads to the question as to which of the anthropometric measures would determine the correct sitting height for each individual . the current methodology used a percentage of sitting height , but it is possible that other elements such as upper limb length may be a contributing factor and therefore this would warrant further investigation . a second reason for the result inconsistencies across both efficiency measures and participants ( 2005 ) suggested that many paddlers arrange their boat cockpit more for comfort than the mechanical advantage it might confer . the introduction of the seat raise was designed to afford the participants in this study with a mechanical improvement . however , this might have been at the cost of comfort as the participants did not rearrange their cockpits between the conditions . this would have had an impact on subsequent contact points in the kinetic chain because an altered gluteal region position would have altered the position of the hips , thighs , knees , and toes ( whiting & varette , 2004 ) . without adjusting the boat setup for these contact points , adding the seat raise could have resulted in full contact not being made at each contact point . this would impact the mechanical improvement provided by the seat raise and affect the ability of the participants to apply propulsive forces to the boat ( ong et al . , 2005 ) . the final reason for these potential inconsistencies could be due to the familiarisation with the seat . it has been found that technique can adapt due to the task constraints placed upon it , such as a change in equipment ( haywood & getchell , 2009 ) . however , due to the belief that it takes 10,000 h of practice to become an expert at a skill ( baker & cobley , 2008 ) , it can be assumed that technique adaptations will not fully occur in one session and therefore with a longer familiarisation period the results may have shown further differences between the two conditions , due to technique having adapted to the new task constraints . it is important to note that despite a lack of consistency amongst the results , patterns were beginning to emerge within this sample of six participants . these patterns can be seen in the fact that all participants improved on at least one third of all of the efficiency measures and each measure showed an improvement for at least two participants . this suggests that although the effects may be individualistic , if a female paddler has had a plateau in performance or is not progressing as quickly as their peers , it is worth introducing a seat raise and using the trial and error method suggested by ong et al . the improvements seen may only be small in terms of the numbers calculated in this study , but when focusing on the most used stroke in white water kayaking ( wassinger et al . , 2011 ) , and when rapids can be potentially fatal ( berry , 2002 ) those small improvements could be the difference between life and death . in conclusion , the introduction of seat raise did return some positive results for each of the participants although these were not consistent . this suggests that a seat raise could have a potential positive impact on efficiency if utilised for female kayakers , however the question of how big the seat raise should be remains . for taller participants , having too big a seat raise may unbalance them due to the centre of gravity being too high and for smaller paddlers , the seat raise may need to be larger in order to elicit better control over the boat . therefore the results seen through using a seat raise will potentially be different for all individuals , dependent on their anthropometric make up and the height of the seat raise selected , and to differing degrees of effectiveness .

abstractthe study compared female white water paddlers over two conditions : with seat raise and with no seat raise . the aim was to determine whether raising the sitting height would improve paddling efficiency . sitting height of each participant was recorded in order to calculate the seat raise height required and three - dimensional kinematic data was collected for six participants over both conditions . twelve measures of efficiency were utilised . the efficiency of all participants improved on the seat condition for 4 of the measures , with three participants showing improvement for 6 of the measures . the stern snaking measure had the highest value of significance ( p = 0.1455 ) and showed an average of 11.98% reduction in movement between no seat and seat conditions . the results indicate that improvements were seen although these were individualistic . therefore it can be concluded that it is worth experimenting with a seat raise for a female kayaker who is lacking efficiency , noting , however , that improvements might depend on anthropometrics and the seat height selected , and therefore could elicit differing results .

Introduction Methods Participants Data collection Data analysis Results Discussion

the aim of white water kayaking is to navigate a river whilst descending rapids ( bcu , 2012 ) , with the forward stroke being the primary stroke used ( wassinger et al . slalom paddling is similar in its aims to white water kayaking but with a competitive element , thus allowing a clear comparison . this is important to appreciate when discussing white water kayakers because there is no data available on the anthropometrics of the population in this recreational sport . ( 2007 ) suggest that , if general population anthropometrics are used , there is still a large difference between male and female sitting height , as seen in the results of their slalom paddlers who did not differ from a general population reference sample . it is identified in other sports that equipment setup is imperative for both comfort and efficient performance ( burke & pruitt , 2003 ) and also that due to anatomical differences seen between males and females , that equipment is becoming more comfortable with more specific function ( jinhua & yun , 2006 ) . for a kayaker to fit their boat effectively a number of contact points within the boat are necessary in order to aid control . the design of the boat with respect to the paddler will affect these contact points and therefore the ability to apply a propulsive force to the boat ( ong et al . in order to improve efficiency , the paddler requires boat control via the contact points previously mentioned ( whiting & varette , 2004 ) . within boats that are too big for females ( manchester , 2008 ) , it can be hypothesised that by raising the sitting height to better reflect male measurements ( ridge et al . if a seat height increase is too much , then the paddler s centre of gravity will be too high , putting the paddler off balance and therefore they will have less control over the boat ( levesque , 2008b ) and therefore , the efficiency measures previously stated will be impacted , thus if the seat raise is not an appropriate height for the individual , either not enough or too much then the paddler will be measured as being inefficient . therefore the aims of this study were to identify the sitting height of the female white water paddlers and to use three - dimensional kinematics and performance measures to identify differences in paddle stroke efficiency when the seat was raised for female white water paddlers . after capturing the calibration frame , on arrival , the participant s anthropometric measures were taken ( table i ) and specifically sitting height was measured to enable their seat raise to be made out of high density foam , and designed to fit the participant s own boat . the seat raise height was 3.5% of the participant s sitting height to the nearest 0.5 cm . , 2007 ) , and the empirical evidence from the canadian freestyle champion suggests that females should raise their seat height by one to one and a half inches ( manchester , 2008 ) . the participants were randomly assigned to start with one of the two conditions ; either with seat or no seat , and then prepared for analysis.table i. anthropometric data for participants and a comparison to slalom paddlers . the boats were marked on the bow , stern , and behind the cockpit on the left and right side using black markers with yellow tips . , 2009 ) was measured from the marker on the bow of the boat . maximum deviation from regression line was calculated.end bouncing : scatter graph for bow movement in the vertical plane against time was translated onto the graph for vertical movement of the stern . maximum difference between bow and stern was recorded.rocking : the vertical movements of the left and right cockpit markers against time were plotted on a scatter graph , and the maximum difference between the two markers was calculated.snaking : lateral movement of the bow and stern were plotted on a scatter graph against time . rocking : the vertical movements of the left and right cockpit markers against time were plotted on a scatter graph , and the maximum difference between the two markers was calculated . snaking : lateral movement of the bow and stern the measures of efficiency : four boat movements , reach , stroke length , average velocity , and velocity standard deviation , were statistically analysed utilising statistics package for the social sciences ( v18.0 ) . a paired samples t - test was carried out for each measure of efficiency , comparing the mean of the seat condition to the mean of the no seat condition . with institutional ethical approval , six female white water kayakers from a uk , south coast kayak club consented to participate in the investigation . after capturing the calibration frame , on arrival , the participant s anthropometric measures were taken ( table i ) and specifically sitting height was measured to enable their seat raise to be made out of high density foam , and designed to fit the participant s own boat . the seat raise height was 3.5% of the participant s sitting height to the nearest 0.5 cm . research identified that the average sitting height of female slalom kayakers was 89.7 cm ( ridge et al . , 2007 ) , and the empirical evidence from the canadian freestyle champion suggests that females should raise their seat height by one to one and a half inches ( manchester , 2008 ) . the participants were randomly assigned to start with one of the two conditions ; either with seat or no seat , and then prepared for analysis.table i. anthropometric data for participants and a comparison to slalom paddlers . there were two left reach measures ( l reach and l reach 2 ) and one right reach measure due to the nature of the stroke cycle analysed . maximum deviation from regression line was calculated.end bouncing : scatter graph for bow movement in the vertical plane against time was translated onto the graph for vertical movement of the stern . maximum difference between bow and stern was recorded.rocking : the vertical movements of the left and right cockpit markers against time were plotted on a scatter graph , and the maximum difference between the two markers was calculated.snaking : lateral movement of the bow and stern were plotted on a scatter graph against time . rocking : the vertical movements of the left and right cockpit markers against time were plotted on a scatter graph , and the maximum difference between the two markers was calculated . snaking : lateral movement of the bow and stern the measures of efficiency : four boat movements , reach , stroke length , average velocity , and velocity standard deviation , were statistically analysed utilising statistics package for the social sciences ( v18.0 ) . a paired samples t - test was carried out for each measure of efficiency , comparing the mean of the seat condition to the mean of the no seat condition . the results from the sitting height of the female white water paddlers ( table i ) show that on average they sit at 84.1 cm , shorter than the female slalom paddlers ( 89.7 cm ) measured by ridge et al . a positive change due to introduction of seat raise was observed for all participants in at least four of the 12 efficiency measures ( table ii ) , for three participants there was an improvement for 6 of the measures . the seat raise also displayed an improvement for each efficiency measure for at least two participants . however , there was limited consistent improvement for either the individual participants or for each measure , with some individuals showing a decrease in efficiency with the seat raise . there was no statistically significant ( p < .05 ) difference between the conditions for any of the measures ( table ii ) . however , the result with the highest value of significance ( p = 0.146 one - tailed ) was for the snaking stern pair where there was a decrease in stern snaking from the no seat condition ( m = 0.1788 , s = 0.053 ) to the seat condition ( m = 0.1574 , s = 0.044 ) . the means for each of the no seat and seat conditions ( 0.1788 and 0.1574 , respectively ) can be used to calculate an average of 11.98% reduction in movement at the stern for the seat condition . the largest difference between the two conditions these results indicate that the seat raise reduced the amount of snaking at the stern on average for the participants . these two participants ( 1 and 5 ) that showed a decrease in efficiency in the seat raise condition were also the lightest paddlers in the sample ( table i ) . efficiency data for each participant.participant123456meanp-value ( effect size)confidence intervals ( lower / upper ) ns centre bounce ( m ) 0.0190.0180.0210.024 0.023 0.0190.021 0.347 0.003 s centre bounce ( m)0.025 0.014 * 0.017 * 0.0220.0230.020 * 0.020 ( 0.18 ) 0.005ns end bounce ( m)0.0280.0200.0650.0270.0170.0260.0300.2740.008s end bounce ( m)0.036 * 0.015 * 0.045 * 0.0240.0200.027 * 0.028(0.28)0.013 ns rock ( m ) 0.017 xxx xxx 0.0580.0300.036 0.035 0.335 0.023 s rock ( m)0.023 xxx xxx0.077 * 0.028 * 0.0250.038 ( 0.21 ) 0.017ns snake stern ( m)0.1640.1710.2140.2630.1070.1540.1790.1460.025s snake stern ( m)0.220 * 0.135 * 0.165 * 0.1960.110 * 0.118 * 0.157(0.47)0.068 ns snake bow ( m ) 0.0900.1270.113 0.160 0.0530.120 0.111 0.475 0.031 s snake bow ( m)0.111 * 0.081 * 0.1040.1750.089 * 0.109 0.111 ( 0.03 ) 0.030ns l stroke length ( m)0.7200.7920.6541.0260.7820.7130.7810.4790.074s l stroke length ( m)*0.781 * 0.872 * 0.6930.9250.7260.6800.780(0.25)0.077 ns r stroke length ( m)0.722 0.6720.8830.962 0.693 0.7270.777 0.395 0.052 s r stroke length ( m ) 0.716 * 0.708 0.771 0.927 * 0.745 * 0.733 0.767 ( 0.18 ) 0.072ns mean velocity ( ms)1.3101.3601.5501.6401.3601.4001.4370.4830.341s mean velocity ( ms)1.310 * 1.4041.510 * 1.680 * 1.4001.3101.436(0.42)0.804 ns sd velocity ( ms ) 0.0600.0700.1600.120 0.060 0.0700.090 0.283 0.267 s sd velocity ( ms)0.080 * 0.069 * 0.11 * 0.090.0700.080 * 0.083 ( 0.37 ) 0.130ns l reach ( m)1.4871.7121.6821.6021.6821.6661.6390.3690.061s l reach ( m)*1.506 * 1.7321.606 * 1.6821.6811.5661.629(0.16)0.080 ns r reach ( m ) 1.5331.7801.7851.728 1.7141.669 1.702 0.382 0.047 s r reach ( m)*1.577 1.770 1.739 1.724 * 1.775 1.655 * 1.707 ( 0.14 ) 0.037ns l reach 2 ( m)1.4281.6631.6021.5871.5721.5961.5750.3220.053s l reach 2 ( m)*1.465 * 1.6941.5781.572 * 1.5971.4671.562(0.21)0.078 note s : bold text indicates larger number for each participant in each measure comparing no seat ( ns ) and seat ( s ) conditions . a negative result denotes a decrease in stern snaking for the no seat condition . note s : bold text indicates larger number for each participant in each measure comparing no seat ( ns ) and seat ( s ) conditions . when looking at the impact of the seat raise on paddling efficiency , it is clear there have been improvements although these are not consistent across the measures or the participants . however for the stern snaking efficiency measure , the paired t - test returned a result with the highest value of significance ( p = 0.1455 ) for the difference between the two conditions . the results show that stern snaking values were higher than the bow snaking values , for all participants , across both conditions . however , with the seat raised , there was an average of 11.98% reduction in the stern movement for all participants . this reduction in stern movement should lead to a reduction in drag at the stern , therefore improving efficiency ( lauder & kemecsey , 1999 ) . this result for the stern snaking suggests a positive result for introducing the seat raise , however the subsequent findings have proven to be more individualistic . the height of each participant s seat raise was based on experiential evidence , recommending that females should raise their seat height by one to one and a half inches this was further supported by the average sitting height data gathered for slalom paddlers ( ridge et al . having since determined the average sitting height of the white water paddlers as shorter than that of the female slalom paddlers , it may be that the seat raise was not sufficiently high to display the changes expected for some participants or to affect the measures used . , 2007 ) and also the male general population ( 91.5 cm sitting height ) ( pheasant , 1996 ) . although the data used to design current white water kayaks was unavailable due to being proprietary information , it can be assumed that this would closer reflect either the male general population or the male slalom population rather than either the female slalom paddlers or female white water kayakers ( levesque , 2008a , 2008b ; manchester , 2008 ) . if this was the case then it can be suggested that the seat raise should have been increased further , whilst not sacrificing balance ( burkett , 2010 ) due to a raised centre of gravity , to be more representative of the male sitting height . despite the possibility that the seat raise height was insufficient , three of the participants in the study did show improvements for 6 of the 12 efficiency measures when paddling with the seat raise , showing that this seat raise height was more effective for some participants than others this is key when looking at some of the boat movements ; it is clear that for some participants there was an efficiency reduction with the introduction of a seat raise , whereas for others there is an efficiency improvement . this could be , as discussed above , due to an insufficient seat raise , or alternatively it could be due to the seat raise being too high . ( 2007 ) also identified that female slalom paddlers had shorter upper limb lengths than their male counterparts , therefore in order to interact with water they would have to lean towards the stroke side causing a moment around the longitudinal axis of the boat if the seat raise was too high , therefore reducing the efficiency measures seen in the boat movements . if the boat movements alone are examined ( table ii ) then it can be seen that for participant 1 ( the tallest participant ) there is no improvement in efficiency seen for any of the boat movements . this would suggest that participant 1 was possibly sitting too high , impacting the centre of gravity and therefore balance ( burkett , 2010 ) , whereas participant 5 was sitting too low , suggesting that the seat raise was not enough to elicit an improvement in the efficiency and control over the boat movements ( whiting & varette , 2004 ) . this leads to the question as to which of the anthropometric measures would determine the correct sitting height for each individual . the current methodology used a percentage of sitting height , but it is possible that other elements such as upper limb length may be a contributing factor and therefore this would warrant further investigation . the introduction of the seat raise was designed to afford the participants in this study with a mechanical improvement . this would impact the mechanical improvement provided by the seat raise and affect the ability of the participants to apply propulsive forces to the boat ( ong et al . however , due to the belief that it takes 10,000 h of practice to become an expert at a skill ( baker & cobley , 2008 ) , it can be assumed that technique adaptations will not fully occur in one session and therefore with a longer familiarisation period the results may have shown further differences between the two conditions , due to technique having adapted to the new task constraints . it is important to note that despite a lack of consistency amongst the results , patterns were beginning to emerge within this sample of six participants . these patterns can be seen in the fact that all participants improved on at least one third of all of the efficiency measures and each measure showed an improvement for at least two participants . this suggests that although the effects may be individualistic , if a female paddler has had a plateau in performance or is not progressing as quickly as their peers , it is worth introducing a seat raise and using the trial and error method suggested by ong et al . the improvements seen may only be small in terms of the numbers calculated in this study , but when focusing on the most used stroke in white water kayaking ( wassinger et al . , 2011 ) , and when rapids can be potentially fatal ( berry , 2002 ) those small improvements could be the difference between life and death . in conclusion , the introduction of seat raise did return some positive results for each of the participants although these were not consistent . this suggests that a seat raise could have a potential positive impact on efficiency if utilised for female kayakers , however the question of how big the seat raise should be remains . for taller participants , having too big a seat raise may unbalance them due to the centre of gravity being too high and for smaller paddlers , the seat raise may need to be larger in order to elicit better control over the boat . therefore the results seen through using a seat raise will potentially be different for all individuals , dependent on their anthropometric make up and the height of the seat raise selected , and to differing degrees of effectiveness .

between may 2006 and august 2007 , 368 patients were screened and 161 were enrolled . supplementary figure s1 online shows a summary of study enrollment and the patients treated at each dose level . the main reasons for screening failure were total sleep time ( tst ) > 6 h and/or latency to persistent sleep ( lps ) < 20 min . overall , eight patients were excluded from the per - protocol analysis : two of these did not complete the second treatment night because of adverse events ( aes ) and six were excluded because of technical difficulties during polysomnography . the per - protocol analysis set therefore consisted of 153 patients . the mean age of all the patients treated was 45.2 years ( 60% in the age group 4160 years ) ; 66.5% were women ; 98.8% were caucasian , 0.6% were hispanic , and 0.6% were asian . the mean values for objective sleep variables on the screening / adaptation night and the almorexant and placebo treatment nights for almorexant 400 , 200 , 100 , and 50 mg are reported in table 1 . se significantly improved after a single 400-mg dose of almorexant , relative to placebo ( mean treatment effect 14.4% ; p < 0.001 ) , thereby demonstrating the efficacy of almorexant at this dose level ( figure 1a ) . a dose - related increase in se was observed with almorexant 100 , 200 , and 400 mg relative to placebo ( figure 1a ) . the lowest effective dose was 100 mg ( mean treatment effect 4.6% ; p = 0.02 ) . no significant treatment effect was observed for a dose of 50 mg ( mean treatment effect 3.3% ; p = 0.23 ) , and enrollment was stopped in accordance with the study protocol . as a result , only six patients were recruited to the 25-mg dose group , and consequently the efficacy data for this dose group are not reported ; however , the data for these patients were included in the safety analysis . the robustness of the main analysis was confirmed by similar findings for the all - treated analysis , which persisted even after adjustment for period and carryover effects ( see supplementary table s1 online ) . lps was significantly reduced after the 400-mg dose of almorexant relative to placebo ( mean treatment effect 18 min ; p = 0.02 ; figure 1b ) . wake after sleep onset ( waso ) declined significantly in response to the 400-mg dose relative to placebo ( mean treatment effect 54 min ; p < 0.001 ) , and dose - related , clinically relevant , and nominally significant improvements were observed after almorexant doses of 200 ( p < 0.001 ) and 100 mg ( p = 0.004 ) ( figure 1c ) . exploratory polysomnography measurements showed dose - related increases in mean tst for almorexant 400 , 200 , and 100 mg as compared with placebo ( figure 1d ) . almorexant 400 mg was associated with a mean of 411.8 min sleep vs. 342.1 min with placebo . higher almorexant doses reduced latencies to s1 ( 400 mg ) , s2 , s3 , and s4 sleep stages ( 200 mg ) ( data not shown ) . latency to rapid eye movement ( rem ) sleep decreased dose - dependently ( figure 1e ) . almorexant 400 mg decreased the time spent in s1 and increased the time spent in s2 , s3 , s4 , and rem sleep stages , as compared with placebo ( table 2 ) . the percentage of tst spent in s1 decreased dose - dependently and the percentage of tst spent in rem increased dose - dependently after 400 and 200 mg as compared with placebo . the percentages of tst spent in s2 , s3 , and s4 were similar after placebo and after almorexant , irrespective of the almorexant dose . the mean values for subjective assessments on the night of the screening / adaptation and on the nights of almorexant ( 400- , 200- , 100- , and 50-mg doses ) and placebo treatments are given in table 3 . self - reported patient assessments showed dose - related increases in subjective se ( figure 2a ) and decreases in subjective sleep latency after almorexant 400 and 200 mg relative to placebo ( figure 2b ) . no treatment differences were observed for mean subjective waso at any almorexant dose relative to placebo ( figure 2c ) . subjective sleep quality improved after almorexant 400 and 200 mg relative to placebo ( figure 2e ) . awakening quality and somatic complaint subscores were similar among treatments ( data not shown ) . overall , no relevant negative residual effects of the previous night 's treatment were observed after any of the almorexant doses relative to placebo with respect to subjective alertness levels ( bond and lader visual analog scale ; figure 3a ) . with respect to mean reaction time ( figure 3b ) , there was a small increase after almorexant 400 mg only ( mean treatment effect 34.7 ms ( 95% confidence interval 4.964.5 ms ) ) . almorexant had no effect on the scores in the fine - motor test ( data not shown ) ; in the digit span test , a lower forward score was observed for almorexant 200 mg as compared with placebo ( mean treatment effect 0.41 ( 95% confidence interval 0.80 to 0.01 ) ) ( data not shown ) . overall , a larger proportion of patients reported at least one ae after almorexant administration than after placebo ( 21.9% vs. 13.8% ) . dizziness , nausea , fatigue , headache , and dry mouth were the most common aes reported after almorexant treatment ( table 4 ) . the proportion of patients with one or more ae was highest after almorexant 400 mg ( 40.0% ) but was markedly lower after doses of 200 , 100 , and 50 mg ( 12.8 , 17.9 , and 13.9% , respectively ) . there was one serious ae ( vasovagal syncope after placebo treatment ) and no deaths . one patient discontinued the study because of a febrile upper respiratory tract infection after receiving almorexant 400 mg ( unrelated to study treatment ) ; another discontinued because of the aforementioned serious ae . no treatment - related changes in laboratory parameters , vital signs , or quantitative electrocardiogram variables were observed , nor did any electrocardiogram abnormalities develop . no narcolepsy or cataplexy events were reported , and no trend or dose relationship was detected by the narcoleptic effects questionnaire . the mean values for objective sleep variables on the screening / adaptation night and the almorexant and placebo treatment nights for almorexant 400 , 200 , 100 , and 50 mg are reported in table 1 . se significantly improved after a single 400-mg dose of almorexant , relative to placebo ( mean treatment effect 14.4% ; p < 0.001 ) , thereby demonstrating the efficacy of almorexant at this dose level ( figure 1a ) . a dose - related increase in se was observed with almorexant 100 , 200 , and 400 mg relative to placebo ( figure 1a ) . the lowest effective dose was 100 mg ( mean treatment effect 4.6% ; p = 0.02 ) . no significant treatment effect was observed for a dose of 50 mg ( mean treatment effect 3.3% ; p = 0.23 ) , and enrollment was stopped in accordance with the study protocol . as a result , only six patients were recruited to the 25-mg dose group , and consequently the efficacy data for this dose group are not reported ; however , the data for these patients were included in the safety analysis . the robustness of the main analysis was confirmed by similar findings for the all - treated analysis , which persisted even after adjustment for period and carryover effects ( see supplementary table s1 online ) . lps was significantly reduced after the 400-mg dose of almorexant relative to placebo ( mean treatment effect 18 min ; p = 0.02 ; figure 1b ) . wake after sleep onset ( waso ) declined significantly in response to the 400-mg dose relative to placebo ( mean treatment effect 54 min ; p < 0.001 ) , and dose - related , clinically relevant , and nominally significant improvements were observed after almorexant doses of 200 ( p < 0.001 ) and 100 mg ( p = 0.004 ) ( figure 1c ) . exploratory polysomnography measurements showed dose - related increases in mean tst for almorexant 400 , 200 , and 100 mg as compared with placebo ( figure 1d ) . almorexant 400 mg was associated with a mean of 411.8 min sleep vs. 342.1 min with placebo . higher almorexant doses reduced latencies to s1 ( 400 mg ) , s2 , s3 , and s4 sleep stages ( 200 mg ) ( data not shown ) . latency to rapid eye movement ( rem ) sleep decreased dose - dependently ( figure 1e ) . almorexant 400 mg decreased the time spent in s1 and increased the time spent in s2 , s3 , s4 , and rem sleep stages , as compared with placebo ( table 2 ) . the percentage of tst spent in s1 decreased dose - dependently and the percentage of tst spent in rem increased dose - dependently after 400 and 200 mg as compared with placebo . the percentages of tst spent in s2 , s3 , and s4 were similar after placebo and after almorexant , irrespective of the almorexant dose . the mean values for subjective assessments on the night of the screening / adaptation and on the nights of almorexant ( 400- , 200- , 100- , and 50-mg doses ) and placebo treatments are given in table 3 . self - reported patient assessments showed dose - related increases in subjective se ( figure 2a ) and decreases in subjective sleep latency after almorexant 400 and 200 mg relative to placebo ( figure 2b ) . no treatment differences were observed for mean subjective waso at any almorexant dose relative to placebo ( figure 2c ) . subjective sleep quality improved after almorexant 400 and 200 mg relative to placebo ( figure 2e ) . awakening quality and somatic complaint subscores were similar among treatments ( data not shown ) . overall , no relevant negative residual effects of the previous night 's treatment were observed after any of the almorexant doses relative to placebo with respect to subjective alertness levels ( bond and lader visual analog scale ; figure 3a ) . with respect to mean reaction time ( figure 3b ) , there was a small increase after almorexant 400 mg only ( mean treatment effect 34.7 ms ( 95% confidence interval 4.964.5 ms ) ) . almorexant had no effect on the scores in the fine - motor test ( data not shown ) ; in the digit span test , a lower forward score was observed for almorexant 200 mg as compared with placebo ( mean treatment effect 0.41 ( 95% confidence interval 0.80 to 0.01 ) ) ( data not shown ) . overall , a larger proportion of patients reported at least one ae after almorexant administration than after placebo ( 21.9% vs. 13.8% ) . dizziness , nausea , fatigue , headache , and dry mouth were the most common aes reported after almorexant treatment ( table 4 ) . the proportion of patients with one or more ae was highest after almorexant 400 mg ( 40.0% ) but was markedly lower after doses of 200 , 100 , and 50 mg ( 12.8 , 17.9 , and 13.9% , respectively ) . there was one serious ae ( vasovagal syncope after placebo treatment ) and no deaths . one patient discontinued the study because of a febrile upper respiratory tract infection after receiving almorexant 400 mg ( unrelated to study treatment ) ; another discontinued because of the aforementioned serious ae . no treatment - related changes in laboratory parameters , vital signs , or quantitative electrocardiogram variables were observed , nor did any electrocardiogram abnormalities develop . no narcolepsy or cataplexy events were reported , and no trend or dose relationship was detected by the narcoleptic effects questionnaire . this is the first study to suggest that the orexin system may play a role in nocturnal sleep regulation in humans and that orexin receptor antagonism results in sleep - enabling effects in patients with primary insomnia . in this two - part clinical study in patients with primary insomnia , a significant and clinically relevant increase in se was observed after almorexant 400 mg , and the lowest effective dose was 100 mg . almorexant 400 mg significantly improved sleep initiation as assessed by lps , and at doses 100 mg almorexant dose - dependently and significantly decreased waso , a measure of sleep maintenance . almorexant decreased latency to rem sleep in a dose - dependent manner and decreased latencies to s1s4 sleep stages at the higher doses , while increasing tst . the drug reduced the time spent in s1 and increased the time spent in rem sleep . at all doses 100 mg , it had similar effects on the percentages of tst spent in the s1 and rem sleep stages . the increase in time spent in rem sleep brought the percentage of tst spent in rem to approximately within the normal range , generally 2025% in normal young adults and decreasing with age to 18% in men and 19% in women ( age range 6170 years ) . these results indicate that the administration of an almorexant dose as low as 100 mg caused an increase in the tst during the night , mainly by improving sleep maintenance . this dose level administered in the daytime did not result in detectable impairment of vigilance , alertness , or visuomotor and motor coordination in healthy subjects . consistent effects were generally observed between objective and subjective assessments of sleep variables , particularly with respect to se , tst , and lps , although differences were apparent at the lower almorexant doses . the drug had no effect on subjective waso at any dose , although objective waso decreased dose - dependently and patients reported improved sleep quality . this absence of an effect on subjective waso warrants further investigation in future studies using other sleep - diary assessments . with respect to all the objective sleep variables , improvements were observed in the mean values from the night of screening / adaptation to that after placebo treatment . this may be due to an improvement in the patients ' comfort with the equipment and sleep laboratory procedures . two residual effects on next - day performance , namely , increased mean reaction time and a lower forward score on the digit span test , were detected in association with the higher almorexant doses ( 400 and 200 mg , respectively ) . given that the major goal for insomnia therapies is to improve sleep maintenance without affecting next - day performance , further investigation of any potential next - day effects after almorexant treatment at the therapeutic dose would be valuable . of the current standard pharmacologic treatments used for sleep maintenance indications , benzodiazepines are associated with next - day effects ; however , nonbenzodiazepine agents such as eszopiclone and controlled - release zolpidem cause minimal or no residual daytime effects . the safety profile of almorexant was similar to that of placebo for the lower doses , and an increase in the incidence of aes was evident only for the 400-mg dose . there was no evidence of narcolepsy or cataplexy as seen from ae reporting and the narcoleptic effects questionnaire . the decrease in latencies to rem after almorexant administration deserves further attention and should be studied in larger trials at the therapeutic doses . shortened rem latency could potentially point to narcolepsy - like changes in sleep architecture or potential direct effects of dual orexin receptor antagonists on sleep ; alternatively , it could indicate rem rebound in individuals susceptible to chronic partial rem deprivation . the strengths of this study include the trial design , which involved the initial administration of a high dose of almorexant followed by decreasing doses in subsequent stages . this design allowed the efficacy of almorexant at a high dose to be established first before determining the lowest effective dose . limitations of the study include the shortness of the treatment duration from which to assess the efficacy , safety , and tolerability of almorexant . the sleep - enabling and -maintaining effects and long - term safety and tolerability of dual orexin receptor antagonist treatment need to be assessed in large , long - term , randomized trials . this step is essential for new insomnia therapies because long - term use of current pharmacologic treatments can be associated with the development of tolerance and dependence , and with rebound insomnia on their withdrawal . the clinical development of almorexant was discontinued in january 2011 for reasons not related to any of the observations in our study . in conclusion , in this study , a single dose of the dual orexin receptor antagonist almorexant enabled initiation and maintenance of sleep in patients with primary insomnia . almorexant treatment was associated with a dose - dependent improvement in se , strongly suggesting that the endogenous orexin system plays an important role in the sleep consistent improvements were observed in sleep time , initiation , and maintenance , and in the patients ' perception of sleep . small effects on next - day performance were observed only after the higher almorexant doses . overall , dual orexin receptor antagonism may offer a potential new treatment approach for primary insomnia . this was a prospective , multicenter , multistage , double - blind , randomized , placebo - controlled , two - way crossover , single - dose study involving almorexant in patients with primary insomnia . the study was conducted at 20 centers across europe and israel ( 6 in germany ; 3 in austria ; 2 each in finland , sweden , and switzerland ; and 1 each in denmark , israel , the netherlands , spain , and the united kingdom ) . the study design incorporated two parts : a first part to determine the efficacy of almorexant at a high dose followed by a second part with dose - descending stages to establish the minimum effective dose . in the first part of the study , the patients received a single dose of 400 mg oral almorexant and a placebo on different treatment nights 1 week apart , in a crossover design . the patients were allocated in a 1:1 ratio to the two treatment sequences ( placebo / almorexant and almorexant / placebo ) on the first treatment night , using a computer - generated randomization list . the study treatments were indistinguishable with respect to packaging , appearance , and number of capsules administered . the duration of the washout period and the timing of study drug administration were based on pharmacodynamic and pharmacokinetic data from a previous ascending single - dose study of almorexant in healthy subjects . almorexant was rapidly absorbed , with a median time to maximum concentration ranging from 0.7 to 2.3 h and rapid decreases to ~20% of peak plasma concentrations over the course of 8 h after time to maximum concentration . the pharmacodynamics of the drug correlated well with the concentration levels and , in general , the onset of pharmacodynamic effects was observed within 1 h after the dose . if the primary end point treatment effect was found to be significant ( p < 0.05 ) , the study continued with the next lower dose ( 200 , 100 , 50 , or 25 mg ) in new patients sequentially until the treatment effect was no longer statistically significant . when the number of patients successfully screened reached the target sample size for a dose level , subsequent patients were assigned to the next dose level . therefore some patients entered the next dose level before the outcome for the previous dose was known . after study enrollment was completed , all the patients were scheduled to complete all periods and assessments . if the primary end point was not significant at 400 mg , 1,000 mg was to be tested against placebo in new patients . for each dose level , patients underwent a screening period ( up to 4 weeks ) , first treatment night , 1-week washout period , second treatment night , and 28-day post - treatment follow - up . patients were required to complete a daily sleep log for at least 1 week before the screening / adaptation night , 1 week before the first treatment night , and during the 1-week washout period before the second treatment night . the sleep log consisted of a day questionnaire and the self - rating sleep and awakening quality questionnaire ( ssa ) . the day questionnaire assessed the patient 's daily habits regarding meals , caffeine - containing beverages , alcoholic drinks , smoking , nonstudy medication , and naps . in addition , information on the patient 's well - being , physical and emotional stress , and expected stressful events was collected . the investigator reviewed the sleep log and assessed the patient 's well - being and compliance before each polysomnography recording . polysomnography was performed for 8 h overnight during the screening / adaptation night and each treatment night . the purpose of the screening / adaptation night was to objectively confirm the subjectively patient - reported pattern of sleep disturbances relating to tst and sleep onset latency . the screening / adaptation night also served to accustom the patient to the sleep laboratory , the polysomnography equipment , and the psychometric tests . all polysomnography results were analyzed in accordance with the rules of rechtschaffen and kales , using the validated somnolyzer 24 7 scoring tool and including a structured expert review . , a single dose of almorexant or placebo was administered 30 min before the start of the polysomnography . the patients were required to remain at the center the morning following the polysomnography until they were free of symptoms possibly related to sleep - enabling medication , as judged by the physician responsible . patients . male and female patients 1865 years of age were eligible for inclusion if they had primary insomnia ( by the criteria of the diagnostic and statistical manual of mental disorders , fourth edition text revision ) and a history ( 3 months ) of subjectively reported usual tst of 36 h , usual sleep disturbance with a subjective sleep onset latency of > 30 min , and daytime complaints associated with poor sleep . other inclusion criteria were polysomnographic confirmation of tst of < 6 h and lps 20 min during the screening / adaptation night ; a body mass index of 1830 kg / m ; no clinically relevant abnormalities as shown by a 12-lead electrocardiogram and by hematology / biochemistry test results ; willingness to refrain from central nervous system - active drugs for five half - lives of the drug ( at least 1 week ) ; and a negative urine test for barbiturates , cannabinoids , amphetamines , and cocaine . psychotropic drugs were not permitted , except hypnotics with a short half - life of 10 h taken 48 h before each treatment night ; other stable medications were allowed . key exclusion criteria included major depressive disorder , severe psychosis , or significant anxiety disorder ; a score > 2 on the symptom assessment questionnaire for diagnosis of apnea , a raw score 50 on the zung self - rating depression or anxiety scale ; and restless legs syndrome or insomnia associated with or caused by sleep apnea or periodic limb movement disorder , as assessed by polysomnography during the screening night ( defined as apnea / hypopnea index > 10/h or periodic limb movement arousal index > 10/h , respectively ) . caffeine consumption of > 7 u/ day was not permitted on a regular basis ( one unit of caffeine was defined as one cup of coffee or two cups of tea ) . women with childbearing potential were administered urine pregnancy tests at predefined time points during the study and were required to use a reliable method of contraception during the entire study duration and for at least 3 months after intake of the study drug . study end points . the primary end point was se as determined by polysomnography , where se ( % ) = ( tst in minutes / total time in bed in minutes ( fixed to 480 min ) ) 100 . secondary end points , determined by polysomnography , were lps ( the time in minutes from the start of recording to the beginning of the first 20 nonwake epochs ) and waso ( the time in minutes spent awake after sleep onset until the end of the recording , where sleep onset is the time of the first occurrence of three consecutive epochs in s1 or first occurrence of s2 ) . exploratory end points measured by polysomnography included tst ( the amount of actual sleep time in minutes in the total sleep period ) , latency to sleep stages ( the time in minutes from the start of the polysomnography recording to the first occurrence of the respective sleep stage ) including rem sleep ( the time in minutes from sleep onset to the first occurrence of rem ) , and time spent ( in minutes ) and percentage of tst for each of the sleep stages ; and subjective measures of se , sleep latency , waso , tst , and sleep quality , assessed using the ssa . next - day performance and alertness after treatment nights were assessed using the bond and lader visual analog scale , which assesses 16 subjective feelings ; fine - motor testing , reaction time testing ; and both forward and backward digit span testing . safety assessments . aes and serious aes occurring within 36 h of administration of study treatment were recorded , irrespective of whether they were considered to be related to the study treatment . any ae that continued for 24 h after the last drug intake was monitored for up to 28 days . clinical laboratory tests , vital signs , 12-lead electrocardiogram , and a subjective narcoleptic effects questionnaire were assessed the morning after study drug administration . the narcoleptic effects questionnaire was specifically designed for this study ; it evaluated symptoms of cataplexy and sleep paralysis seen in narcolepsy with a series of yes / no questions on muscle relaxation / weakness and dreams . statistical analysis . this dose - ranging study was powered to detect a placebo - corrected mean difference in se of 6.5% . se was assumed to be normally distributed , with a standard deviation of 9.8% ; no period or carryover effects were expected . the desired power for each dose level ( 1,000 , 400 , 200 , 100 , 50 , and 25 mg ) was 98% , 98% , 96% , 94% , 94% , and 94% , requiring 39 , 39 , 34 , 31 , 31 , and 31 patients , respectively . this approach was used to maximize the power for the first dose tested ( 400 mg ) and to have 80% actual power at the 50 mg dose level . by this calculation , a minimum of 78 patients ( 400 and 1,000 mg dose levels ) and a maximum of 166 patients ( 400 , 200 , 100 , 50 , and 25 mg dose levels ) were required for the study . the null hypothesis of no difference between each dose and placebo was tested using a two - sided paired t - test on the per - protocol analysis set , and rejected when p < 0.05 . if the null hypothesis was rejected , secondary end points were to be sequentially tested ( i.e. , first lps , then waso ) using a two - sided paired t - test . robustness analyses of the primary and secondary end points included the wilcoxon signed - rank test , and analysis of the all - treated set using all available data . for the primary end point , carryover and period effects were investigated using mixed modeling . if the carryover effect was significant ( at the = 0.10 level ) exploratory end points were analyzed in the same manner as the main analysis of the primary end point , but any statistical inferences had no confirmatory value . statistical analyses were performed using sas software version 8.2 ( sas institute , cary , nc ) . all materials were reviewed and approved by the appropriate independent ethics committees before the study began . the study was conducted in accordance with the declaration of helsinki , followed the international conference on harmonization guidelines for good clinical practice , and was registered at clinicaltrials.gov ( nct00640848 ) . written informed consent was obtained from each patient before any study procedure and after adequate explanation of the aims , methods , objectives , and potential hazards of the study . it was made clear to each patient that he or she was completely free to refuse to enter the study or to withdraw from it at any time for any reason . the authors had access to the data , and they vouch for the accuracy and completeness of the data . g.d . wrote the manuscript , designed research , analyzed data , and contributed new reagents / analytical tools . h . wrote the manuscript , performed research , and contributed new reagents / analytical tools .

the orexin system is a key regulator of sleep and wakefulness . in a multicenter , double - blind , randomized , placebo - controlled , two - way crossover study , 161 primary insomnia patients received either the dual orexin receptor antagonist almorexant , at 400 , 200 , 100 , or 50 mg in consecutive stages , or placebo on treatment nights at 1-week intervals . the primary end point was sleep efficiency ( se ) measured by polysomnography ; secondary end points were objective latency to persistent sleep ( lps ) , wake after sleep onset ( waso ) , safety , and tolerability . dose - dependent almorexant effects were observed on se , lps , and waso . se improved significantly after almorexant 400 mg vs. placebo ( mean treatment effect 14.4% ; p < 0.001 ) . lps ( 18 min ( p = 0.02 ) ) and waso ( 54 min ( p < 0.001 ) ) decreased significantly at 400 mg vs. placebo . adverse - event incidence was dose - related . almorexant consistently and dose - dependently improved sleep variables . the orexin system may offer a new treatment approach for primary insomnia .

Results Efficacy Safety Discussion Methods Author Contributions Supplementary Material

the main reasons for screening failure were total sleep time ( tst ) > 6 h and/or latency to persistent sleep ( lps ) < 20 min . the mean values for objective sleep variables on the screening / adaptation night and the almorexant and placebo treatment nights for almorexant 400 , 200 , 100 , and 50 mg are reported in table 1 . se significantly improved after a single 400-mg dose of almorexant , relative to placebo ( mean treatment effect 14.4% ; p < 0.001 ) , thereby demonstrating the efficacy of almorexant at this dose level ( figure 1a ) . a dose - related increase in se was observed with almorexant 100 , 200 , and 400 mg relative to placebo ( figure 1a ) . the lowest effective dose was 100 mg ( mean treatment effect 4.6% ; p = 0.02 ) . no significant treatment effect was observed for a dose of 50 mg ( mean treatment effect 3.3% ; p = 0.23 ) , and enrollment was stopped in accordance with the study protocol . as a result , only six patients were recruited to the 25-mg dose group , and consequently the efficacy data for this dose group are not reported ; however , the data for these patients were included in the safety analysis . lps was significantly reduced after the 400-mg dose of almorexant relative to placebo ( mean treatment effect 18 min ; p = 0.02 ; figure 1b ) . wake after sleep onset ( waso ) declined significantly in response to the 400-mg dose relative to placebo ( mean treatment effect 54 min ; p < 0.001 ) , and dose - related , clinically relevant , and nominally significant improvements were observed after almorexant doses of 200 ( p < 0.001 ) and 100 mg ( p = 0.004 ) ( figure 1c ) . exploratory polysomnography measurements showed dose - related increases in mean tst for almorexant 400 , 200 , and 100 mg as compared with placebo ( figure 1d ) . almorexant 400 mg was associated with a mean of 411.8 min sleep vs. 342.1 min with placebo . higher almorexant doses reduced latencies to s1 ( 400 mg ) , s2 , s3 , and s4 sleep stages ( 200 mg ) ( data not shown ) . latency to rapid eye movement ( rem ) sleep decreased dose - dependently ( figure 1e ) . almorexant 400 mg decreased the time spent in s1 and increased the time spent in s2 , s3 , s4 , and rem sleep stages , as compared with placebo ( table 2 ) . the percentage of tst spent in s1 decreased dose - dependently and the percentage of tst spent in rem increased dose - dependently after 400 and 200 mg as compared with placebo . the percentages of tst spent in s2 , s3 , and s4 were similar after placebo and after almorexant , irrespective of the almorexant dose . the mean values for subjective assessments on the night of the screening / adaptation and on the nights of almorexant ( 400- , 200- , 100- , and 50-mg doses ) and placebo treatments are given in table 3 . self - reported patient assessments showed dose - related increases in subjective se ( figure 2a ) and decreases in subjective sleep latency after almorexant 400 and 200 mg relative to placebo ( figure 2b ) . no treatment differences were observed for mean subjective waso at any almorexant dose relative to placebo ( figure 2c ) . subjective sleep quality improved after almorexant 400 and 200 mg relative to placebo ( figure 2e ) . with respect to mean reaction time ( figure 3b ) , there was a small increase after almorexant 400 mg only ( mean treatment effect 34.7 ms ( 95% confidence interval 4.964.5 ms ) ) . almorexant had no effect on the scores in the fine - motor test ( data not shown ) ; in the digit span test , a lower forward score was observed for almorexant 200 mg as compared with placebo ( mean treatment effect 0.41 ( 95% confidence interval 0.80 to 0.01 ) ) ( data not shown ) . overall , a larger proportion of patients reported at least one ae after almorexant administration than after placebo ( 21.9% vs. 13.8% ) . dizziness , nausea , fatigue , headache , and dry mouth were the most common aes reported after almorexant treatment ( table 4 ) . the proportion of patients with one or more ae was highest after almorexant 400 mg ( 40.0% ) but was markedly lower after doses of 200 , 100 , and 50 mg ( 12.8 , 17.9 , and 13.9% , respectively ) . there was one serious ae ( vasovagal syncope after placebo treatment ) and no deaths . one patient discontinued the study because of a febrile upper respiratory tract infection after receiving almorexant 400 mg ( unrelated to study treatment ) ; another discontinued because of the aforementioned serious ae . no treatment - related changes in laboratory parameters , vital signs , or quantitative electrocardiogram variables were observed , nor did any electrocardiogram abnormalities develop . the mean values for objective sleep variables on the screening / adaptation night and the almorexant and placebo treatment nights for almorexant 400 , 200 , 100 , and 50 mg are reported in table 1 . se significantly improved after a single 400-mg dose of almorexant , relative to placebo ( mean treatment effect 14.4% ; p < 0.001 ) , thereby demonstrating the efficacy of almorexant at this dose level ( figure 1a ) . a dose - related increase in se was observed with almorexant 100 , 200 , and 400 mg relative to placebo ( figure 1a ) . the lowest effective dose was 100 mg ( mean treatment effect 4.6% ; p = 0.02 ) . no significant treatment effect was observed for a dose of 50 mg ( mean treatment effect 3.3% ; p = 0.23 ) , and enrollment was stopped in accordance with the study protocol . as a result , only six patients were recruited to the 25-mg dose group , and consequently the efficacy data for this dose group are not reported ; however , the data for these patients were included in the safety analysis . lps was significantly reduced after the 400-mg dose of almorexant relative to placebo ( mean treatment effect 18 min ; p = 0.02 ; figure 1b ) . wake after sleep onset ( waso ) declined significantly in response to the 400-mg dose relative to placebo ( mean treatment effect 54 min ; p < 0.001 ) , and dose - related , clinically relevant , and nominally significant improvements were observed after almorexant doses of 200 ( p < 0.001 ) and 100 mg ( p = 0.004 ) ( figure 1c ) . exploratory polysomnography measurements showed dose - related increases in mean tst for almorexant 400 , 200 , and 100 mg as compared with placebo ( figure 1d ) . almorexant 400 mg was associated with a mean of 411.8 min sleep vs. 342.1 min with placebo . higher almorexant doses reduced latencies to s1 ( 400 mg ) , s2 , s3 , and s4 sleep stages ( 200 mg ) ( data not shown ) . latency to rapid eye movement ( rem ) sleep decreased dose - dependently ( figure 1e ) . almorexant 400 mg decreased the time spent in s1 and increased the time spent in s2 , s3 , s4 , and rem sleep stages , as compared with placebo ( table 2 ) . the percentage of tst spent in s1 decreased dose - dependently and the percentage of tst spent in rem increased dose - dependently after 400 and 200 mg as compared with placebo . the percentages of tst spent in s2 , s3 , and s4 were similar after placebo and after almorexant , irrespective of the almorexant dose . the mean values for subjective assessments on the night of the screening / adaptation and on the nights of almorexant ( 400- , 200- , 100- , and 50-mg doses ) and placebo treatments are given in table 3 . self - reported patient assessments showed dose - related increases in subjective se ( figure 2a ) and decreases in subjective sleep latency after almorexant 400 and 200 mg relative to placebo ( figure 2b ) . no treatment differences were observed for mean subjective waso at any almorexant dose relative to placebo ( figure 2c ) . subjective sleep quality improved after almorexant 400 and 200 mg relative to placebo ( figure 2e ) . with respect to mean reaction time ( figure 3b ) , there was a small increase after almorexant 400 mg only ( mean treatment effect 34.7 ms ( 95% confidence interval 4.964.5 ms ) ) . almorexant had no effect on the scores in the fine - motor test ( data not shown ) ; in the digit span test , a lower forward score was observed for almorexant 200 mg as compared with placebo ( mean treatment effect 0.41 ( 95% confidence interval 0.80 to 0.01 ) ) ( data not shown ) . overall , a larger proportion of patients reported at least one ae after almorexant administration than after placebo ( 21.9% vs. 13.8% ) . dizziness , nausea , fatigue , headache , and dry mouth were the most common aes reported after almorexant treatment ( table 4 ) . the proportion of patients with one or more ae was highest after almorexant 400 mg ( 40.0% ) but was markedly lower after doses of 200 , 100 , and 50 mg ( 12.8 , 17.9 , and 13.9% , respectively ) . there was one serious ae ( vasovagal syncope after placebo treatment ) and no deaths . one patient discontinued the study because of a febrile upper respiratory tract infection after receiving almorexant 400 mg ( unrelated to study treatment ) ; another discontinued because of the aforementioned serious ae . no treatment - related changes in laboratory parameters , vital signs , or quantitative electrocardiogram variables were observed , nor did any electrocardiogram abnormalities develop . this is the first study to suggest that the orexin system may play a role in nocturnal sleep regulation in humans and that orexin receptor antagonism results in sleep - enabling effects in patients with primary insomnia . in this two - part clinical study in patients with primary insomnia , a significant and clinically relevant increase in se was observed after almorexant 400 mg , and the lowest effective dose was 100 mg . almorexant 400 mg significantly improved sleep initiation as assessed by lps , and at doses 100 mg almorexant dose - dependently and significantly decreased waso , a measure of sleep maintenance . almorexant decreased latency to rem sleep in a dose - dependent manner and decreased latencies to s1s4 sleep stages at the higher doses , while increasing tst . consistent effects were generally observed between objective and subjective assessments of sleep variables , particularly with respect to se , tst , and lps , although differences were apparent at the lower almorexant doses . the drug had no effect on subjective waso at any dose , although objective waso decreased dose - dependently and patients reported improved sleep quality . with respect to all the objective sleep variables , improvements were observed in the mean values from the night of screening / adaptation to that after placebo treatment . the safety profile of almorexant was similar to that of placebo for the lower doses , and an increase in the incidence of aes was evident only for the 400-mg dose . shortened rem latency could potentially point to narcolepsy - like changes in sleep architecture or potential direct effects of dual orexin receptor antagonists on sleep ; alternatively , it could indicate rem rebound in individuals susceptible to chronic partial rem deprivation . limitations of the study include the shortness of the treatment duration from which to assess the efficacy , safety , and tolerability of almorexant . the sleep - enabling and -maintaining effects and long - term safety and tolerability of dual orexin receptor antagonist treatment need to be assessed in large , long - term , randomized trials . this step is essential for new insomnia therapies because long - term use of current pharmacologic treatments can be associated with the development of tolerance and dependence , and with rebound insomnia on their withdrawal . in conclusion , in this study , a single dose of the dual orexin receptor antagonist almorexant enabled initiation and maintenance of sleep in patients with primary insomnia . almorexant treatment was associated with a dose - dependent improvement in se , strongly suggesting that the endogenous orexin system plays an important role in the sleep consistent improvements were observed in sleep time , initiation , and maintenance , and in the patients ' perception of sleep . overall , dual orexin receptor antagonism may offer a potential new treatment approach for primary insomnia . this was a prospective , multicenter , multistage , double - blind , randomized , placebo - controlled , two - way crossover , single - dose study involving almorexant in patients with primary insomnia . the study design incorporated two parts : a first part to determine the efficacy of almorexant at a high dose followed by a second part with dose - descending stages to establish the minimum effective dose . in the first part of the study , the patients received a single dose of 400 mg oral almorexant and a placebo on different treatment nights 1 week apart , in a crossover design . the patients were allocated in a 1:1 ratio to the two treatment sequences ( placebo / almorexant and almorexant / placebo ) on the first treatment night , using a computer - generated randomization list . the study treatments were indistinguishable with respect to packaging , appearance , and number of capsules administered . if the primary end point treatment effect was found to be significant ( p < 0.05 ) , the study continued with the next lower dose ( 200 , 100 , 50 , or 25 mg ) in new patients sequentially until the treatment effect was no longer statistically significant . if the primary end point was not significant at 400 mg , 1,000 mg was to be tested against placebo in new patients . for each dose level , patients underwent a screening period ( up to 4 weeks ) , first treatment night , 1-week washout period , second treatment night , and 28-day post - treatment follow - up . patients were required to complete a daily sleep log for at least 1 week before the screening / adaptation night , 1 week before the first treatment night , and during the 1-week washout period before the second treatment night . the sleep log consisted of a day questionnaire and the self - rating sleep and awakening quality questionnaire ( ssa ) . the day questionnaire assessed the patient 's daily habits regarding meals , caffeine - containing beverages , alcoholic drinks , smoking , nonstudy medication , and naps . in addition , information on the patient 's well - being , physical and emotional stress , and expected stressful events was collected . the purpose of the screening / adaptation night was to objectively confirm the subjectively patient - reported pattern of sleep disturbances relating to tst and sleep onset latency . male and female patients 1865 years of age were eligible for inclusion if they had primary insomnia ( by the criteria of the diagnostic and statistical manual of mental disorders , fourth edition text revision ) and a history ( 3 months ) of subjectively reported usual tst of 36 h , usual sleep disturbance with a subjective sleep onset latency of > 30 min , and daytime complaints associated with poor sleep . key exclusion criteria included major depressive disorder , severe psychosis , or significant anxiety disorder ; a score > 2 on the symptom assessment questionnaire for diagnosis of apnea , a raw score 50 on the zung self - rating depression or anxiety scale ; and restless legs syndrome or insomnia associated with or caused by sleep apnea or periodic limb movement disorder , as assessed by polysomnography during the screening night ( defined as apnea / hypopnea index > 10/h or periodic limb movement arousal index > 10/h , respectively ) . the primary end point was se as determined by polysomnography , where se ( % ) = ( tst in minutes / total time in bed in minutes ( fixed to 480 min ) ) 100 . secondary end points , determined by polysomnography , were lps ( the time in minutes from the start of recording to the beginning of the first 20 nonwake epochs ) and waso ( the time in minutes spent awake after sleep onset until the end of the recording , where sleep onset is the time of the first occurrence of three consecutive epochs in s1 or first occurrence of s2 ) . exploratory end points measured by polysomnography included tst ( the amount of actual sleep time in minutes in the total sleep period ) , latency to sleep stages ( the time in minutes from the start of the polysomnography recording to the first occurrence of the respective sleep stage ) including rem sleep ( the time in minutes from sleep onset to the first occurrence of rem ) , and time spent ( in minutes ) and percentage of tst for each of the sleep stages ; and subjective measures of se , sleep latency , waso , tst , and sleep quality , assessed using the ssa . next - day performance and alertness after treatment nights were assessed using the bond and lader visual analog scale , which assesses 16 subjective feelings ; fine - motor testing , reaction time testing ; and both forward and backward digit span testing . this dose - ranging study was powered to detect a placebo - corrected mean difference in se of 6.5% . the desired power for each dose level ( 1,000 , 400 , 200 , 100 , 50 , and 25 mg ) was 98% , 98% , 96% , 94% , 94% , and 94% , requiring 39 , 39 , 34 , 31 , 31 , and 31 patients , respectively . this approach was used to maximize the power for the first dose tested ( 400 mg ) and to have 80% actual power at the 50 mg dose level . by this calculation , a minimum of 78 patients ( 400 and 1,000 mg dose levels ) and a maximum of 166 patients ( 400 , 200 , 100 , 50 , and 25 mg dose levels ) were required for the study . the null hypothesis of no difference between each dose and placebo was tested using a two - sided paired t - test on the per - protocol analysis set , and rejected when p < 0.05 . if the null hypothesis was rejected , secondary end points were to be sequentially tested ( i.e. , first lps , then waso ) using a two - sided paired t - test . robustness analyses of the primary and secondary end points included the wilcoxon signed - rank test , and analysis of the all - treated set using all available data . for the primary end point , carryover and period effects were investigated using mixed modeling . if the carryover effect was significant ( at the = 0.10 level ) exploratory end points were analyzed in the same manner as the main analysis of the primary end point , but any statistical inferences had no confirmatory value . the study was conducted in accordance with the declaration of helsinki , followed the international conference on harmonization guidelines for good clinical practice , and was registered at clinicaltrials.gov ( nct00640848 ) . written informed consent was obtained from each patient before any study procedure and after adequate explanation of the aims , methods , objectives , and potential hazards of the study . the authors had access to the data , and they vouch for the accuracy and completeness of the data .

over the past decades , the development of new antibiotics has plummeted while antimicrobial resistance ( amr ) has increased . only a limited number of new antimicrobials have been developed in the last decade and overtreatment with the available antibiotics has led to the emergence of amr . despite big advances in antimicrobial therapies and strategies in counteracting infections , the emergence of amr represents an emergency situation . a high percentage of hospital - acquired infections are caused by highly resistant bacteria such as methicillin - resistant staphylococcus aureus and vancomycin - resistant enterococci . further , infections caused by resistant microorganisms often fail to respond to the conventional treatment , resulting in prolonged illness and higher death risk . considering the scenario , the world health organization has necessitated an urgent and consolidated effort to avoid regressing to the preantibiotic era . therefore , discovery of novel antimicrobial molecules and their rational use are crucial to combat microbial infections . in recent years triazine based compounds are proven to possess potent anticancer , antiretroviral , antimalarial , and antitubercular activity . additionally , it has also been reported that certain s - triazine derivatives possess potent antimicrobial activity [ 1012 ] and have been explored by researchers successfully to yield potent antimicrobial agents . for example , compounds having thiazole nucleus attached with this structural motif displayed promising antifungal activity . furthermore , we have reported certain s - triazine derivative substituted with diethylamino side chain and para - methoxyphenyl ring that have demonstrated broad spectrum antibacterial activity comparable to streptomycin . it is noteworthy that several s - triazines coupled with piperazine have shown promising antimicrobial activity . in particular , 1-(2-pyridyl ) piperazine attached to the s - triazine nucleus containing 8-hydroxy quinoline in addition to 4-amino benzonitrile linkage showed potent activity against escherichia coli compared to other analogues . recently , 4-benzyl piperazine derivatives of s - triazine were also found to be active against some gram positive and gram negative bacterial strains . the importance of piperazine moiety has been substantiated by the fact that several n - alkyl and n - aryl piperazine derivatives showed potent antibacterial activity against resistant microbial strains [ 17 , 18 ] . additionally , thiosemicarbazones of piperazine bearing arylmethylidene also displayed potent antimicrobial activity compared to ciprofloxacin . thus considering the above factors and with a goal to explore and improve the antimicrobial profile of s - triazines , we carried out the synthesis of a series of new 2,4,6-trisubstituted - s - triazines . all the compounds are sequentially substituted with aryl , diethylamino , and/or morpholinyl substituent at 2nd , 4th , and 6th position , respectively , of s - triazine . in addition , the compounds are invariably substituted with piperazine , n - methylpiperazine or aryl substituted piperazine as one of its substituents of s - triazine scaffold . the precursor for the compounds is cyanuric chloride and the synthetic route involves temperature controlled nucleophilic substitution reaction . the reaction relies on differential reactivity of the substituted chlorine atoms and the easy displacement of the chloride with nucleophiles for nonsymmetrical trisubstituted functionalization . the newly synthesized compounds were screened for their possible antimicrobial activity to identify new candidates as antibacterial and antifungal agents . all the chemicals used in this experiment were of analytical grade and procured from merck ( india ) and hi - media ( india ) . cyanuric chloride was obtained from lonza ltd . , switzerland , as a gift sample . melting points were determined in open capillaries using veego vmp1 melting point apparatus , veego instruments corporation ( mumbai , india ) , and are uncorrected . the progress of the reactions was monitored by thin layer chromatography developed with n - hexane / diethyl ether ( 1 : 1 ) and performed on merck silica gel 60 f254 aluminium sheets and products were purified by recrystallization . ir spectra were recorded on a perkin elmer - spectrum rx-1 spectrophotometer , perkinelmer , inc . ( california , usa ) , on potassium bromide pellets and were recorded in cm . h nmr and c nmr spectra were performed on a jeol al300 ft - nmr ( 300 mhz ) , jeol ltd . , japan , in dmso - d6/cdcl3 using tetramethylsilane as internal standard and the chemical shifts are reported in ppm ( ) . elemental analysis ( c , h , and n ) were performed on exeter ce-440 elemental analyzer ( exeter analytical inc . scheme 1 outlines the general classic three - step temperature controlled method of synthesis of 2,4,6-trisubstituted - s - triazine derivatives ( table 1 ) as mentioned in the literature . cyanuric chloride ( 1 ) ( 0.01 mol ) was dissolved in acetone ( 25 ml ) and different substituted nucleophiles ( r1h ) ( table 1 ) ( 0.02 mol ) were added to it at 05c and stirred for 2 h. thereafter , sodium carbonate solution ( 10% ) was added to neutralize the hydrochloric acid evolved during the reaction . the reaction mixture was then poured into crushed ice and the solid separated out was filtered , washed with water , and recrystallized from ethanol . 2-substituted-4,6-dichloro - s - triazine derivatives ( 2a2o ) were added to 100 ml of acetone and different substituted nucleophiles ( r2h ) ( table 1 ) ( 0.02 mol ) were added to it and stirred at room temperature for 3 h. sodium carbonate solution ( 10% ) was added to neutralize the hydrochloric acidevolved during the reaction and the product was filtered and washed with cold water and recrystallized from ethanol . 2,4-disubstituted-6-chloro - s - triazine derivatives ( 3a3o ) were added to 20 ml of 1,4dioxane and different substituted nucleophiles ( r3h ) ( 0.02 mol ) ( table 1 ) were refluxed at 6065c for 68 h during which anhydrous sodium carbonate ( 10% ) was added in fractions ( 0.2 g ) over 2 hr . the content was then poured into crushed ice ; precipitate obtained was dried and recrystallized from ethanol to afford the target compounds ( 4a4o ) . pale brown solid , yield : 80% , mp 155157c ; ir cm ( kbr ) : 3315 ( 2 nh stretch amines ) ; 2965 ( methyl ch asymmetric stretch ) ; 2833 ( methoxy ch stretch ) ; 1575 ( c = n ring stretch ) ; 1331 , 1523 ( no stretch ) , 805 ( cn stretch s - triazine ) . h nmr ( 300 mhz , dmso - d6 ) : 1.98 ( s , 1h , piperazinyl nh ) , 2.56 ( s , 3h , ch3 ) , 3.21 ( t , 4h , piperazinyl ) , 3.53 ( t , 4h , piperazinyl ) , 3.78 ( s , 3h , och3 ) , 6.457.71 ( m , 7h , arh ) , 8.36 ( s , 2h , nh ) . c nmr ( 75 mhz , dmso - d6 ) : 181.3 ( c6 s - triazine , c n 4-methyl-2-nitrophenyl linkage ) , 153.3 , 137.5 , 134.2 , 129.8 , 117.2 ( 12c aryl carbons ) , 56.4 ( och3 ) , 51.2 , 46.3 ( 4c piperazinyl carbons ) , 23.5 ( n4 aryl 4ch3 ) . yellow solid , yield : 86% , mp 160162c ; ir cm ( kbr ) : 3321 ( 2 nh stretch ) ; 2966 ( methyl ch asymmetric stretch ) ; 1577 ( c = n ring str ) ; 1334 , 1527 ( no stretch ) ; 1117 ( morpholinyl c o c stretch ) ; 809 ( cn stretch s - triazine ) . h nmr ( 300 mhz , cdcl3 ) : 1.87 ( s , 1h , piperazinyl nh ) , 2.62 ( s , 3h , ch3 ) , 3.18 ( m , 4h , piperazinyl ) , 3.47 ( m , 4h , piperazinyl ) , 3.65 ( t , 4h , morpholinyl ) , 3.81 ( t , 4h , morpholinyl ) , 6.607.74 ( m , 3h , arh ) , 8.47 ( s , 1h , nh ) . c nmr ( 75 mhz , cdcl3 ) : 183.2 ( c4 s - triazine , c n 4-methyl-2-nitrophenyl linkage ) , 139.4 , 136.0 , 127.1 , 117.2 ( 6c aryl carbons ) , 65.2 , 51.7 ( 4c morpholino carbons ) , 46.7 , 44.9 ( 4c piperazinyl carbons ) , 23.1 ( c2 aryl 4ch3 ) . calcd for c18h24n8o3 : c , 53.99 ; h , 6.04 ; n , 27.98 ; found : c , 53.95 ; h , 6.02 ; n , 28.01 . pale brown solid , yield : 76% , mp 181183c ; ir cm ( kbr ) : 3317 ( 2 nh stretch ) ; 2962 ( methyl ch asymmetric stretch ) ; 2831 ( methoxy ch stretch ) ; 1570 ( c = n ring stretch ) ; 1333 , 1525 ( no stretch ) , 805 ( cn stretch s - triazine ) . h nmr ( 300 mhz , dmso - d6 ) : 2.25 ( s , 3h , ch3 ) , 2.49 ( s , 3h , ch3 ) , 2.98 ( t , 4h , piperazinyl ) , 3.36 ( t , 4h , piperazinyl ) , 3.75 ( t , 3h , och3 ) , 6.377.78 ( m , 7h , arh ) , 8.63 ( s , 2h , nh ) . c nmr ( 75 mhz , dmso - d6 ) : 180.6 ( c6 s - triazine , c n 4-methoxyphenyl linkage ) , 138.5 , 136.1 , 134.5 , 130.3 , 124.9 117.1 , 114.1 ( 12c aryl carbons ) , 56.7 ( n2 aryl 4 och3 ) , 52.2 , 47.7 ( 4c piperazinyl carbons ) , 42.5 ( c6 piperazinyl 4ch3 ) , 23.4 ( n4 aryl 4ch3 ) . anal . calcd for : c22h26n8o3 : c , 58.65 ; h , 5.82 ; n , 24.87 ; found : c : 57.48 ; h : 5.83 ; n : 24.85 . pale brown solid , yield : 78% , mp 171173c ; ir cm ( kbr ) : 3321 ( 2 nh stretch ) ; 1575 ( c h nmr ( 300 mhz , dmso - d6 ) : 2.25 ( s , 3h , ch3 ) , 3.13 ( t , 4h , piperazinyl ) , 3.31 ( t , 4h , piperazinyl ) , 6.357.74 ( m , 8h , arh ) , 8.54 ( s , 2h , nh ) . c nmr ( 75 mhz , dmso - d6 ) : 181.2 ( c6 s - triazine , c n 4chlorophenyl linkage ) , 139.3 , 136.0 , 135.4 , 133.7 , 130.3 , 126.1 118.5 , 116.9 ( 12c aryl carbons ) , 54.7 , 45.8 ( 4c piperazinyl carbons ) , 43.1 ( c6 piperazinyl 4ch3 ) . anal . calcd for : c20h21brcln7 : c , 50.59 ; h , 4.46 ; n , 20.65 found : c : 50.44 ; h : 4.45 ; n : 20.60 . pale brown solid , yield : 82% , mp 210212c ; ir cm ( kbr ) : 3315 ( 2 nh stretch ) ; 2965 ( methyl ch asymmetric stretch ) ; 2835 ( methoxy ch stretch ) ; 1575 ( c = n ring stretch ) ; 1334 , 1530 ( n o stretch ) ; 805 ( cn stretch s - triazine ) . h nmr ( 300 mhz , dmso - d6 ) : 2.08 ( s , 1h , nh ) , 2.20 ( s , 3h , ch3 ) , 2.55 ( s , 3h , ch3 ) , 2.793.23 ( m , 7h , piperazinyl ) , 3.73 ( s , 3h , och3 ) , 6.567.81 ( m , 7h , ar c nmr ( 75 mhz , dmso - d6 ) : 181.2 ( c6 s - triazine , c n 4methoxyphenyl linkage ) , 138.5 , 136.6 , 134.8 , 135.2 , 130.3 , 124.9 , 117.1 , 115.1 ( 12c aryl carbons ) , 59.5 , 57.6 ( piperazinyl ) , 55.9 ( n2 aryl 4 och3 ) , 50.4 , 47.1 ( piperazinyl ) , 23.4 ( n4 aryl 4ch3 ) , 16.5 ( c6 piperazinyl 2ch3 ) . calcd for : c22h26n8o3 : c , 58.65 ; h , 5.82 ; n , 24.87 ; found : c : 58.63 ; h : 5.84 ; n : 24.85 . brown solid , yield : 82% , mp 207209c ; ir cm ( kbr ) : 3318 ( 2 nh stretch ) ; 2830 ( methoxy ch stretch ) ; 1570 ( c = n ring stretch ) ; 1333 , 1528 ( no stretch ) ; 808 ( cn stretch s - triazine ) . h nmr ( 300 mhz , dmso - d6 ) : 1.98 ( s , 1h , nh ) , 2.21 ( s , 3h , ch3 ) , 2.753.31 ( m , 7h , piperazinyl ) , 3.74 ( s , 3h , och3 ) , 6.367.93 ( s , 8h , arh ) , 8.33 ( s , 2h , nh ) . c nmr ( 75 mhz , dmso - d6 ) : 180.1 ( c6 s - triazine , c n 4methoxyphenyl linkage ) , 149.2 , 138.4 , 134.6 , 129.5 , 121.8 , 117.6 ( 12c aryl carbons ) , 61.4 ( piperazinyl ) , 56.5 ( n2 aryl 4 och3 ) , 51.7 , 49.3 , 46.2 ( piperazinyl ) , 16.7 ( c6 piperazinyl 2ch3 ) . calcd for : c21h24n8o3 : c , 57.79 ; h , 5.54 ; n , 25.67 ; found : c : 57.92 ; h : 5.53 ; n : 25.59 . pale yellow solid , yield : 84% , mp 190192c ; ir cm ( kbr ) : 3324 ( 2 nh stretch ) ; 2963 ( methyl ch asymmetric stretch ) ; 2837 ( methoxy ch stretch ) ; 1577 ( c = n ring stretch ) ; 1335 , 1531 ( no stretch ) ; 809 ( cn stretch s - triazine ) . h nmr ( 300 mhz , dmso - d6 ) : 2.37 ( s , 3h , ch3 ) , 3.22 ( t , 4h , piperazinyl ) , 3.61 ( t , 4h , piperazinyl ) , 3.75 ( s , 3h , och3 ) , 6.388.01 ( s , 11h , arh ) , 8.36 ( s , 2h , nh ) . c nmr ( 75 mhz , dmso - d6 ) : 179.8 ( c6 s - triazine , c n 4methoxyphenyl linkage ) , 156.5 , 151.3 , 137.8 , 136.1 , 134.9 , 130.3 , 124.6 , 119.3 , 116.2 ( 18c aryl carbons ) , 56.3 ( n2 aryl 4 och3 ) , 50.9 , 46.4 ( 4c piperazinyl carbons ) , 23.2 ( n4 aryl 4ch3 ) . calcd for : c27h27n9o5 : c , 58.16 ; h , 4.88 ; n , 22.61 ; found : c : 58.21 ; h : 4.87 ; n : 22.67 . pale yellow solid , yield : 84% , mp 213215c ; ir cm ( kbr ) : 3319 ( 2 nh stretch ) ; 2963 ( methyl ch asymmetric stretch ) ; 1575 ( c = n ring stretch ) ; 1331 , 1528 ( no stretch ) ; 809 ( cn stretch s - triazine ) . h nmr ( 75 mhz , dmso - d6 ) : 2.37 ( s , 3h , ch3 ) , 3.24 ( t , 4h , piperazinyl ) , 3.45 ( t , 4h , piperazinyl ) , 6.658.18 ( m , 11h , arh ) , 8.31 ( s , 2h , nh ) . c nmr ( 75 mhz , dmso - d6 ) : 180.6 ( c6 s - triazine , c n 4-nitrophenyl linkage ) , 156.4 , 148.8 , 136.5 , 127.6 , 122.0 , 118.9 116.2 ( 18c aryl carbons ) , 49.6 , 46.7 ( 4c piperazinyl carbons ) , 23.5 ( n2 aryl 4ch3 ) . calcd for : c26h24n10o6 : c , 54.54 ; h , 4.23 ; n , 24.46 ; found : c : 54.73 ; h : 4.22 ; n : 24.51 . pale yellow solid , yield : 84% , mp 165167c ; ir cm ( kbr ) : 3323 ( 2 nh stretch ) ; 1573 ( c = n ring stretch ) ; 1119 ( morpholinyl c o c stretch ) ; 1336 , 1527 ( no stretch ) ; 810 ( cn stretch s - triazine ) . h nmr ( 300 mhz , dmso - d6 ) : 3.21 ( t , 4h , piperazinyl ) , 3.41 ( t , 4h , piperazinyl ) , 3.56 ( t , 4h , morpholino ) , 3.84 ( t , 4h , morpholino ) , 6.558.16 ( m , 8h , arh ) , 8.48 ( s , 1h , nh ) . c nmr ( 75 mhz , dmso - d6 ) : 182.4 ( c6 s - triazine , c n 4-chlorophenyl linkage ) , 151.3 , 146.2 , 130.8 , 126.5 , 119.1 , 115.2 ( 12c aryl carbons ) , 66.4 ( morpholino ) , 50.7 , 48.3 ( 4c piperazinyl carbons ) , 46.5 ( morpholino ) . calcd for : c23h25cln8o3 : c , 55.59 ; h , 5.07 ; n , 22.55 ; found : c : 55.61 ; h : 5.18 ; n : 22.48 . yellow solid , yield : 84% , mp 208210c ; ir cm ( kbr ) : 3321 ( 2 nh stretch ) ; 2835 ( methoxy ch stretch ) ; 2964 ( methyl ch asymmetric stretch ) ; 1577 ( c = n ring stretch ) ; 1336 , 1528 ( no stretch ) ; 809 ( cn stretch s - triazine ) . h nmr ( 300 mhz , dmso - d6 ) : 2.42 ( s , 3h , ch3 ) , 3.18 ( t , 4h , piperazinyl ) , 3.51 ( t , 4h , piperazinyl ) , 3.73 ( s , 3h , och3 ) , 6.357.87 ( m , 11h , arh ) , 8.43 ( s , 2h , nh).c nmr ( 75 mhz , dmso - d6 ) : 180.8 ( c6 s - triazine , c n 4-methoxyphenyl linkage ) , 155.4 , 138.0 , 135.6 , 134.9 , 128.7 , 117.2 , 114.7 ( 18c aryl carbons ) , 56.9 ( n2 aryl 4 och3 ) , 49.6 , 46.2 ( 4c piperazinyl carbons ) , 23.5 ( n4 aryl 4ch3 ) . calcd for : c27h27cln8o3 : c , 59.28 ; h , 4.98 ; n , 20.48 ; found : c : 59.19 ; h : 4.97 ; n : 20.61 . yellow solid , yield : 84% , mp 211213c ; ir cm ( kbr ) : 3316 ( 2 nh stretch ) ; 1570 ( c = n ring stretch ) ; 1337 , 1528 ( no stretch ) ; 1314 ( 3cn stretch ) ; 805 ( cn stretch s - triazine ) . h nmr ( 300 mhz , dmso - d6 ) : 2.23 ( s , 4h , ch2 ) , 3.18 ( t , 4h , piperazinyl ) , 3.33 ( s , 6h , ch3 ) , 3.54 ( t , 4h , piperazinyl ) , 6.498.05 ( s , 8h , arh ) , 8.52 ( s , 1h , nh ) . c nmr ( 75 mhz , dmso - d6 ) : 181.5 ( c6 s - triazine , c n 4-nitrophenyl linkage ) , 151.8 , 147.7 , 145.2 , 136.3 , 130.8 , 117.5 , 115.2 ( 12c aryl carbons ) , 51.9 , 49.4 ( 4c piperazinyl carbons ) , 44.6 ( 4c diethyl ch2 ) , 18.3 ( 6c diethyl ch3 ) . calcd for : c23h27cln8o2 : c , 57.20 ; h , 5.63 ; n , 23.20 ; found : c : 57.36 ; h : 5.61 ; n : 23.25 . pale yellow solid , yield : 85% , mp 205207c ; ir cm ( kbr ) : 3324 ( 2 nh stretch ) ; 1576 ( c = n ring stretch ) ; 1118 ( morpholinyl c o c stretch ) ; 807 ( cn stretch s - triazine ) . h nmr ( 300 mhz , dmso - d6 ) : 3.19 ( t , 4h , piperazinyl ) , 3.44 ( t , 4h , piperazinyl ) , 3.53 ( t , 4h , morpholinyl ) , 3.80 ( t , 4h , morpholinyl ) , 6.657.72 ( m , 8h , ar c nmr ( 75 mhz , dmso - d6 ) : 183.1 ( c4 s - triazine , c n 4chlorophenyl linkage ) , 148.2 , 144.5 , 130.5 , 124.3 , 118.2 , 116.1 ( 12c aryl carbons ) , 65.3 ( morpholino ) , 51.4 , 49.2 ( 4c piperazinyl carbons ) , 46.9 ( morpholino ) . calcd for : c23h25cl2n7o : c , 56.79 ; h , 5.18 ; n , 20.16 ; found : c : 56.62 ; h : 5.20 ; n : 20.23 . yellow solid , yield : 84% , mp 184186c ; ir cm ( kbr ) : 3321 ( 2 nh stretch ) ; 2961 ( methyl ch asymmetric stretch ) ; 2830 ( methoxy ch stretch ) ; 1575 ( c h nmr ( 300 mhz , dmso - d6 ) : 2.49 ( s , 3h , ch3 ) , 3.16 ( t , 4h , piperazinyl ) , 3.55 ( t , 4h , piperazinyl ) , 3.71 ( s , 3h , och3 ) , 6.397.96 ( m , 11h , arh ) , 8.41 ( s , 2h , nh ) . c nmr ( 75 mhz , dmso - d6 ) : 181.5 ( c6 s - triazine , c n 4-methoxyphenyl linkage ) , 152.4 , 137.3 , 135.1 , 133.4 , 129.2 , 116.3 , 113.9 ( 18c aryl carbons ) , 56.4 ( n2 aryl 4 och3 ) , 49.6 , 48.5 ( 4c piperazinyl carbons ) , 23.6 ( n4 aryl 4ch3 ) . calcd for : c27h27fn8o3 : c , 61.12 ; h , 5.13 ; n , 21.12 ; found : c : 61.22 ; h : 5.14 ; n : 21.19 . pale yellow solid , yield : 80% , mp 202204c ; ir cm ( kbr ) : 3323 ( 2 nh stretch ) ; 1577 ( c = n ring stretch ) ; 1312 ( 3 cn stretch ) ; 1335 , 1526 ( no stretch ) ; 805 ( cn stretch s - triazine ) . h nmr ( 300 mhz , dmso - d6 ) : 2.17 ( s , 6h , ch3 ) , 3.21 ( t , 4h , piperazinyl ) , 3.26 ( s , 4h , ch2 ) , 3.52 ( t , 4h , piperazinyl ) , 6.568.13 ( m , 8h , arh ) , 8.45 ( s , 1h , nh ) . c nmr ( 75 mhz , dmso - d6 ) : 180.3 ( c6 s - triazine , c n 4-nitrophenyl linkage ) , 150.7 , 148.6 , 144.1 , 137.4 , 129.7 , 117.7 , 114.9 ( 12c aryl carbons ) , 50.4 , 48.7 ( 4c piperazinyl carbons ) , 43.2 ( 4c diethyl ch2 ) , 18.3 ( 6c diethyl ch3 ) . calcd for : c23h27fn8o2 : c , 59.22 ; h , 5.83 ; n , 24.02 ; found : c : 59.40 ; h : 5.85 ; n : 20.07 . pale yellow solid , yield : 80% , mp 194196c ; ir cm ( kbr ) : 3317 ( 2 nh stretch ) ; 1573 ( c = n ring stretch ) ; 1121 ( morpholinyl c o c ) , 810 ( cn stretch s - triazine).h nmr ( 75 mhz , dmso - d6 ) : 3.23 ( m , 4h , piperazinyl ) , 3.46 ( m , 4h , piperazinyl ) , 3.51 ( t , 4h , morpholinyl ) , 3.83 ( t , 4h , morpholinyl ) , 6.397.44 ( m , 8h , arh ) , 8.42 ( s , 1h , nh ) . c nmr ( 300 mhz , dmso - d6 ) : 181.6 ( c4 s - triazine , c n 4-bromophenyl linkage ) , 153.2 , 143.4 , 131.5 , 119.5 , 117.6 , 115.9 ( 12c aryl carbons ) , 65.6 ( morpholino ) , 50.2 , 49.8 ( 4c piperazinyl carbons ) , 46.5 ( mopholino ) . calcd for : c23h25brfn7o : c , 53.70 ; h , 4.90 ; n , 19.06 ; found : c : 53.50 ; h : 4.88 ; n : 19.11 . the antimicrobial potency of all the synthesized compounds was assessed in vitro in accordance with the guidelines of clinical laboratory and standards institute against four representative gram positive bacteria , namely , bacillus subtilis ( atcc 6633 , ncim 2063 ) , bacillus cereus ( atcc 10876 , ncim 2156 ) , staphylococcus epidermidis ( atcc 12228 , ncim 2493 ) , and staphylococcus aureus ( ncim 2079 ) ; gram negative bacteria , namely , escherichia coli ( atcc 8739 , ncim 2065 ) , klebsiella pneumoniae ( ncim 2706 ) , proteus mirabilis ( ncim 2241 ) , and pseudomonas aeruginosa ( atcc 19429 , ncim 2036 ) ; and two fungal strains candida albicans ( atcc 2091 , ncim 3102 ) and candida tropicalis ( atcc 13803 , ncim 3556 ) . the microbial strains employed for the activity were procured from national collection of industrial microorganisms ( ncim ) , pune , india . ciprofloxacin was used as the standard antibacterial drug and fluconazole as the standard antifungal drug . 13.0 g of nutrient broth medium was suspended in 1000 ml distilled water . then it was boiled to dissolve the medium completely and filtered from 5 sintered glass filter . a set of test tubes with nutrient broth ( 5 ml ) was capped with cotton plugs and sterilized by autoclaving at 15 psig pressure ( 121c ) for 15 min . then after cooling , a loop of organism was inoculated into liquid broth and incubated at 37 1c and used within 12 h. the inoculum size for test strain was adjusted to 10 cfu / ml ( colony forming unit per milliliter ) by comparing the turbidity . petri plates containing 15 ml of mueller hinton agar media ( hi - media ) were used for all the bacterial strains and sabouraud dextrose agar ( sda ) media ( hi - media ) were used for the fungal strains . 0.1 ml of seed of various organisms was aseptically inoculated over the sterile solid agar medium . 1 filter paper discs ( 6 mm in diameter ) impregnated with the synthesized compound ( 10 g / disc ) dissolved in dimethyl sulfoxide ( dmso ) were placed on the plates . all the sensitivity plates were then incubated at 37 1c for 24 h for bacterial strain and 72 h at 25c for fungal strain . ciprofloxacin ( 10 g / disc , hi - media ) and fluconazole ( 10 g / disc , hi - media ) were used as positive control for the assessment of antibacterial and antifungal activity , respectively . a paper disc impregnated with dmso the zone of inhibition on agar plate was measured in millimeters ( mm ) and the test was performed in triplicate and the average was taken as final reading ( table 2 ) . compounds that displayed favorable zone of inhibition were considered for further assessment of minimum inhibitory concentration ( mic ) . mic of a compound is defined as the lowest concentration at which it completely inhibits visible growth ( turbidity on liquid media ) . solutions of the test compounds , ciprofloxacin and fluconazole , were prepared in dmso at a concentration of 100 g / ml . from this stock solution , serial broth dilutions of the compounds ( 3.125 , 6.25 , 12.5 , 25 , 50 , and 100 g / ml ) were prepared to determine the mic . all the determinations were done in triplicate and the average was taken as the final reading . the standard antibiotics , ciprofloxacin and fluconazole ( 100 g / ml ) , were used as positive controls and 100 g / ml of dmso was used as a negative control . at the end of the incubation period ( 37 1c for 24 h ) , the mic values were determined ( table 3 ) . synthesis of the intermediates and target compounds was accomplished according to the steps illustrated in scheme 1 . the structure of the synthesized compounds was confirmed on the basis of ir , h nmr , c nmr , and elemental analysis and the spectral data are in accordance with the structures of the compounds ( 4a4o ) . only significant bands from ir the derivatives showed characteristic band in the range of 15701577 cm for c = n stretching of triazine nucleus . further , strong absorption band in the range of 805810 cm indicates cn stretch of s - triazine moiety . a characteristic band appearing in the range of 11171121 cm corresponds to the c o c stretching of morpholine in the ir spectra of compounds 4b , 4i , and 4o . the compounds also displayed nh stretch for secondary amines that appears in the region 33153324 cm . the h nmr spectra of all the derivatives ( 4a4o ) displayed two triplets in the range 2.983.46 and 3.213.61 attributable to ch2 protons of piperazine ring and each integrating for four protons except for compounds 4e and 4f that showed multiplet in the range of 2.753.31 . a singlet peak in the range of 8.318.63 integrating for two protons corresponding to 2 nh linkage was observed for compounds 4a , 4c4h , 4j , and 4 m while a singlet in the range 8.428.52 integrating for one proton corresponding to nh attached with s - triazine was observed for rest of the compounds . all the other protons belonging toaromatic ring , diethylamino , methyl , and methoxy group were seen accordingly to the expected chemical shift . the c nmr depicted the peaks of s - triazine nucleus that appeared at the lowest field in the range of 179.8183.2 ; 164.6179.7 ; and 157.5167.6 for three nonequivalent carbons . the peaks for two equivalent carbons of piperazine moiety appeared in the range of 49.454.7 and 44.649.8 ppm for all the compounds except 4e and 4f . the newly synthesized compounds ( 4a4o ) were evaluated for their antibacterial and antifungal activity against various gram positive , gram negative bacteria , and two fungal strains using agar disc diffusion method . the zone of inhibition of all the fifteen 2,4,6-trisubstituted - s - triazine derivatives indicates varying degree of inhibition . it was observed that compound 4d and 4h displayed equipotent antibacterial efficacy against b. subtilis ( mic 3.125 gml ) and s. aureus ( mic 6.25 gml ) under similar experimental conditions with reference to the standard drug . considering the compounds 4d and 4h that bear n - methylpiperazine and n-(4-nitrophenyl ) piperazine moiety , respectively , the compounds also possess a substituted phenyl ring at n4 position of s - triazine nucleus . therefore , it may be suggested that irrespective of the bulkiness of piperazine ring , it is the electron withdrawing group at para - position of n4 phenyl ring that imparts the activity against gram positive organism . this is further substantiated by the fact that the compounds substituted with electron releasing groups at para - position of phenyl ring ( 4a , 4c , 4e , 4f , 4 g , 4j , and 4 m ) and compounds with morpholinyl ( 4b , 4i , 4l , and 4o ) and dimethylamino side chain ( 4k and 4n ) have showed either no inhibition or minimal inhibition against gram positive organism . alternatively , few of the aryl piperazine substituted s - triazine ( 4 g , 4k and 4n ) showed moderate to good potency against gram negative bacteria . in particular , the para - fluorophenylpiperazine substituted s - triazine ( 4n ) was found to be the most active and exhibited broad spectrum antibacterial activity . this compound was active against s. epidermidis , k. pneumoniae , and p. aeruginosa with an mic of 6.25 gml whereas for e. coli it showed an mic of 3.125 gml equipotent with reference to the standard drug . the compound 4 g was found to be active against e. coli and p. aeruginosa while compound 4k , wherein the para - fluorophenylpiperazine of 4n has been replaced by para - chlorophenylpiperazine , displayed specific activity against gram negative strain such as e. coli and k. pneumoniae . most of the compounds were incapable of showing effectiveness against the fungal strains under study . however , few compounds ( 4c , 4 g , and 4n ) have demonstrated antifungal potency comparable to standard fluconazole . replacement of diethylamino group ( 4n ) with n2 para - methoxyphenyl ring ( 4c and 4 g ) makes the compounds more active against c. albicans and 4n was active against c. tropicalis that showed equipotent antifungal activity ( mic 6.25 gml ) with reference to fluconazole . nevertheless , it was not possible to substantially correlate between the structures and antifungal activity as other compounds having phenyl ring substituted with electron releasing group were unable to elicit favourable activity ( 4a , 4e , 4f , 4j , and 4 m ) . among all the compounds under investigation , compound 4 g demonstrated most significant antifungal activity against c. albicans ( mic 3.125 gml , zone of inhibition 22.2 mm ) . in summary , various 2,4,6-trisubstituted - s - triazine derivatives with varying degree of substitution were synthesized and evaluated for their in vitro antimicrobial activity . the simplicity of their preparation and better yield potentially make s - triazines useful and attractive antimicrobial agents . all the compounds were invariably substituted with piperazine , n - methylpiperazine or aryl substituted piperazine as one of the substituents of s - triazine scaffold . the tested compounds , 4d , 4 g , 4h , 4k , and 4n , have shown considerable in vitro antibacterial efficacy at the range of 3.1256.25 gml with reference to the standard drug ciprofloxacin . irrespective of the bulkiness of piperazine ring , it is the electron withdrawing group at para - position of n4 phenyl ring that imparts good activity against gram positive organism . from the present study , it may be concluded that the para - fluorophenylpiperazine derivatives of s - triazine ( 4n ) were found to be the most active and exhibited broad spectrum antibacterial activity . among all the compounds under investigation , compound 4 g demonstrated most significant antifungal activity ( 3.125 gml ) against c. albicans .

a series of new 2,4,6-trisubstituted - s - triazine was synthesized , assessed for antimicrobial activity , and characterized by ftir , 1hnmr , 13cnmr , and elemental analysis . the tested compounds , 4d , 4 g , 4h , 4k , and 4n , have shown considerable in vitro antibacterial efficacy with reference to the standard drug ciprofloxacin ( mic 3.125 gml1 against b. subtilis , e. coli , and k. pneumoniae ) . it was observed that compounds 4d and 4h displayed equipotent antibacterial efficacy against b. subtilis ( mic 3.125 gml1 ) and s. aureus ( mic 6.25 gml1 ) . the studies demonstrated that the para - fluorophenylpiperazine substituted s - triazine ( 4n ) was potent and exhibited broad spectrum antibacterial activity against s. epidermidis , k. pneumoniae , and p. aeruginosa with mic of 6.25 gml1 and for e. coli , it showed an mic of 3.125 gml1 equipotent with reference to the standard drug . among all the compounds under investigation , compound 4 g also demonstrated significant antifungal activity ( 3.125 gml1 ) against c. albicans .

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

additionally , it has also been reported that certain s - triazine derivatives possess potent antimicrobial activity [ 1012 ] and have been explored by researchers successfully to yield potent antimicrobial agents . furthermore , we have reported certain s - triazine derivative substituted with diethylamino side chain and para - methoxyphenyl ring that have demonstrated broad spectrum antibacterial activity comparable to streptomycin . it is noteworthy that several s - triazines coupled with piperazine have shown promising antimicrobial activity . in particular , 1-(2-pyridyl ) piperazine attached to the s - triazine nucleus containing 8-hydroxy quinoline in addition to 4-amino benzonitrile linkage showed potent activity against escherichia coli compared to other analogues . thus considering the above factors and with a goal to explore and improve the antimicrobial profile of s - triazines , we carried out the synthesis of a series of new 2,4,6-trisubstituted - s - triazines . all the compounds are sequentially substituted with aryl , diethylamino , and/or morpholinyl substituent at 2nd , 4th , and 6th position , respectively , of s - triazine . in addition , the compounds are invariably substituted with piperazine , n - methylpiperazine or aryl substituted piperazine as one of its substituents of s - triazine scaffold . elemental analysis ( c , h , and n ) were performed on exeter ce-440 elemental analyzer ( exeter analytical inc . scheme 1 outlines the general classic three - step temperature controlled method of synthesis of 2,4,6-trisubstituted - s - triazine derivatives ( table 1 ) as mentioned in the literature . 2-substituted-4,6-dichloro - s - triazine derivatives ( 2a2o ) were added to 100 ml of acetone and different substituted nucleophiles ( r2h ) ( table 1 ) ( 0.02 mol ) were added to it and stirred at room temperature for 3 h. sodium carbonate solution ( 10% ) was added to neutralize the hydrochloric acidevolved during the reaction and the product was filtered and washed with cold water and recrystallized from ethanol . 2,4-disubstituted-6-chloro - s - triazine derivatives ( 3a3o ) were added to 20 ml of 1,4dioxane and different substituted nucleophiles ( r3h ) ( 0.02 mol ) ( table 1 ) were refluxed at 6065c for 68 h during which anhydrous sodium carbonate ( 10% ) was added in fractions ( 0.2 g ) over 2 hr . h nmr ( 300 mhz , dmso - d6 ) : 1.98 ( s , 1h , piperazinyl nh ) , 2.56 ( s , 3h , ch3 ) , 3.21 ( t , 4h , piperazinyl ) , 3.53 ( t , 4h , piperazinyl ) , 3.78 ( s , 3h , och3 ) , 6.457.71 ( m , 7h , arh ) , 8.36 ( s , 2h , nh ) . c nmr ( 75 mhz , cdcl3 ) : 183.2 ( c4 s - triazine , c n 4-methyl-2-nitrophenyl linkage ) , 139.4 , 136.0 , 127.1 , 117.2 ( 6c aryl carbons ) , 65.2 , 51.7 ( 4c morpholino carbons ) , 46.7 , 44.9 ( 4c piperazinyl carbons ) , 23.1 ( c2 aryl 4ch3 ) . pale brown solid , yield : 76% , mp 181183c ; ir cm ( kbr ) : 3317 ( 2 nh stretch ) ; 2962 ( methyl ch asymmetric stretch ) ; 2831 ( methoxy ch stretch ) ; 1570 ( c = n ring stretch ) ; 1333 , 1525 ( no stretch ) , 805 ( cn stretch s - triazine ) . c nmr ( 75 mhz , dmso - d6 ) : 180.1 ( c6 s - triazine , c n 4methoxyphenyl linkage ) , 149.2 , 138.4 , 134.6 , 129.5 , 121.8 , 117.6 ( 12c aryl carbons ) , 61.4 ( piperazinyl ) , 56.5 ( n2 aryl 4 och3 ) , 51.7 , 49.3 , 46.2 ( piperazinyl ) , 16.7 ( c6 piperazinyl 2ch3 ) . pale yellow solid , yield : 84% , mp 190192c ; ir cm ( kbr ) : 3324 ( 2 nh stretch ) ; 2963 ( methyl ch asymmetric stretch ) ; 2837 ( methoxy ch stretch ) ; 1577 ( c = n ring stretch ) ; 1335 , 1531 ( no stretch ) ; 809 ( cn stretch s - triazine ) . h nmr ( 300 mhz , dmso - d6 ) : 2.37 ( s , 3h , ch3 ) , 3.22 ( t , 4h , piperazinyl ) , 3.61 ( t , 4h , piperazinyl ) , 3.75 ( s , 3h , och3 ) , 6.388.01 ( s , 11h , arh ) , 8.36 ( s , 2h , nh ) . c nmr ( 75 mhz , dmso - d6 ) : 182.4 ( c6 s - triazine , c n 4-chlorophenyl linkage ) , 151.3 , 146.2 , 130.8 , 126.5 , 119.1 , 115.2 ( 12c aryl carbons ) , 66.4 ( morpholino ) , 50.7 , 48.3 ( 4c piperazinyl carbons ) , 46.5 ( morpholino ) . h nmr ( 300 mhz , dmso - d6 ) : 2.42 ( s , 3h , ch3 ) , 3.18 ( t , 4h , piperazinyl ) , 3.51 ( t , 4h , piperazinyl ) , 3.73 ( s , 3h , och3 ) , 6.357.87 ( m , 11h , arh ) , 8.43 ( s , 2h , nh).c nmr ( 75 mhz , dmso - d6 ) : 180.8 ( c6 s - triazine , c n 4-methoxyphenyl linkage ) , 155.4 , 138.0 , 135.6 , 134.9 , 128.7 , 117.2 , 114.7 ( 18c aryl carbons ) , 56.9 ( n2 aryl 4 och3 ) , 49.6 , 46.2 ( 4c piperazinyl carbons ) , 23.5 ( n4 aryl 4ch3 ) . yellow solid , yield : 84% , mp 211213c ; ir cm ( kbr ) : 3316 ( 2 nh stretch ) ; 1570 ( c = n ring stretch ) ; 1337 , 1528 ( no stretch ) ; 1314 ( 3cn stretch ) ; 805 ( cn stretch s - triazine ) . h nmr ( 300 mhz , dmso - d6 ) : 2.23 ( s , 4h , ch2 ) , 3.18 ( t , 4h , piperazinyl ) , 3.33 ( s , 6h , ch3 ) , 3.54 ( t , 4h , piperazinyl ) , 6.498.05 ( s , 8h , arh ) , 8.52 ( s , 1h , nh ) . c nmr ( 75 mhz , dmso - d6 ) : 181.5 ( c6 s - triazine , c n 4-nitrophenyl linkage ) , 151.8 , 147.7 , 145.2 , 136.3 , 130.8 , 117.5 , 115.2 ( 12c aryl carbons ) , 51.9 , 49.4 ( 4c piperazinyl carbons ) , 44.6 ( 4c diethyl ch2 ) , 18.3 ( 6c diethyl ch3 ) . pale yellow solid , yield : 85% , mp 205207c ; ir cm ( kbr ) : 3324 ( 2 nh stretch ) ; 1576 ( c = n ring stretch ) ; 1118 ( morpholinyl c o c stretch ) ; 807 ( cn stretch s - triazine ) . h nmr ( 300 mhz , dmso - d6 ) : 3.19 ( t , 4h , piperazinyl ) , 3.44 ( t , 4h , piperazinyl ) , 3.53 ( t , 4h , morpholinyl ) , 3.80 ( t , 4h , morpholinyl ) , 6.657.72 ( m , 8h , ar c nmr ( 75 mhz , dmso - d6 ) : 183.1 ( c4 s - triazine , c n 4chlorophenyl linkage ) , 148.2 , 144.5 , 130.5 , 124.3 , 118.2 , 116.1 ( 12c aryl carbons ) , 65.3 ( morpholino ) , 51.4 , 49.2 ( 4c piperazinyl carbons ) , 46.9 ( morpholino ) . c nmr ( 75 mhz , dmso - d6 ) : 181.5 ( c6 s - triazine , c n 4-methoxyphenyl linkage ) , 152.4 , 137.3 , 135.1 , 133.4 , 129.2 , 116.3 , 113.9 ( 18c aryl carbons ) , 56.4 ( n2 aryl 4 och3 ) , 49.6 , 48.5 ( 4c piperazinyl carbons ) , 23.6 ( n4 aryl 4ch3 ) . pale yellow solid , yield : 80% , mp 202204c ; ir cm ( kbr ) : 3323 ( 2 nh stretch ) ; 1577 ( c = n ring stretch ) ; 1312 ( 3 cn stretch ) ; 1335 , 1526 ( no stretch ) ; 805 ( cn stretch s - triazine ) . pale yellow solid , yield : 80% , mp 194196c ; ir cm ( kbr ) : 3317 ( 2 nh stretch ) ; 1573 ( c = n ring stretch ) ; 1121 ( morpholinyl c o c ) , 810 ( cn stretch s - triazine).h nmr ( 75 mhz , dmso - d6 ) : 3.23 ( m , 4h , piperazinyl ) , 3.46 ( m , 4h , piperazinyl ) , 3.51 ( t , 4h , morpholinyl ) , 3.83 ( t , 4h , morpholinyl ) , 6.397.44 ( m , 8h , arh ) , 8.42 ( s , 1h , nh ) . the antimicrobial potency of all the synthesized compounds was assessed in vitro in accordance with the guidelines of clinical laboratory and standards institute against four representative gram positive bacteria , namely , bacillus subtilis ( atcc 6633 , ncim 2063 ) , bacillus cereus ( atcc 10876 , ncim 2156 ) , staphylococcus epidermidis ( atcc 12228 , ncim 2493 ) , and staphylococcus aureus ( ncim 2079 ) ; gram negative bacteria , namely , escherichia coli ( atcc 8739 , ncim 2065 ) , klebsiella pneumoniae ( ncim 2706 ) , proteus mirabilis ( ncim 2241 ) , and pseudomonas aeruginosa ( atcc 19429 , ncim 2036 ) ; and two fungal strains candida albicans ( atcc 2091 , ncim 3102 ) and candida tropicalis ( atcc 13803 , ncim 3556 ) . ciprofloxacin ( 10 g / disc , hi - media ) and fluconazole ( 10 g / disc , hi - media ) were used as positive control for the assessment of antibacterial and antifungal activity , respectively . from this stock solution , serial broth dilutions of the compounds ( 3.125 , 6.25 , 12.5 , 25 , 50 , and 100 g / ml ) were prepared to determine the mic . the structure of the synthesized compounds was confirmed on the basis of ir , h nmr , c nmr , and elemental analysis and the spectral data are in accordance with the structures of the compounds ( 4a4o ) . a singlet peak in the range of 8.318.63 integrating for two protons corresponding to 2 nh linkage was observed for compounds 4a , 4c4h , 4j , and 4 m while a singlet in the range 8.428.52 integrating for one proton corresponding to nh attached with s - triazine was observed for rest of the compounds . all the other protons belonging toaromatic ring , diethylamino , methyl , and methoxy group were seen accordingly to the expected chemical shift . the peaks for two equivalent carbons of piperazine moiety appeared in the range of 49.454.7 and 44.649.8 ppm for all the compounds except 4e and 4f . the newly synthesized compounds ( 4a4o ) were evaluated for their antibacterial and antifungal activity against various gram positive , gram negative bacteria , and two fungal strains using agar disc diffusion method . the zone of inhibition of all the fifteen 2,4,6-trisubstituted - s - triazine derivatives indicates varying degree of inhibition . it was observed that compound 4d and 4h displayed equipotent antibacterial efficacy against b. subtilis ( mic 3.125 gml ) and s. aureus ( mic 6.25 gml ) under similar experimental conditions with reference to the standard drug . considering the compounds 4d and 4h that bear n - methylpiperazine and n-(4-nitrophenyl ) piperazine moiety , respectively , the compounds also possess a substituted phenyl ring at n4 position of s - triazine nucleus . therefore , it may be suggested that irrespective of the bulkiness of piperazine ring , it is the electron withdrawing group at para - position of n4 phenyl ring that imparts the activity against gram positive organism . this is further substantiated by the fact that the compounds substituted with electron releasing groups at para - position of phenyl ring ( 4a , 4c , 4e , 4f , 4 g , 4j , and 4 m ) and compounds with morpholinyl ( 4b , 4i , 4l , and 4o ) and dimethylamino side chain ( 4k and 4n ) have showed either no inhibition or minimal inhibition against gram positive organism . alternatively , few of the aryl piperazine substituted s - triazine ( 4 g , 4k and 4n ) showed moderate to good potency against gram negative bacteria . in particular , the para - fluorophenylpiperazine substituted s - triazine ( 4n ) was found to be the most active and exhibited broad spectrum antibacterial activity . this compound was active against s. epidermidis , k. pneumoniae , and p. aeruginosa with an mic of 6.25 gml whereas for e. coli it showed an mic of 3.125 gml equipotent with reference to the standard drug . the compound 4 g was found to be active against e. coli and p. aeruginosa while compound 4k , wherein the para - fluorophenylpiperazine of 4n has been replaced by para - chlorophenylpiperazine , displayed specific activity against gram negative strain such as e. coli and k. pneumoniae . however , few compounds ( 4c , 4 g , and 4n ) have demonstrated antifungal potency comparable to standard fluconazole . replacement of diethylamino group ( 4n ) with n2 para - methoxyphenyl ring ( 4c and 4 g ) makes the compounds more active against c. albicans and 4n was active against c. tropicalis that showed equipotent antifungal activity ( mic 6.25 gml ) with reference to fluconazole . nevertheless , it was not possible to substantially correlate between the structures and antifungal activity as other compounds having phenyl ring substituted with electron releasing group were unable to elicit favourable activity ( 4a , 4e , 4f , 4j , and 4 m ) . among all the compounds under investigation , compound 4 g demonstrated most significant antifungal activity against c. albicans ( mic 3.125 gml , zone of inhibition 22.2 mm ) . in summary , various 2,4,6-trisubstituted - s - triazine derivatives with varying degree of substitution were synthesized and evaluated for their in vitro antimicrobial activity . all the compounds were invariably substituted with piperazine , n - methylpiperazine or aryl substituted piperazine as one of the substituents of s - triazine scaffold . the tested compounds , 4d , 4 g , 4h , 4k , and 4n , have shown considerable in vitro antibacterial efficacy at the range of 3.1256.25 gml with reference to the standard drug ciprofloxacin . irrespective of the bulkiness of piperazine ring , it is the electron withdrawing group at para - position of n4 phenyl ring that imparts good activity against gram positive organism . from the present study , it may be concluded that the para - fluorophenylpiperazine derivatives of s - triazine ( 4n ) were found to be the most active and exhibited broad spectrum antibacterial activity . among all the compounds under investigation , compound 4 g demonstrated most significant antifungal activity ( 3.125 gml ) against c. albicans .